cloning research paper topics

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Cloning articles within Scientific Reports

Article 29 February 2024 | Open Access

Generation of Fel d 1 chain 2 genome-edited cats by CRISPR-Cas9 system

• Sang Ryeul Lee
• , Kyung-Lim Lee
•  &  Il-Keun Kong

Article 01 July 2022 | Open Access

Insights from one thousand cloned dogs

• P. Olof Olsson
• , Yeon Woo Jeong
•  &  Woo Suk Hwang

Article 12 August 2021 | Open Access

shRNA transgenic swine display resistance to infection with the foot-and-mouth disease virus

• , Haixue Zheng
•  &  Ning Li

Article 12 July 2021 | Open Access

Blastocyst formation, embryo transfer and breed comparison in the first reported large scale cloning of camels

• P. O. Olsson
• , A. H. Tinson
•  &  W. S. Hwang

Article 28 April 2021 | Open Access

In vivo enrichment of busulfan-resistant germ cells for efficient production of transgenic avian models

• Young Min Kim
• , Kyung Je Park
•  &  Jae Yong Han

Article 17 February 2021 | Open Access

Embryonic fate after somatic cell nuclear transfer in non-enucleated goldfish oocytes is determined by first cleavages and DNA methylation patterns

• Alexandra Depincé
• , Pierre-Yves Le Bail
•  &  Catherine Labbé

Article 10 February 2020 | Open Access

Melatonin Protects Rabbit Somatic Cell Nuclear Transfer (SCNT) Embryos from Electrofusion Damage

• Pengxiang Qu
• , Chong Shen
•  &  Enqi Liu

Article 12 September 2019 | Open Access

Aminopeptidase N-null neonatal piglets are protected from transmissible gastroenteritis virus but not porcine epidemic diarrhea virus

• , Shaohua Wang
•  &  Kun Zhang

Article 28 August 2019 | Open Access

Somatic cell nuclear transfer in non-enucleated goldfish oocytes: understanding DNA fate during oocyte activation and first cellular division

• Charlène Rouillon
• , Alexandra Depincé

Article 06 August 2019 | Open Access

Successful cloning of a superior buffalo bull

• Naresh L. Selokar
• , Papori Sharma
•  &  Prem Singh Yadav

Article 18 July 2019 | Open Access

A newly developed cloning technique in sturgeons; an important step towards recovering endangered species

• Effrosyni Fatira
• , Miloš Havelka
•  &  Taiju Saito

Article 18 October 2018 | Open Access

A Real-Time PCR based assay for determining parasite to host ratio and parasitaemia in the clinical samples of Bovine Theileriosis

• Debabrata Dandasena
• , Vasundhra Bhandari
•  &  Paresh Sharma

Article 03 May 2018 | Open Access

Efficient Generation of Transgenic Buffalos (Bubalus bubalis) by Nuclear Transfer of Fetal Fibroblasts Expressing Enhanced Green Fluorescent Protein

•  &  Deshun Shi

Article 16 April 2018 | Open Access

Application of interspecific Somatic Cell Nuclear Transfer (iSCNT) in sturgeons and an unexpectedly produced gynogenetic sterlet with homozygous quadruple haploid

Article 31 January 2018 | Open Access

Comparison of a PCR assay using novel selective primers with current methods in terms of ABO blood phenotyping in rhesus macaques

• Yun-Jung Choi
• , Rae Hyung Ryu
•  &  Jae-Il Lee

Article 19 December 2017 | Open Access

Correction of a Disease Mutation using CRISPR/Cas9-assisted Genome Editing in Japanese Black Cattle

• Mitsumi Ikeda
• , Shuichi Matsuyama
•  &  Misa Hosoe

Article 23 November 2017 | Open Access

Radiographic assessment of the skeletons of Dolly and other clones finds no abnormal osteoarthritis

• , D. S. Gardner
•  &  K. D. Sinclair

Article 10 November 2017 | Open Access

Birth of clones of the world’s first cloned dog

• Min Jung Kim
• , Hyun Ju Oh
•  &  Byeong Chun Lee

Article 11 September 2017 | Open Access

Melatonin enhances the developmental competence of porcine somatic cell nuclear transfer embryos by preventing DNA damage induced by oxidative stress

• Shuang Liang
• , Yong-Xun Jin
•  &  Nam-Hyung Kim

Article 24 April 2017 | Open Access

In vitro developmental ability of ovine oocytes following intracytoplasmic injection with freeze-dried spermatozoa

• Maite Olaciregui
• , Victoria Luño
•  &  Lydia Gil

Article 13 December 2016 | Open Access

Altered DNA methylation associated with an abnormal liver phenotype in a cattle model with a high incidence of perinatal pathologies

• Hélène Kiefer
• , Luc Jouneau
•  &  Hélène Jammes

Article 23 November 2016 | Open Access

Increased gene dosage for β- and κ-casein in transgenic cattle improves milk composition through complex effects

• Götz Laible
• , Grant Smolenski
•  &  Brigid Brophy

Article 21 October 2016 | Open Access

Production and verification of a 2 nd generation clonal group of Japanese flounder, Paralichthys olivaceus

• , Guixing Wang
•  &  Haijin Liu

Article 22 September 2016 | Open Access

Generation of biallelic knock-out sheep via gene-editing and somatic cell nuclear transfer

•  &  Hong-Jiang Wei

Article 17 August 2016 | Open Access

Isozygous and selectable marker-free MSTN knockout cloned pigs generated by the combined use of CRISPR/Cas9 and Cre/LoxP

• , Zaidong Hua
•  &  Xinmin Zheng

Article 29 June 2016 | Open Access

Efficient production of multi-modified pigs for xenotransplantation by ‘combineering’, gene stacking and gene editing

• Konrad Fischer
• , Simone Kraner-Scheiber
•  &  Angelika Schnieke

Article 27 April 2016 | Open Access

Generation of exogenous germ cells in the ovaries of sterile NANOS3 -null beef cattle

• Atsushi Ideta
• , Shiro Yamashita
•  &  Yutaka Sendai

Article 14 April 2016 | Open Access

Generation of heterozygous fibrillin-1 mutant cloned pigs from genome-edited foetal fibroblasts

• Kazuhiro Umeyama
• , Kota Watanabe
•  &  Morio Matsumoto

Article 01 April 2016 | Open Access

The first description of complete invertebrate arginine metabolism pathways implies dose-dependent pathogen regulation in Apostichopus japonicus

• , Li Chenghua
•  &  Lv Zhimeng

Article 10 March 2016 | Open Access

Large-scale production of functional human lysozyme from marker-free transgenic cloned cows

Article 08 February 2016 | Open Access

Generation of TALE nickase-mediated gene-targeted cows expressing human serum albumin in mammary glands

• , Yongsheng Wang
•  &  Yong Zhang

Article 02 November 2015 | Open Access

Generation of hypoxanthine phosphoribosyltransferase gene knockout rabbits by homologous recombination and gene trapping through somatic cell nuclear transfer

• , Weihua Jiang
•  &  Shangang Li

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115 Cloning Essay Topic Ideas & Examples

Inside This Article

Cloning has always been a controversial topic that sparks debates and discussions worldwide. The concept of creating an identical copy of an organism, whether it be a plant, animal, or even a human being, has both fascinated and frightened people for decades. If you have been assigned an essay on cloning and are looking for some inspiration, here are 115 cloning essay topic ideas and examples to help you get started:

• The history and evolution of cloning.
• The ethical implications of cloning.
• The science behind cloning and how it works.
• The benefits and potential applications of cloning in medicine.
• The disadvantages and risks associated with cloning.
• The role of cloning in genetic engineering.
• The cloning of extinct animals: should we bring them back to life?
• The moral dilemma of cloning endangered species.
• The social and psychological impact of human cloning.
• The legal and regulatory challenges of cloning.
• The religious perspectives on cloning.
• The impact of cloning on biodiversity.
• The role of cloning in agriculture and food production.
• The cloning of pets: a luxury or a necessity?
• The cloning of celebrities: the pursuit of immortality?
• The role of cloning in organ transplantation.
• The cloning debate: nature vs. nurture.
• The cloning of body parts: a solution for amputees?
• The cloning of animals for food production: ethical concerns.
• The cloning of endangered plants: preserving biodiversity.
• The cloning of plants for improved crop yield.
• The cloning of athletes: enhancing performance or cheating?
• The cloning of animals for scientific research.
• The potential risks of cloning humans: health and safety concerns.
• The cloning of Neanderthals: ethical considerations.
• The psychological impact on cloned individuals: identity and self-perception.
• The cloning of celebrities: a violation of privacy?
• The cloning of extinct plants: restoring ecosystems.
• The cloning of insects: controlling pests or disrupting ecosystems?
• The cloning of bacteria: implications for antibiotic resistance.
• The cloning of animals for entertainment purposes: ethical considerations.
• The cloning of endangered animals: saving species from extinction.
• The cloning of humans: the quest for immortality.
• The cloning of body parts for transplantation: ethical concerns.
• The cloning of plants for pharmaceutical purposes.
• The potential impact of cloning on global food security.
• The cloning of animals for military purposes: ethical considerations.
• The cloning of humans for organ harvesting: ethical dilemmas.
• The cloning of animals for cosmetic purposes: vanity or necessity?
• The cloning of animals for companionship: ethical considerations.
• The cloning of animals for scientific testing: ethical concerns.
• The cloning of humans for reproductive purposes: ethical dilemmas.
• The cloning of animals for zoos and wildlife conservation.
• The cloning of plants for environmental restoration.
• The cloning of animals for therapeutic purposes: ethical considerations.
• The cloning of humans for research purposes: ethical dilemmas.
• The cloning of animals for military applications: ethical concerns.
• The cloning of humans for genetic enhancement: ethical considerations.
• The cloning of animals for entertainment purposes: ethical dilemmas.
• The cloning of humans for cosmetic purposes: ethical concerns.
• The cloning of animals for agricultural purposes: ethical considerations.
• The cloning of humans for therapeutic purposes: ethical dilemmas.
• The cloning of animals for reproductive purposes: ethical concerns.
• The cloning of humans for military applications: ethical considerations.
• The cloning of animals for genetic enhancement: ethical dilemmas.
• The cloning of humans for entertainment purposes: ethical concerns.
• The cloning of animals for cosmetic purposes: ethical considerations.
• The cloning of humans for agricultural purposes: ethical dilemmas.
• The cloning of animals for therapeutic purposes: ethical concerns.
• The cloning of humans for reproductive purposes: ethical considerations.
• The cloning of animals for military applications: ethical dilemmas.
• The cloning of humans for genetic enhancement: ethical concerns.
• The cloning of humans for cosmetic purposes: ethical dilemmas.
• The cloning of animals for agricultural purposes: ethical concerns.
• The cloning of humans for therapeutic purposes: ethical considerations.
• The cloning of animals for reproductive purposes: ethical dilemmas.
• The cloning of humans for military applications: ethical concerns.
• The cloning of animals for genetic enhancement: ethical considerations.
• The cloning of humans for entertainment purposes: ethical dilemmas.
• The cloning of animals for cosmetic purposes: ethical concerns.
• The cloning of humans for agricultural purposes: ethical considerations.
• The cloning of animals for therapeutic purposes: ethical dilemmas.
• The cloning of humans for reproductive purposes: ethical concerns.
• The cloning of animals for military applications: ethical considerations.
• The cloning of humans for genetic enhancement: ethical dilemmas.
• The cloning of animals for entertainment purposes: ethical concerns.
• The cloning of humans for cosmetic purposes: ethical considerations.
• The cloning of animals for agricultural purposes: ethical dilemmas.
• The cloning of humans for therapeutic purposes: ethical concerns.
• The cloning of animals for reproductive purposes: ethical considerations.
• The cloning of humans for military applications: ethical dilemmas.
• The cloning of animals for genetic enhancement: ethical concerns.
• The cloning of humans for entertainment purposes: ethical considerations.
• The cloning of animals for cosmetic purposes: ethical dilemmas.
• The cloning of humans for agricultural purposes: ethical concerns.

These 115 cloning essay topic ideas and examples should provide you with a solid foundation to start your essay. Remember to choose a topic that interests you the most and conduct thorough research to support your arguments. Good luck!

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Article Contents

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The Cloning Debates and Progress in Biotechnology

• Article contents
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Paul L Wolf, George Liggins, Dan Mercola, The Cloning Debates and Progress in Biotechnology, Clinical Chemistry , Volume 43, Issue 11, 1 November 1997, Pages 2019–2020, https://doi.org/10.1093/clinchem/43.11.2019

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The perception by humans of what is doable is itself a great determiner of future events. Thus, the successful sheep cloning experiment leading to “Dolly” by Ian Wilmut and associates at Roslin Institute, Midlothian, UK, compels us to look in the mirror and consider the issue of human cloning. Should it occur, and if not, how should that opposing mandate be managed? If human cloning should have an acceptable role, what is that role and how should it be monitored and supervised?

In the February 27, 1997, issue of Nature , Ian Wilmut et al. reported that they cloned a sheep (which they named “Dolly”) by transferring the nuclear DNA from an adult sheep udder cell into an egg whose DNA had been removed ( 1 ). Their cloning experiments have led to widespread debate on the potential application of this remarkable technique to the cloning of humans. Following the Scottish researchers’ startling report, President Clinton declared his opposition to using this technique to clone humans. He moved swiftly to order that federal funds not be used for such an experiment and asked an independent panel of experts, the National Bioethics Advisory Commission (NBAC), chaired by Princeton University President Harold Shapiro, to report to the White House with recommendations for a national policy on human cloning. According to recommendations by the NBAC, human cloning is likely to become a crime in the US in the near future. The Commission’s main recommendation is to enact federal legislation to prohibit any attempts, whether in a research or a clinical setting, to create a human through somatic cell nuclear transfer cloning.

The concept of genetic manipulation is not new and has been a general practice for more than a century, through practices ranging from selective cross-pollination in plants to artificial insemination in domestic farm animals.

Wilmut and his colleagues made 277 attempts before they succeeded with Dolly. Previously, investigators had reported successful cloning in frogs, mice, and cattle ( 2 )( 3 )( 4 )( 5 ), and 1 week after Wilmut’s report, Don Wolf and colleagues at the Oregon Regional Primate Research Center reported their cloning of two rhesus monkeys by utilizing embryonic cells. The achievement of Wilmut’s team shocked nucleic acid experts, who thought it would be an impossible feat. They believed that the DNA of adult cells could not perform similarly to the DNA formed when a spermatozoa’s genes mingle with those of an ovum.

On July 25, 1997, the Roslin team also reported the production of lambs that contained human genes ( 6 ). Utilizing techniques similar to those they had used in Dolly, they inserted a human gene into the nuclei of sheep cells. These cells were next inserted into the ova of sheep from which the DNA had been removed. The resulting lambs contained the human gene in every cell. In this new procedure the DNA had been inserted into skin fibroblast cells, which are specialized cells, unlike previous procedures in which DNA was introduced into a fertilized ovum. The new lamb has been named “Polly” because she is a Poll Dorset sheep. The goal of this new genetically engineered lamb is for these lambs to produce human proteins necessary for the treatment of human genetic diseases, such as factor VIII for hemophiliacs, cystic fibrosis transmembrane conductance regulator (CFTR) substance for patients with cystic fibrosis, tissue plasminogen activator to induce lysis of acute coronary and cerebral artery thrombi, and human growth factor.

Charles Darwin was frightened when he concluded that humans were not specifically separated from all other animals. Not until 20 years after his discovery did he have the courage to publish his findings, which changed the way humans view life on earth. Wilmut’s amazing investigations have also created worldwide fear, misunderstanding, and ethical shock waves. Politicians and a few scientists are proposing legislation to outlaw human cloning ( 7 ). Although the accomplishment of cloning clearly could provide many benefits to medicine and to conservation of endangered species of animals, politicians and a few scientists fear that the cloning procedure will be abused.

The advantages of cloning are numerous. The ability to clone dairy cattle may have a larger impact on the dairy industry than artificial insemination. Cloning might be utilized to produce multiple copies of animals that are especially good at producing meat, milk, or wool. The average cow makes 13 000 pounds (5800 kg) of milk a year. Cloning of cows that are superproducers of milk might result in cows producing 40 000 pounds (18 000 kg) of milk a year.

Wilmut’s recent success in cloning “Polly” represents his main interest in cloning ( 8 ). He believes in cloning animals able to produce proteins that are or may prove to be useful in medicine. Cloned female animals could produce large amounts of various important proteins in their milk, resulting in female animals that serve as living drug factories. Investigators might be able to clone animals affected with human diseases, e.g., cystic fibrosis, and investigate new therapies for the human diseases expressed by these animals.

Another possibility of cloning could be to change the proteins on the cell surface of heart, liver, kidney, or lung, i.e., to produce organs resembling human organs and enhancing the supply of organs for human transplantation. The altered donor organs, e.g., from pigs, would be less subject to rejection by the human recipient. The application of cloning in the propagation of endangered species and conservation of gene pools has been proposed as another important use of the cloning technique ( 9 )( 10 ).

The opponents of cloning have especially focused on banning the cloning of humans ( 11 ). The UK, Australia, Spain, Germany, and Denmark have implemented laws barring human cloning. Opponents of human cloning have cited potential ethical and legal implications. They emphasize that individuals are more than a sum of their genes. A clone of an individual might have a different environment and thus might be a different person psychologically and have a different “soul.” Cloning of a human is replication and not procreation.

Morally questionable uses of genetic material transfer and cloning obviously exist. For example, infertility experts might be especially interested in the cloning technique to produce identical twins, triplets, or quadruplets. Parents of a child who has a terminal illness might wish to have a clone of the child to replace the dying child. The old stigma, eugenics, also raises its ugly head if infertile couples wish to use the nuclear transfer techniques to ensure that their “hard-earned” offspring will possess excellent genes. Moral perspectives will differ tremendously in these cases. Judgments about the appropriateness of such uses are outside the realm of science.

Opponents of animal cloning are concerned that cloning will negate genetic diversity of livestock. This also applies to human cloning, which could negate genetic diversity of humans. Cloning creates, by definition, a second class of human, a human with a determined genotype called into existence, however benevolently, at the behest of another. The insulation of selection-of-mate is lost, and the second class is created. Few contrasts could be so clear. Selection-of-mate is so imprecise that, at present, would-be parents have to accept a complete new genome for the sake of including or excluding one or a few traits; cloning, in contrast, is the precise determination of all genes. If we acknowledge that the creation of a second class of humans is unethical, then we preempt any argument that some motivations for human cloning may be acceptable.

The opponents of cloning also fear that biotechnically cloned foods might increase the risk of humans acquiring some malignancies or infections such as “mad cow disease,” a prion spongiform dementia encephalopathy (human Jakob–Creutzfeldt disease).

The technological advances associated with manipulation of genetic materials now permit us to envision replacement of defective genes with “good” genes. Although current progress is not sufficient to make this practical today for human diseases, any efforts to stop such research as a result of cloning hysteria would preclude the development of true cures for many hereditary human diseases. Unreasonable restrictions on the use of human tissues in gene transfer research will have the inevitable consequences of delaying, if not preventing, the development of strategies to combat defective genes.

Wise legislation will enable humankind to realize the benefits of gene transfer technologies without risking the horrors that could arise from misuse of these technologies. Our hope is that such wise legislation is what will be enacted. In our view, the controversy surrounding human cloning must not lead to prohibitions that would prevent advances similar to those described here.

Wilmut I, Schnieke AE, McWhire J, Kind AJ, Campbell KHS. Viable offspring derived from fetal and adult mammalian cells. Nature 1997 ; 385 : 810 -813.

Pennisi E, Williams N. Will Dolly send in the clones?. Science 1997 ; 275 : 1415 -1416.

Gurdon JB, Laskey RA, Reeves OR. The developmental capacity of nuclei transplanted from keratinized skin cells of adult frogs. J Embryol Exp Morphol 1975 ; 34 : 93 -112.

Prather RS. Nuclei transplantation in the bovine embryo. Assessment of donor nuclei and recipient oocyte. Biol Reprod 1987 ; 37 : 859 -866.

Kwon OY, Kono T. Production of identical sextuplet mice by transferring metaphase nuclei from 4-cell embryos. J Reprod Fert Abst Ser 1996 ; 17 : 30 .

Kolata G. Lab yields lamb with human gene. NY Times 1997;166:July 25;A12..

Specter M, Kolta G. After decades of missteps, how cloning succeeded. NY Times 1997;166:March 3;B6–8..

Ibrahim YM. Ian Wilmut. NY Times 1997;166:February 24;B8..

Ryder OA, Benirschke K. The potential use of “cloning” in the conservation effort. Zoo Biol 1997 ; 16 : 295 -300.

Cohen J. Can cloning help save beleaguered species?. Science 1997 ; 276 : 1329 -1330.

Williams N. Cloning sparks calls for new laws. Science 1997;275:141-5..

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117 Cloning Essay Topic Ideas & Examples

🏆 best cloning topic ideas & essay examples, 💡 most interesting cloning topics to write about, 📌 simple & easy cloning essay titles, 👍 good essay topics on cloning, ❓ cloning questions.

• Cloning in Terms of Society and Theology The aim of this paper is to establish the implications of cloning on society and understand the theologians are saying about cloning.
• The Cloning Controversy Considering the fact that most of the controversy about cloning arises from misinformation or ignorance about the matter, this study shall set out to conclusively research on cloning and its merits so as to attest […]
• Whether or Not Human Cloning Should Be Allowed One of the benefits of cloning is the fact that it is able to provide children to people with fertility problems. It is no wonder that the process of cloning cells to form embryos is […]
• The State of Cloning in 2062 One of the concerns of those who are against cloning is that it is inhuman to collect, store and freeze the surplus embryos in order to use them later.
• Cloning of Plants at the Botanic Garden Cloning is now considered to be an efficient means to grow plants in being the result of vegetative propagation while seeds are the result of the natural reproductive phenomenon of plants.
• Molecular Cloning of GFP Gene Molecular cloning is a set of methods in molecular biology that is used to obtain multiple copies of the target DNA fragment. Bacterial transformation is a process of recombinant DNA insertion into a host bacterial […]
• Human Cloning Considerations Analysis The multitude of the biologically born have no way of knowing their fathers and mothers and have all the rights in human society, and no one can even afford to think about violating them.
• Aspects of Cloning for Medical Purposes The second reason for the industry’s support is the cloning of vital organs for use in medicine, as it is known that there is a shortage of donor organs in the world.
• Human Cloning and Kantian Ethics The current paper will define the issue of human cloning through the prism of Kantian ethics and support the idea of reproductive cloning being a contravention of human dignity and fundamental biological principles.
• The Human Cloning Issue and Ethics Additionally, as expressed by Ayala, “the biological endowment of mankind is rapidly deteriorating,” and cloning allows us to resolve such issues. As seen in the example of Frankenstein, “breatheless horror and disgust” are followed by […]
• Cloning, 3D Printing, and Artificial Parts: Replacement Strategies The possibility to turn such cells into any other is the main advantage of the method. This is 3D printing, apparently; as mentioned, it continues to grow more popular in medicine, which calls for studying […]
• Cloning: Genetically Identical Copy The clone develops in the womb and eventually, the adult female gives birth, with the new clone having an identical genetic makeup to the organism from which the somatic cell originated.
• Human Cloning and the Challenge of Regulation Cloning is, therefore, a highly beneficial process from a scientific standpoint, and it has the potential to usher in a new era of technological progress.
• Should Cloning Be 100% Legal or Illegal? After all, an embryo is recognized as a living organism, and for cloning experiments, embryo cells would have to be killed in the research.
• Genetic Modification and Cloning Even though it is hard to predict all the outcomes of genetic modification and cloning, I would suggest using CRISPR Cas9 in treating retinal diseases such as the one described in the case study.
• Religious Perspective on Human Cloning The cloning of embryos exposes little humans to the danger of death. The article evaluates the position of religions in the world of technological reproduction.
• DNA Cloning and Sequencing: The Experiment The plasmid vector pTTQ18 and the GFP PCR product will be digested with restriction enzymes and the desired DNA fragments obtained thereof will be purified by Polyacrylamide gel electrophoresis and ligated with DNA ligase resulting […]
• Cloning Humans as a Controversial Question In this case, the offender is likely to cheat on the police to make the innocent be imprisoned and then continue to break the law.
• Genetics, Reproductive and Cloning Technology in “Frankenstein” If Mary Shelley was for the idea of cloning technology, I think her novel would have ended up with Frankenstein creating a female companion for the monster to compliment the theme of love in the […]
• The Case of Human Cloning at Kyunghee University The objective of the KUMC in the research was to conduct in vitro fertilization of the ova but the researchers went ahead and performed human cloning using some of the ova that they had obtained […]
• Counterarguments to Human Cloning One of the most controversial is the attempt to reproduce an exact replica of a human being through the process of cloning.
• Ethical Debate on Human Cloning Cloning refers to the scientific multiplication and production of new cells to reproduce individuals that resemble their natural counterparts. These proponents insist that cloning will lead to the production of individuals that are resistant to […]
• Controversies in Therapeutic Cloning The embryonic cells have a potential to transform into any type of cell in the body and because of this, opponents of therapeutic cloning assert that the procedure equates murder.
• The Concept of DNA Cloning In the approach based on cells both the replicating molecule or the biological vehicle known as the vector and the foreign DNA fragment are cut using the same restriction enzyme to produce compatible cohesive or […]
• Can Cloning Technology Be Useful for Endangered Species? This is because animal cloning is popularly understood as the creation of a copy of another animal, much the same way as the capability to create twins but in the laboratory.
• Animal and Reproductive Cloning: Current Events It is the nucleus that contains the DNA of the donor. Its DNA structure is similar to the donor.”The blastocyst is then transferred to the uterus of a surrogate mother”.
• Cloning and the Principles That Should Regulate It Since the research of the possibilities of cloning, as well as the opportunities that it opens for humankind, is still in process, it is worth stressing that the existing ethical principles have not been shaped […]
• Medical Ethics. Reproductive and Therapeutic Cloning I suppose that cloning is one of the breakthroughs that need the system of counterbalance providing a holistic approach to the problem.
• Definition, Benefits and Legislations on Human Cloning There are a number of ways in which the human cloning is beneficial to mankind the examples include: Better Understanding of Genetic Diseases.
• Animal Cloning and Engineering Another issue of especial importance to people is the preservation of endangered species of animals and breeding perfect samples of a kind since the achievement of the desired objective in purely biological ways is more […]
• Cloning of Organisms and Its Approaches Artificial embryo twining is the traditional way of cloning and can be said to be the lowest technology in the art of cloning.
• Subsequent Cloning of PARK2 Gene The following description is a series of important events that led to the identification and subsequent cloning of the PARK2 gene responsible for Parkinson’s disease.
• Understanding the Human Cloning Concept All the religions of the world admit that the human beings were created by the God, and it is not in the human power to duplicate God’s creatures.
• Human Cloning Technology and Its Justification Since human cloning is still in the experimental stage and the criticism for and against the subject is replete with valid reasons rational thinkers will be put to the dilemma in agreeing with either of […]
• Therapeutic and Reproductive Cloning, Ethical Issues However, a common problem is that though the person may have consented to the use of his biological samples for genetic research, he may not be aware of the future developments of genetic research to […]
• No to Cloning for Medical Research Those who do not subscribe to cloning for biomedical research believe that the embryo is in fact “one of us”; a human life in process an equal member of the species “Homo Sapiens” in the […]
• Cloning Impact of Science & Technology on Society Technically speaking, cloning is a means of isolating particular parts of the genome in small fragments of DNA and making copies of and studying the sequence in another organism. And they should be open to […]
• Cloning, Expression, and Crystallisation of Pectate Lyase The emergence of molecular cloning has enhanced the application of pectate lyases in industrial processes of manufacturing natural fibres and fruit juices.
• Debate on Human Reproductive Cloning According to Baird, human cloning should be prohibited for the simple reason that the onus of justification will be placed on the shoulders of those performing the cloning rather than those who want the cloning […]
• Ethical Issues on Human Therapeutic and Reproductive Cloning The two types of cloning differ in the procedure involved and the objective of the process. In the case of reproductive cloning, the egg is already fertilized and its failure to develop into a complete […]
• Human and Animal Production Cloning Concepts This research paper thus seeks to examine the concept of human and animal reproductive cloning with an aim of investigating the tenets of this concept and the perspective of society on the issue from ethical, […]
• Ethics of Cloning It is important to understand that cloning is not associated with the production of a clone that has the same size and age as its donor, but rather, it is a form of twinning referred […]
• The Human Cloning Debates Nonetheless, the scientists opposed reproductive cloning claiming that the practice undermines the uniqueness of humankind and that it is unethical to put the lives of clones in a condition of being susceptible to harm or […]
• The New Advancements in Cloning and the Ethical Debate Surrounding It Cellular cloning involves use of somatic cells to produce a cell line identical to the original cell, and this can be used to produce therapies like those of molecular cloning.
• The Concept of Human Cloning Human cloning on the other hand refers to the process of creation of genetically copy of a human. The Adult DNA cloning is the process that entails removing the DNA from the embryo and replacing […]
• The Issue of Cloning as Described in Aldous Huxley’s Brave New World
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• The Analysis of Genetic Engineered Cloning in Modern Society and Alterations to the DNA
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• Upgrading Cybercafé and Installing Cloning Software
• The Positive, Negative and Ethical Aspects of Human Cloning
• The Description of Cloning and the Scientific Advancement Toward Human Cloning
• The Potential Benefits of Cloning and Genetic Engineering to the Future of Society
• The Electric Potential of the Female Body Liquids and the Effectiveness of Cloning
• The Sensitivity of the Subject of Cloning
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• The New Breakthrough in Cloning Is a Great Advance in Biotechnology
• The Several Compelling Reasons Why Cloning Should Not Be Legalized
• The Deficiencies of Artificial Cloning for Realistic Medical and Scientific Purposes
• The Important Points in the Controversial Ethical Issue of Human Cloning
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• The Science And The Laws Impacting Human Cloning
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• The Moral and Ethical Implications of Human Cloning
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• Has Cloning Been Accomplished in Humans?
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• How Has the Media Trained People on the Ethics of Cloning?
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• Does Religion Really Allow Cloning?
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• What Is the Future of Human Cloning?
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Articles on Cloning

Displaying 1 - 20 of 21 articles.

Meet the world’s largest plant: a single seagrass clone stretching 180 km in Western Australia’s Shark Bay

Elizabeth Sinclair , The University of Western Australia ; Gary Kendrick , The University of Western Australia ; Jane Edgeloe , The University of Western Australia , and Martin Breed , Flinders University

Can we resurrect the thylacine? Maybe, but it won’t help the global extinction crisis

Corey J. A. Bradshaw , Flinders University

Why Barbra Streisand’s cloned dogs aren’t identical to the original pet

Russell Bonduriansky , UNSW Sydney

The Force of biology is strong in Star Wars

Allison E. McDonald , Wilfrid Laurier University

Dolly the sheep didn’t develop premature arthritis after all – and that’s good news for cloning

Kevin Sinclair , University of Nottingham

20 years after Dolly: Everything you always wanted to know about the cloned sheep and what came next

George Seidel , Colorado State University

More lessons from Dolly the sheep: Is a clone really born at age zero?

José Cibelli , Michigan State University

Human genome editing report strikes the right balance between risks and benefits

Merlin Crossley , UNSW Sydney

Dolly’s ‘sisters’ show cloned animals don’t grow old before their time

What does it mean for researchers, journalists and the public when secrecy surrounds science?

Jeff Bessen , Harvard University

Why the case against designer babies falls apart

Hugh McLachlan , Glasgow Caledonian University

The curse of Frankenstein: how archetypal myths shape the way people think about science

Alan Levinovitz , James Madison University

If you could clone yourself, would you still have sex?

Angela Crean , UNSW Sydney ; Nathan W Burke , UNSW Sydney , and Russell Bonduriansky , UNSW Sydney

Produce mammoth stem cells, says creator of Dolly the sheep

Ian Wilmut , The University of Edinburgh

Mammoth cloning: the ethics

Julian Savulescu , Monash University and Russell Powell , Boston University

De-extinction is about as sensible as  de-death

Corey J. A. Bradshaw , University of Adelaide

Seeds without sex – some racy findings on the cloning of plants

John Bowman , Monash University

Caveman ethics? The rights and wrongs of cloning Neanderthals

Neil Levy , Florey Institute of Neuroscience and Mental Health

Women sought for Neandertal surrogacy? Not Yeti, thankfully

Darren Curnoe , UNSW Sydney

Cheek cells maintain immune system status quo

Cardiff University

Related Topics

• Animal health
• Dolly the sheep
• Reproduction
• Woolly mammoth

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Professor of Developmental Biology, University of Nottingham

Professor of evolutionary biology, UNSW Sydney

Matthew Flinders Professor of Global Ecology and Models Theme Leader for the ARC Centre of Excellence for Australian Biodiversity and Heritage, Flinders University

Emeritus Professor at the MRC Centre for Regenerative Medicine, The University of Edinburgh

Professor of Biology, Wilfrid Laurier University

Professor of Genetics, Monash University

Scientific Director LARCEL-BIONAND, Spain and Professor of Animal Biotechnology, Michigan State University

Visiting Professor in Biomedical Ethics, Murdoch Children's Research Institute; Distinguished Visiting Professor in Law, University of Melbourne; Uehiro Chair in Practical Ethics, University of Oxford

Honorary Associate Professor, UNSW Sydney

Ph.D. Candidate in Chemical Biology, Harvard University

Deputy Vice-Chancellor Academic Quality and Professor of Molecular Biology, UNSW Sydney

Assistant Professor of Religion, James Madison University

Research fellow, University of Sydney

Professor Emeritus of Applied Philosophy, Glasgow Caledonian University

Senior Research Fellow, Uehiro Centre for Practical Ethics, University of Oxford

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74 Cloning Essay Topics

🏆 best essay topics on cloning, ✍️ cloning essay topics for college, 🎓 most interesting cloning research titles, 🌶️ hot cloning ideas to write about.

• Negative Effects of Human Cloning
• Should Human Cloning Be Allowed?
• Cloning Moral and Ethical Issues
• Cloning in Never Let Me Go by Kazuo Ishiguro
• Cloning Discussion: Pros and Cons
• Ethical Issues in Human Cloning: Response
• Human Reproductive Cloning: Benefits and Drawbacks
• Aspects of Human Embryos Cloning The paper describes and addresses the prompts involved in reproductive cloning, such as the overview of the events, the parties involved, values and motivations.
• Genetic Engineering: Cloning With Pet-28A Embedding genes into plasmid vectors is an integral part of molecular cloning as part of genetic engineering. An example is the cloning of the pectate lyase gene.
• Human Embryo Cloning in the United States Human embryo cloning should not be allowed in the United States because it is an affront to human life and dignity.
• Ethical Issues in Animal Cloning: Acceptable Risk? Engineered animals suffer because of low efficiency thus causing huge amounts of deaths and pregnancy-related predicaments including miscarriage.
• An Experiment in DNA Cloning and Sequencing The aim of this experiment is to clone a fragment of DNA that includes the Green Fluorescent Protein (GFP) gene into the vector pTTQ18, which is an expression vector.
• Analysis of Articles by Kevles and Krauthammer on Cloning This article analyzes two articles by Kevles and Krauthammer with very different views on cloning, and points out their strengths and weaknesses.
• DNA Cloning and Sequencing: The Vector pTTQ18 This study will use a cell-based process approach where the vector pTTQ18 will be used for expression and use an ampicillin resistance gene to conduct the cloning process.
• Cloning: Issues and Moral Aspects Thesis Cloning is unpredictable and uncertain, so it should be limited to general research only involving any human embryos
• Cloning Influenced by Mobile Cell phones and SIM card cloning cause lots of problems not only to police but also to cell phone companies who suffer financial expenses because of illegal phone system access.
• Genetic Engineering and Cloning Controversy Genetic engineering and cloning are the most controversial issues in modern science. The benefits of cloning are the possibility to treat incurable diseases and increase longevity.
• “Human Cloning” by Rudolf Jaenisch This reading summary essay focuses on the reading “Human Cloning – The Science and Ethics of Nuclear Transplantation” by Rudolf Jaenisch.
• Cloning Research Ethics: Ethical Dispute and Issues Cloning research is one of the most discussed issues in the health care system development. While admitting its benefits, the specialists scrutinize its legal and ethical aspects.
• The Cloning of a DNA Fragment, and a Southern Blot Southern blotting can either be used in the determination of small fragment of a single gene or a large DNA sequence such as part of the genome of an organism.
• Is Cloning “Playing God”? Several types of cloning are practiced among human beings: these are reproductive cloning, therapeutic cloning, and replacement cloning.
• Moral Grounds of the Cloning Cloning is a strategy to reproduce and develop a living organism by retaining all its identical features. This would mean obtaining a Photostat copy of the original one.
• Animal Rights: What of Animal Cloning? Animal cloning is a subject that has attracted substantial controversy, especially after scientists revealed that it is also possible to clone humans.
• Human Cloning: A Socio-Legal and Ethical Appraisal
• Brave New Beef: Animal Cloning and Its Impacts
• Nuclear Reprogramming of Cloned Embryos and Its Implications for Therapeutic Cloning
• Arguing That Cloning Is an Affront to Human Dignity
• Genuine Fakes: Cloning Extinct Species as Science and Spectacle
• Animal Transgenesis and Cloning: Combined Development and Future Perspectives
• Islamic Perspective on Human Cloning and Stem Cell Research
• Cloning: New Breakthroughs Leading to Commercial Opportunities
• Apelin Signaling: A Promising Pathway From Cloning to Pharmacology
• What Religion Has to Say About Cloning
• Reproductive Cloning: Useful Technology or an Unethical Experiment
• The Benefits of Cloning and Where to Draw the Line
• Molecular Cloning of DNA: An Introduction to Techniques and Problems
• Exploring the Many Potential Problems With Cloning Human Beings
• Marshall Barber and the Century of Microinjection: From Cloning of Bacteria to Cloning of Everything
• Nutritional Value of Milk and Meat Products Derived From Cloning
• The Issues Involved in Cloning: Sociology and Bioethics
• Using Therapeutic Cloning to Fight Human Disease: A Conundrum or Reality?
• Cloning Techniques and Applications in Human Health
• National Legislation Concerning Human Reproductive and Therapeutic Cloning
• Therapeutic Cloning Applications for Organ Transplantation
• The Impact of New Cloning Techniques on the Diagnosis and Treatment of Infectious Diseases
• Advances in Maize Genomics: The Emergence of Positional Cloning
• Cloning Adult Farm Animals: Possibilities & Problems Associated With Somatic Cell Nuclear Transfer
• Potential Uses of Cloning in Breeding Schemes: Dairy Cattle
• Controversial Arguments For and Against the Case of Cloning
• Governing Cloning: United Nations’ Debates and the Institutional Context of Standards
• The US FDA and Animal Cloning: Risk and Regulatory Approach
• Bioprospecting Through Cloning of Whole Natural Product Biosynthetic Gene Clusters
• How Reproductive Cloning Can Save Our Lives in the Future
• Variations and Voids: The Regulation of Human Cloning Around the World
• Cloning From a Gene Database: Bioinformatics
• An Anti-Human Cloning Perspective on Why Genetic Manipulation Should Be Banned
• Criminal Investigation Into Korean Human Cloning
• The Balance Between the Risks and Benefits of Cloning in the Modern World
• Catholic Debate on Stem Cell Research and Embryonic Cloning
• Two Important Issues in Environmental Ethics: Cloning and Genetic Engineering
• Legislative Approaches to Human Cloning in the United States
• Moral and Ethical Issues of Genetic Immortality: To Clone or Not to Clone
• The Impact of Cloning Technology on Biomedical Uses of Livestock
• Views on Cloning: How It Is Wrong to Play God and Create Another Life
• Molecular Cloning as a Powerful Tool for Studying Genes
• Legal Approach to Stem Cell and Cloning Research: A Comparative Analysis of Policies Around the World
• The Cloning of a Self-Replicating RNA Molecule
• Can Artificial Parthenogenesis Sidestep Ethical Pitfalls in Human Therapeutic Cloning?
• Strategies for Cloning and Manipulating Natural and Synthetic Chromosomes
• Cloning the Mammoth: A Complicated Task or Just a Dream?
• Approaches to Cloning Plant Genes Conferring Resistance to Fungal Pathogens
• Global Governance of Human Cloning: The Case of UNESCO
• Reproductive Cloning: Advantages, Disadvantages, and Ethical Issues

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This essay topic collection was updated on June 20, 2024 .

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Recent Advances in Strategies for the Cloning of Natural Product Biosynthetic Gene Clusters

Wenfang wang.

1 College of Life Sciences, Shanghai Normal University, Shanghai, China

Guosong Zheng

2 Shanghai Engineering Research Center of Plant Germplasm Resources, College of Life Sciences, Shanghai Normal University, Shanghai, China

Microbial natural products (NPs) are a major source of pharmacological agents. Most NPs are synthesized from specific biosynthetic gene clusters (BGCs). With the rapid increase of sequenced microbial genomes, large numbers of NP BGCs have been discovered, regarded as a treasure trove of novel bioactive compounds. However, many NP BGCs are silent in native hosts under laboratory conditions. In order to explore their therapeutic potential, a main route is to activate these silent NP BGCs in heterologous hosts. To this end, the first step is to accurately and efficiently capture these BGCs. In the past decades, a large number of effective technologies for cloning NP BGCs have been established, which has greatly promoted drug discovery research. Herein, we describe recent advances in strategies for BGC cloning, with a focus on the preparation of high-molecular-weight DNA fragment, selection and optimization of vectors used for carrying large-size DNA, and methods for assembling targeted DNA fragment and appropriate vector. The future direction into novel, universal, and high-efficiency methods for cloning NP BGCs is also prospected.

Introduction

Natural products (NPs) produced by microbes are a major source of pharmacological agents and industrially useful compounds. With the spread of drug-resistant pathogens rendering widely used antibiotics ineffective, the discovery of new NPs has become an urgent necessity ( Atanasov et al., 2021 ). The development of next-generation sequencing technology has led to the genomes of a vast array of culturable microorganisms being sequenced in recent years. Through analysis of sequenced microbial genomes, a remarkably large number of orphan biosynthetic gene clusters (BGCs) have been discovered, which represent a treasure trove of novel bioactive compounds with potential pharmacological relevance ( Kang and Kim, 2021 ). However, translating these putative BGCs into specialized compounds is a challenge, as the majority of NP BGCs are either poorly or not at all expressed in native hosts under defined conditions ( Li et al., 2021 ). Further, it has been estimated that over 99% of environmental microbes are recalcitrant to culture under laboratory conditions ( Daniel, 2005 ). Now, metagenomics has emerged as a strategic approach to explore uncultivated microbes from environment ( Daniel, 2005 ), which also revealed the presence of a vast array of NP BGCs. In addition, to facilitate the exploration of NP sources from uncultured microbes, many innovative techniques for targeted or high-throughput cultivation of novel microorganisms are emerging rapidly. Nevertheless, further development of cultivation technologies is still required ( Lewis et al., 2021 ).

In the past decades, efforts have been committed to explore this treasure trove and a number of efficient strategies for activating silent gene clusters have been developed, among which the heterologous expression of NP BGCs has been most widely used ( Kang and Kim, 2021 ). An advantage of this strategy is that once a novel metabolite appears in the surrogate host cell wherein the BGC has been introduced, it can be ascribed to the gene cluster with a high degree of confidence ( Hu et al., 2016 ). A prerequisite for heterologous expression is to clone the target BGC into a suitable vector. Traditional library construction method is sequence-independent and has been proven to be efficient for cloning NP BGCs. Recently, it has been successfully employed for cloning NP BGCs larger than 150 kb using the bacterial artificial chromosome (BAC) ( Zhang L. et al., 2017 ; Hashimoto et al., 2018 ; Sun et al., 2018 ). However, it requires considerable screening, which is time-consuming and laborious. In order to directly clone the target BGCs, researchers have developed a variety of DNA cloning or assembly methods, including in vitro DNA assembly (restriction enzyme-mediated assembly, recombination-based assembly, enzyme-independent DNA assembly), as well as in vivo direct cloning methods, such as Red/ET-mediated recombination in Escherichia coli and transformation-associated recombination (TAR) cloning in Saccharomyces cerevisiae ( Abbasi et al., 2020 ; Lin et al., 2020 ).

The previously reported in vitro , in vivo , or even vitro / vivo hybrid technologies for cloning large DNA fragments have distinct mechanisms, advantages, as well as drawbacks. However, regardless of the methods employed, it is necessary to prepare high-quality and high-molecular-weight DNA as well as to select suitable vectors. Further, efficient strategies for assembling large DNA fragments and vectors are required ( Figure 1 ). The cloned BGCs usually have to be refactored in order to become more compatible with the heterologous host. In this review, we will focus on recent developments of the process for high-molecular-weight DNA fragment preparation, vectors used for carrying large-size DNA and methods for assembling target BGCs and vectors, and have a prospect on novel, universal and high-efficiency cloning methods for large-size DNA.

The general workflow for the cloning of natural product BGCs.

Preparation of High Quality and High-Molecular-Weight DNA

Microbial NP BGCs normally range from 10 to 150 kb in length. Thus, methods for preparing high-quality and high-molecular-weight DNA are critical for successful cloning of intact BGCs. Fundamentally, genomic DNA extraction from microorganisms mainly involves three steps: (1) lysis of the cell wall or membrane using chemical disruption [e.g., SDS (sodium dodecyl sulfate)], enzymatic lysis (e.g., lysozyme and proteinase K), or physical disruption (e.g., manually grinding, sonication); (2) removal of all other unwanted cell components including cell wall debris, proteins, polysaccharides, and other metabolic substances by CTAB (cetyltrimethylammonium bromide) and/or phenol-chloroform; (3) recovery of the pure genomic DNA by ethanol precipitation, spin column-based technique, or magnetic bead-based strategy ( Park, 2007 ; Varma et al., 2007 ). Integrity of genomic DNA may be mainly affected by the mechanical shearing and endogenous DNases. In these methods, proteinase K, phenol, and EDTA could suppress DNase activity, to a certain extent, enhancing the integrity of genomic DNA ( Varma et al., 2007 ). To prevent mechanical shearing of DNA, microbial cells (e.g., protoplasts) can be embedded in low-melting-point agarose gels in the form of plugs, resulting in the preparation of megabase-sized DNA ( Zhang M. et al., 2012 ). However, this process takes 3 days, and the operation is complex. A novel method for genomic DNA extraction, which involves cell grinding in liquid nitrogen, lysis with SDS-base buffer, and purification using carboxylated magnetic beads, was recently developed. Using this method, up to 80 kb DNA fragments could be prepared rapidly (∼1 h) and efficiently ( Mayjonade et al., 2016 ). By fine-tuning three critical parameters, including the grinding duration and vibrational frequency, as well as lysis temperature and duration, the sizes of genomic DNA fragments ranging from 79 to 145 kb can be obtained ( Penouilh-Suzette et al., 2020 ). More detailed information of the methods for genome DNA extraction can refer to a recent review ( Gomez-Acata et al., 2019 ). However, no method can be universally applicable to all microorganisms. In general, researchers need to modify or blend different methods to obtain DNA of desired quality ( Varma et al., 2007 ).

In the case where the genome sequence information of native hosts is under-characterized (e.g., environmental DNA), target BGCs can only be obtained through the construction of a genomic DNA library and subsequent screening via PCR or the identification of corresponding products via heterologous expression. Currently, three methods are available for DNA fragmentation for the construction of large-sized fragment libraries, including enzymatic digestion, sonication, and hydrodynamic shearing ( Ignatov et al., 2019 ). Among them, sonication and hydrodynamic methods randomly disrupt the genome, which may cause the shearing of intact BGCs into different segments ( Ignatov et al., 2019 ). So, enzymatic digestion is the most widely used for DNA fragmentation of library construction.

Currently, more and more microbial genome sequences are being published. For cloning small- to mid-sized BGCs, long-amplicon PCR could be used to amplify BGCs fragments, and then entire BGCs were obtained by DNA assembly ( Greunke et al., 2018 ). However, as the length of NP BGCs increases, the probability of mutations introduced by PCR also increases. Fortunately, the development of genome editing tool CRISPR-Cas (clustered regularly interspaced short palindromic repeat-CRISPR-associated protein) system has made it possible to isolate the exact sequence of target BGCs ( Lee et al., 2015 ; Jiang and Zhu, 2016 ). With the aid of Cas9 endonuclease, DNA segment of desired sizes can be obtained through generating the double strand breaks (DSBs) at specific sites within the genome guided by sgRNA. For example, bacterial cultures (e.g., Escherichia coli ) was embedded in a low-melting-point agarose gel plug, treated with lysozyme and proteinase K, and subsequently washed to remove cellular components, leaving behind genomic DNA. Finally, the plug was transferred into cleavage buffer containing Cas9 and corresponding sgRNA pairs, which were designed to target genome segments of different lengths (50, 75, 100, 150, 200 kb). Clear DNA bands at the expected lengths were observed using pulsed-field gel electrophoresis (PFGE) assessment ( Jiang et al., 2015 ). Recently, CISMR (CRISPR-mediated isolation of specific megabase-sized regions of the genome), which enables the targeted isolation of contiguous megabase-sized segments of the mouse genome, has also been developed by improving in vitro CRISPR specificity with the aid of both Target Finder and ZiFIT Targeter software to design 17 base sgRNA other than traditional 20 base target sequences ( Bennett-Baker and Mueller, 2017 ). Further, a highly sensitive novel method for the simultaneous separation and concentration of high-molecular-weight DNA fragments was established by optimizing the formulation of viscoelastic liquids and engineering a capillary system. It was successfully used to isolate a 31.5 kb DNA fragment from the complicated 450 Mb Medicago truncatula genome with the aid of Cas9 cleavage ( Milon et al., 2019 ).

The quality of DNA fragments can be analyzed via fragment analyzer or horizontal agarose gel electrophoresis. DNA fragments of desired sizes can be separated and extracted through multiple rounds of PFGE with different ramped pulse times ( Clos and Zander-Dinse, 2019 ). Regardless of the cloning method used, sufficient amounts of DNA fragments are indispensable. Therefore, the preparation of high-quality and high-molecular-weight DNA fragments is recognized as a critical step in gene cluster cloning ( Sapojnikova et al., 2017 ).

Vectors for BGC Cloning

Given that most NP BGCs are of relatively large in length, appropriate vector systems capable of carrying the entire gene cluster as well as shuffling these genetic segments between different hosts are necessary. Since the first generation of general cloning vectors was introduced in 1973, a variety of high-capacity vectors have been developed so far ( Bajpai, 2014 ). Despite the dazzling choice of commercial and other available vectors, cloning vector selection can be determined by several key criteria, such as the BGC size and GC content, vector copy number, host compatibility of different vectors, selection markers, and multiple cloning sites. Several types of high-capacity vectors are available for cloning large DNA fragments, including cosmid and artificial chromosomes, such as the fungal artificial chromosome (FAC), yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), and P1 phage artificial chromosome (PAC) ( Monaco and Larin, 1994 ; Bajpai, 2014 ; Bok et al., 2015 ; Clevenger et al., 2017 ).

Cosmid vectors, the first generation of high-capacity vectors used in genome research, are hybrids of plasmid and phage λ vector. As such, the cosmid vector encodes cos sequences required for packaging large fragments into the λ capsid and propagates their DNA as a virus or plasmid in the host cell. Since the possibility for cloning large fragments in cosmid vectors was first confirmed in 1979, they have been widely used for the construction of genomic libraries of various biological species, including Drosophila, mice, and humans. The construction of cosmid library is relatively simple and has been widely applied for cloning various NP BGCs. However, as the cosmid library requires tedious screening, it is necessary to combine high-throughput screening and sequencing methods. For example, the anisomycin BGC from Streptomyces hygrospinosus was identified using a bioactivity-guided high-throughput method for cosmid library screening ( Zheng et al., 2017 ). Recently, CONKAT-seq (co-occurrence network analysis of targeted sequences) was used to uncover the potential of the rare BGCs from millions of cosmid clones harboring metagenomic DNA inserts ( Libis et al., 2019 ).

Cosmid vectors can accommodate up to approximately 40 kb of DNA. They are multicopy plasmids in E. coli that facilitate DNA isolation and in vitro manipulation. However, loss of inserted DNA sometimes occurs within cosmid clones, which may be indicative of sequences that are instability in E. coli or of the transcription/translation products of the sequences are toxic to E. coli , particularly at a high copy number.

An F factor cosmid (fosmid), which contains a replicon derived from F factor and exists at a low/single copy number in E. coli , is more stable than its conventional cosmid counterpart ( Kim et al., 1992 ). In addition, fosmid has an inducible oriV replication start point for high copy propagation, if necessary. Recently, a fosmid library containing 10,656 clones of metagenomic DNA isolated from the ATII (the Red Sea brine pool, Atlantis II Deep) lower convective layer (LCL) was functionally screened, and the products of two putative NP BGCs were detected to exhibit antibacterial and anticancer effects ( Ziko et al., 2019 ). Typically, a cosmid or fosmid vector can only accept relatively small BGCs (up to 45 kb), which greatly hampers their application in cloning large NP BGCs.

Artificial Chromosome

To address the limitation of cosmids, artificial chromosomal vectors, including YAC, PAC, BAC, and FAC, which harbor the carrying capacity of 100∼350 kb, have been used for cloning NP BGCs.

Yeast Artificial Chromosome

YAC vectors contain two copies of yeast telomeres for chromosomal stability, a yeast centromere for segregation, a yeast ARS (autonomously replicating sequence) for replication, and appropriate markers for the selection of recombinant molecules ( Burke et al., 1987 ). YAC provides the largest DNA insert capacity among all cloning vector types. Exogenous DNA fragments with sizes up to several hundred kilobase pairs or even as much as 2 Mb can be cloned into YAC vectors ( Monaco and Larin, 1994 ). A cornerstone of the Human Genome Project (HGP) is the cloning of large chromosomal fragments using YAC vectors. However, problems are frequently observed during the use of YAC clones, including chimerics, deletions, and rearrangements. Furthermore, the isolation of YAC clones is challenging because of large sizes ( Ramsay, 1994 ). As a result, each YAC clone must be carefully analyzed to ensure that no DNA rearrangements occur. In addition, the YAC system is established from eukaryotes and mainly used to study eukaryotic genomes, in which randomly distributed ARS sequences of 20∼30 kb, while being rarely in prokaryotic genomes ( Stinchcomb et al., 1980 ).

Phage Artificial Chromosome

The first PAC vector pCYPAC1 combining the characteristics of P1-phage and F factor was developed in 1994 ( Loannou et al., 1994 ). It can be efficiently transformed into E. coli via electrotransformation. Foreign DNA inserted in the PAC exhibits almost no chimerism or rearrangement. The PAC vector can carry DNA fragments of up to approximately 300 kb. The recombinant PAC can stably exist as a single copy and propagate efficiently. To facilitate the use of PAC vectors in Streptomyces strains, the ΦC31 attP - int elements required for chromosomal integration in Streptomcyes was incorporated into a pCYPAC1-derivative vector ( Ioannou et al., 1996 ), generating so-called ESAC ( E. coli – Streptomyces artificial chromosome, pESAC) vectors. Using these pESAC vectors, up to 140 kb segments of Actinomyces DNA can be cloned and introduced into genetically accessible Streptomyces lividans via protoplast transformation, stably maintaining the vector in an integrative form ( Sosio et al., 2000 ). Using PAC vector pESAC13 (a derivative of pESAC) harboring an oriT site, which allows for conjugal transfer instead of time-consuming protoplast transformation, a genomic library of Streptomyces tsukubaensis was generated, and the entire 83.5 kb FK506 (tacrolimus) gene cluster was then identified ( Jones et al., 2013 ). The PAC library of Stretomyces sp. PCS3-D2 was also constructed and analyzed in silico . Two clones containing 130 and 140 kb DNA inserts were identified to harbor Type I and Type III PKS (polyketide synthase) gene clusters, respectively ( Bayot Custodio and Alcantara, 2019 ). The positive rates of recombinant clones containing DNA inserts can be greatly improved by introducing the sacB or URA3 gene into PAC vectors as counter-screening markers, which can catalyze the production of toxicants in the presence of sucrose or 5-fluoroorotic acid (5-FOA), respectively ( Noskov et al., 2003 ; Tang X. et al., 2015 ).

Bacterial Artificial Chromosome

In 1992, the first BAC vector pBAC108L was constructed based on the well-studied E. coli F factor. This BAC vector retained the oriS , repE, parA , and parB of the F factor for replication and copy number control, while also harboring a chloramphenicol resistance marker as well as the bacteriophage λ cosN and Pl loxP sites for specific cleavage by terminase and Cre enzymes, respectively. This BAC vector has been reported to carry human genomic DNA fragments approaching 300 kb ( Shizuya et al., 1992 ). Further, it enables the cloning of large-sized DNA fragments from complex genomic sources into bacteria, where they remain stable and are easily manipulated. However, normally, only 10–50% of the clones carry DNA inserts, depending upon the batch of the vector and insert DNA used ( Shizuya et al., 1992 ). To facilitate the screening of positive clones, the pBeloBAC11 BAC vector contains an additional component, β-galactosidase (encoded by lacZ ), which allows clones with DNA inserts to be readily identified based on an X-gal color change. Additionally, the plndigoBAC vector displays a much faster and deeper X-gal color change as a result of a point mutation in the 3’ end of lacZ ( Shizuya and Kouros-Mehr, 2001 ). Various BAC vectors, such as pStreptoBAC and pSBAC, have been extensively used for library construction with the purpose of cloning target large-sized NP BGCs ( Sosio et al., 2000 ; Martinez et al., 2004 ; Miao et al., 2005 ; Liu et al., 2009 ). These BAC vectors harbor two replication origins. One is ori that is essential for the initiation of single-copy replication in E. coli , which is crucial for stability when large DNA fragments were inserted. The other is oriV , which can be induced to increase DNA yield.

Thus far, when compared to YAC and PAC, the BAC vectors are more commonly employed for NP BGC cloning. When the genomic sequence information of BGCs is unknown (e.g., metagenome), BAC-based library construction strategies for NP discovery are always employed. Recently, using this strategy, several large NP BGCs, such as an aminopolyol polyketide BGC over 150 kb, and a quinolidomicin BGC over 200 kb, have been successfully cloned and heterologously expressed ( Zhang L. et al., 2017 ; Hashimoto et al., 2018 ; Sun et al., 2018 ). However, due to the low positive rates, laborious screening is necessary ( Lin et al., 2020 ). Therefore, high-throughput screening methods have received considerable attention. Recently, the MAPLE (Microfluidic automated plasmid library enrichment) method, which combines BAC libraries with single-cell droplet microfluidic techniques for discovering functional biosynthetic pathways, was developed. Using MAPLE, a type I PKS gene cluster from an Antarctic soil metagenome was isolated and sequenced ( Xu et al., 2020 ). In addition, when the genome sequence is available, the pSBAC vector can be inserted into the flanking regions of target BGCs within the chromosome in advance and the entire target BGCs can then be captured into pSBAC through specific restriction enzyme digestion and self-ligation. Using this method, the meridamycin (MER, ∼95 kb), tautomycetin (TMC, ∼80 kb), pikromycin (PIK, ∼60 kb), and daptomycin (DPT, ∼65 kb) BGCs have been successfully cloned ( Liu et al., 2009 ; Nah et al., 2015 ; Pyeon et al., 2017 ; Choi et al., 2019 ). However, a major drawback is the problematic identification of naturally existing unique restriction enzyme recognition sites on both sides of the target BGCs. Therefore, artificial insertion of a specific DNA sequence into the genome via homologous recombination (HR) is a prerequisite, limiting the application of this method in intractable strains.

Fungal Artificial Chromosome

Besides bacterial strains (especially actinomycetes), fungi are also prolific producers of NPs. However, despite the abundance of available fungal genome data that encode a large number of NP BGCs, the majority of them are silent in laboratory growth conditions and most fungi are not genetically amendable. To efficiently discover fungal NPs, Bok and colleagues created a novel Aspergillus / E. coli shuttle FAC expression vector, which is modified from the BAC vector via inserting the fungal autonomously replicating element AMA1 ( Bok et al., 2015 ). Using FAC and metabolomic scoring (MS), 56 recombinant FACs containing uncharacterized BGCs from diverse fungal species were constructed and expressed in Aspergillus nidulans . Finally, 15 new metabolites were discovered and assigned with confidence to their BGCs ( Clevenger et al., 2017 ). It could be anticipated that the development of FAC will facilitate NP research of fungi in the future.

Standardized and Orthogonal Vectors

With the rapid development of synthetic biology, standardized and orthogonal vectors, which follow uniform and modular standards, have been developed. They enable the rapid and easy exchange of modules and boost the interoperability of genetic devices among different users ( Martinez-Garcia et al., 2020 ). However, within the field of specialized NP synthetic biology, even though there are multifarious vectors for large DNA fragment cloning, few such standard vectors have been constructed. It is well known that the size (from a few kb to more than 100 kb) of NP BGCs, the genomic GC content, and the repeat or similar sequence in the PKS or NRPS (non-ribosomal peptide synthase) genes can affect the choice of vectors for BGC cloning ( Aubry et al., 2019 ). Thus, vectors that are flexible and adapted to various assembly methods are preferred. Recently, a suite of standardized, orthogonal integration vectors based on three site-specific integration systems (ΦBT1, ΦC31, and VWB), four antibiotic resistance genes (conferring resistance against apramycin, spectinomycin, thiostrepton, and ampicillin, respectively), and 14 promoters were constructed in order to characterize heterologous genes in Streptomyces species. However, these vectors were mainly used for monocistronic gene expression ( Phelan et al., 2017 ). A set of 12 standardized and modular (three different resistance markers and four orthogonal integration systems) vectors based on model SEVA plasmids were designed to allow for the assembly of NP BGCs through various cloning methods in Streptomyces species ( Aubry et al., 2019 ). In these vectors, the FLP (flippase) recombination system was also incorporated for the recycling of antibiotic markers and for reducing unwanted homologous recombination when several vectors are used simultaneously ( Aubry et al., 2019 ). It can be expected that through the modularization and orthogonalization of key vector elements, including orthogonal integration systems, origins of replication, antibiotic selection markers, and a variety of cargoes with specific applications, a suitable vector can be quickly designed to efficiently assemble or clone large DNA fragments. It is worth to note that so far, many laboratories have designed and constructed a large number of multifarious vectors according to their own needs. To further promote NP research, laboratories should make their vectors freely available to other research groups.

Assembly/Cloning Methods

High fidelity, effective and seamless assembly of large DNA fragments and appropriate vectors is the pivotal step for obtaining entire NP BGCs for heterologous expression. With the rapid development of synthetic biology, various DNA cloning and assembly methods have been established and successfully utilized for cloning NP BGCs. Depending on the experimental setting, assembly methods can be divided into two categories: in vitro and in vivo DNA assembly ( Juhas and Ajioka, 2017 ; Li et al., 2017 ; Aubry et al., 2019 ; Kang and Kim, 2021 ).

In vitro cloning and assembly approaches include three main types: (1) restriction enzyme-mediated methods, e.g., BioBrick, Golden Gate, and MASTER ligation ( Engler et al., 2009 ; Chen et al., 2013 ); (2) recombination-based assembly methods, such as Gibson assembly ( Gibson et al., 2009 ), ligase cycling reaction (LCR) ( Schlichting et al., 2019 ), direct pathway cloning (DiPaC) ( Greunke et al., 2018 ), and DNA assembly methods based on the use of site-specific integrases (e.g., ΦC31, ΦBT1) ( Li et al., 2017 ); (3) enzyme-independent DNA assembly, including enzyme-free cloning (EFC) and twin primer non-enzymatic DNA assembly (TPA) ( Liang et al., 2017 ). NP BGCs can also be directly captured using several in vivo methods, including the use of Red/ET system-mediated cloning tools, such as linear-linear homologous recombination (LLHR) or linear-circular-homologous recombination (LCHR) ( Fu et al., 2012 ), exonuclease combined with Red/ET (ExoCET) ( Wang et al., 2018 ), transformation-associated recombination (TAR) cloning method based on the natural recombination capability of S. cerevisiae ( Noskov et al., 2011 ; Kouprina et al., 2020 ), and site-specific recombination (SSR) system-based tools ( Du et al., 2015 ; Gao et al., 2020 ).

In vitro DNA Assembly Approaches

Restriction enzyme-mediated methods.

The classic method for DNA assembly is via the use of enzymes for the cutting and ligation of DNA fragments and vectors. However, these will leave scars at the restriction site. To address this problem, type IIs restriction enzymes (e.g., Bbs I, Bsa I, and Bpi I), which cut outside of the recognition sites and generate single-stranded DNA overhangs, are employed. The DNA overhangs can be appropriately designed to guide the corresponding DNA fragments for ligation in a designated order. This method was named Golden Gate ( Figure 2A ), which reflects the concept of modular assembly ( Mitchell et al., 2015 ). Recently, it was employed for refactoring carotenoid biosynthetic pathways. In particular, each biosynthetic gene equipped with different promoters and terminators was assembled, resulting in various expression cassettes. A library containing 96 combinatorial refactored carotenoid pathways was then successfully generated by assembling these cassettes ( Ren et al., 2017 ). Based on type IIs restriction enzymes, a Golden Gate shuffling method was developed, which can achieve the assembly of at least nine DNA fragments in a single step with high efficiency (90%) ( Engler et al., 2009 ). A similar method named MASTER (methylation-assisted tailorable ends rational) ligation based on MspJI, a specific type IIs endonuclease, was also developed for sequence-independent hierarchical DNA assembly. Using the MspJI-mediated method, the blue-colored antibiotic actinorhodin (ACT) BGC (29 kb) from Streptomyces coelicolor was successfully assembled and expressed in a fast-growing Streptomyces sp. ( Chen et al., 2013 ). To be appropriate for Golden Gate cloning, special care should be taken to ensure that the type IIs restriction site is present in opposite orientation at the ends of the vector and DNA fragments but absent in internal sequences ( Marillonnet and Grutzner, 2020 ). However, type IIs enzymes are relatively rare, and thus few options are available. Usually, internal type IIs restriction sites should be removed by silent mutations. In addition, the number of DNA fragments that can be simultaneously assembled is still limited ( Schmid-Burgk et al., 2013 ; Marillonnet and Grutzner, 2020 ).

The main cloning methods for BGC capturing. In vitro DNA assembly method including: (A) restriction enzyme-mediated digestion and ligation (e.g., Golden gate), (B) recombination-based assembly methods (e.g., Gibson assembly, DiPaC, CATCH, and CCTL), (C) enzyme-independent DNA assemble method. In vivo assembly approaches including: (D) phage-recombinase-mediated HR in E. coli , (E) TAR cloning in yeast. (F) Site-specific integrase (e.g., Cre/loxP, ΦC31, or ΦBT1) mediated cloning.

Recombination-Based Assembly Methods

Although traditional restriction cutting and ligation methods are still widely used, their low efficiency and enzyme site-dependence do not meet the increasing demand for assembling large DNA fragments. Thus, recombination-based assembly methods ( Figure 2B ) based on the existence of short homologous regions at the extremities of DNA fragments and vectors are attracting more attention. These methods include ligation-independent cloning (LIC) ( Aslanidis and de Jong, 1990 ), sequence and ligation-independent cloning (SLiC) ( Li and Elledge, 2012 ), seamless ligation cloning extract (SliCE) ( Zhang Y. et al., 2012 ), circular polymerase extension cloning (CPEC) ( Quan and Tian, 2014 ), Gibson assembly, and Cas9-associated targeting of chromosome segments (CATCH) ( Jiang et al., 2015 ) and so on. Recombination-based DNA assembly usually employs one to three enzymes in the in vitro reaction, wherein DNA polymerase, exonuclease, and ligase are the most commonly used. Hereafter, we provide a brief introduction to the above-mentioned assembly methods.

Ligation-independent cloning mediates the assembly between a DNA fragment and a PCR-amplified vector with a 12-nt tail complementary to the DNA fragment’s end. It does not require the use of restriction enzymes and T4 DNA ligase. The 3′-terminal sequence can be removed via the (3′→5′) exonuclease activity of T4 DNA polymerase, leading to DNA fragments with a 5′-overhang (10–12 nt in length), which results in annealing and circularization between vector molecules and DNA fragments mediated by the 10–12-nt cohesive ends ( Aslanidis and de Jong, 1990 ). Based on this method, SLIC was developed, which can achieve the assembly of multiple DNA fragments in a single reaction by combining in vitro HR and single-strand annealing. SLIC is more efficient at very low DNA concentrations, especially in the presence of the HR protein RecA ( Li and Elledge, 2007 ). SLiCE is a highly cost-effective method, which utilizes cell extracts from E. coli with overexpression of the λ phage Red recombination system for DNA assembly in vitro . This method provides an effective strategy for directional seamless DNA cloning from BAC or complex genomes ( Zhang Y. et al., 2012 ).

The Gibson assembly method uses three commercial enzymes (T5 exonuclease, Phusion DNA polymerase, and Taq DNA ligase) for the assembly of DNA fragments with short homologous ends in vitro . Unlike the T4 DNA polymerase in LIC, which produces a 5’-overhang, T5 exonuclease chews back the homologous ends to generate 3’-overhangs, which anneal to each other, followed by Phusion DNA polymerase and Taq DNA ligase, which fill the gap and covalently link the fragments, respectively. However, Gibson assembly cannot be efficiently employed for the assembly of DNA fragments with high GC content due to high vector self-ligation background. Recently, a modified Gibson assembly method was developed by adding a pair of universal terminal overhangs with high AT content (21 bp) to the ends of the BAC vector, greatly reducing vector self-ligation ( Li L. et al., 2015 ). Using this method, a 67 kb pristinamycin II (PII) BGC from Streptomyces pristinaespiralis was hierarchically assembled from 15 PCR-amplified fragments ( Li L. et al., 2015 ). In addition, a T5 exonuclease-mediated DNA assembly (TEDA) method was established, in which homologous ends were treated with T5 exonuclease alone. After annealing, the reaction sample was transformed into E. coli to repair the gap and form a phosphodiester bond to link the fragment and vector with the endogenous DNA repair enzymes. The results indicated that the cloning efficiency of TEDA was higher than that of the traditional Gibson assembly tool ( Xia et al., 2019 ). The development of CRISPR technology has greatly promoted recombination-mediated DNA assembly. A novel DNA assembly method named CATCH was developed by combining the in vitro CRISPR/Cas9 endonuclease-mediated genome treatment and Gibson assembly, which could achieve the direct cloning of large bacterial genomic segments (up to 100 kb) ( Jiang et al., 2015 ). Using this tool, the 78-kb bacillaene BGC from Bacillus subtilis was cloned into a BAC vector at a ∼12% positive rate. In addition, the 36 kb jadomycin BGC from Streptomyces venezuelae and the 32 kb chlortetracycline BGC from Streptomyces aureofaciens , were also successfully captured with a ∼90% positive rate, highlighting the versatility of CATCH for cloning large BGCs ( Jiang et al., 2015 ). It should be noted that these recombination-based methods (e.g., Gibson assembly, CATCH) might be inefficient when homologous regions of the fragment extremities have complicated DNA sequences, such as secondary hairpin structure formation or high GC content ( Casini et al., 2014 ; Li L. et al., 2015 ).

Unlike Cas9 which introduces double-strand breaks (DSBs) and produces blunt ends, Cpf1 (Cas12a) cleaves target DNA and produces sticky overhangs, which makes Cpf1 an alternative tool for DNA assembly in vitro ( Lei et al., 2017 ). Recently, a method named CCTL (Cpf1-assisted Cutting and Taq DNA ligase mediated Ligation) was developed, with the 8-nt sticky end produced by Cpf1 cleavage for ligation instead of homologous sequences in recombination-based methods, which therefore make the CCTL is suitable for cloning complicated DNA sequences ( Lei et al., 2017 ). However, the requirement of specific PAM limits its application. To address this limitation, the PAM specificity of Cas12a was expanded via specific structure-guided mutagenesis and two engineered Cas12a variant EP15 and EP16 were obtained, which increased the targeting range by fourfold. Based on this modified Cas12a, the iCOPE (improved Cas12a-assisted one-pot DNA Editing) method was developed, which can avoid many of the DNA sequence constraints ( Wang et al., 2019 ).

In addition to the methods described above, various in vitro novel DNA assembly methods for BGCs cloning have been designed and established. These include the assembly of fragment ends after PCR (AFEAP cloning) ( Zeng et al., 2017 ), versatile genetic assembly system (VEGAS) ( Mitchell et al., 2015 ), uracil-specific excision reagent cloning (USER) ( Salomonsen et al., 2014 ), LCR ( de Kok et al., 2014 ), DNA assembly with thermostable exonuclease and ligase (DATEL) ( Jin et al., 2016 ), overlap extension PCR and recombination (OEPR Cloning) ( Liu C. J. et al., 2017 ), direct pathway cloning (DiPaC) ( Greunke et al., 2018 ), as well as DiPaC combination of SLIC cloning ( D’Agostino and Gulder, 2018 ), which provide alternatives for large fragment cloning under different experimental conditions.

Enzyme-Independent DNA Assembly

Enzyme-mediated DNA assembly methods are efficient and straightforward. As mentioned above, Golden Gate assembly is robust and suitable for assembling over 15 DNA fragments with high efficiency and fidelity. However, due to limited commercially available Type IIs endonucleases, it is not always possible to find suitable restriction enzymes that avoid the naturally occurring Type IIs sites within BGCs ( Liang et al., 2017 ). Therefore, additional efforts are needed to modify the sequences of BGCs in order to eliminate the undesired cut sites. Gibson assembly is versatile, but its efficiency and fidelity drop sharply when the number of fragments is more than four. Furthermore, essential components such as promoters, ribosomal binding sites, and terminators are notoriously difficult for Gibson assembly because of their secondary structures ( Liang et al., 2017 ). Enzyme-independent DNA assembly methods can realize DNA assembly without enzymes, which saves costs and is especially suitable for high-throughput settings. These approaches mainly include enzyme-free cloning (EFC), polymerase incomplete primer extension (PIPE), and Twin primer non-enzymatic DNA assembly (TPA).

A highly efficient EFC procedure for DNA assembly was previously established, utilizing tailed PCR primer sets to generate complementary staggered overhangs on both fragments and vectors via a denaturation-hybridization reaction ( Tillett and Neilan, 1999 ). This approach enables directional cloning in a ligase-free manner. Therefore, it is not constrained by the requirement for appropriate enzyme sites. However, this method is mainly used for the assembly of two DNA fragments, and its efficiency is low. TPA for the efficient assembly of multiple PCR fragments was recently developed ( Figure 2C ), allowing for the successful construction of a 31 kb plasmid harboring an n -butanol production pathway (∼26 kb) from five fragments with ∼50% fidelity ( Liang et al., 2017 ). TPA cloning is also seamless and sequence-independent, and its performance rivals even the best in vitro assembly methods. Although these enzyme-free cloning tools provide a number of advantages over other cloning strategies, they still have limitations. For example, these methods usually require a number of specially designed primers, and the assembly capability as well as fidelity drop sharply with increasing fragment size ( Tillett and Neilan, 1999 ; Yuan et al., 2016 ; Liang et al., 2017 ; Richter et al., 2019 ).

In vivo Assembly Approaches

In vitro assembly methods provide flexible cloning of DNA fragments, wherein the DNA fragments can be produced through multiple rounds of PCR or direct chemical synthesis. However, random mutations cannot be entirely ruled out. In addition, incorrect pairing of DNA fragments during assembly may also cause unanticipated mutations, especially in the PKS or NRPS genes, which contain numerous repeat sequences. In vivo DNA cloning methods for direct capture of the target DNA fragment, which are based on the strong homologous recombination ability of E. coli expressing the Red/ET system or that of yeast, have previously been developed. They represent an alternative strategy for BGC cloning. These methods mainly include phage recombinase-mediated homologous recombination cloning in E. coli such as LLHR ( Figure 2D ), transformation-associated recombination-mediated cloning (TAR) in yeast ( Figure 2E ) and site-specific recombination (SSR)-mediated cloning in Streptomyces ( Figure 2F ; Li et al., 2017 ; Abbasi et al., 2020 ; Kang and Kim, 2021 ).

Phage-Recombinase-Mediated HR in E. coli

The endogenous HR system in E. coli is mainly mediated by the chromosome-encoded recombinases RecA/RecBCD ( Abbasi et al., 2020 ). Many cloning strategies based on the endogenous HR have been created, such as in vivo cloning (IVC), in which PCR products containing terminal sequences identical to the two terminals of the linearized vector were co-transfected into E. coli to incorporate PCR fragments into the vector via the high HR ability of E. coli ( Oliner et al., 1993 ). However, due to its strong exonuclease activity, the RecBCD complex can rapidly degrade exogenous linear DNA molecules. The PCR products and linear vector can be introduced and stably maintained only in RecBCD-deficient E. coli strains ( Abbasi et al., 2020 ). In addition, RecA-dependent recombination requires a much longer homologous region (approximately 500 bp). To develop a more efficient and reliable HR system in E. coli , Red/ET recombineering was developed, which depends on phage-recombinases, either RecE/RecT from the Rac prophage or Redα/Redβ from the λ phage ( Zhang et al., 1998 ). RecE and Redα are 5′→3′ ATP-independent exonucleases, while RecT and Redβ are DNA annealing proteins. Another protein, Redγ, identified only in the λ phage, was found to significantly promote the recombination efficiency of Redα/Redβ. It was later identified as an inhibitor of the RecB subunit of RecBCD. This protein protects linear DNA from degradation by endogenous nucleases ( Abbasi et al., 2020 ).

Red/ET recombineering has been established as an efficient in vivo homologous recombination strategy for E. coli ( Wang et al., 2016 ). This technology was first used to reconstitute an entire 43 kb myxochromide BGC from two overlapping cosmids ( Wenzel et al., 2005 ). Subsequently, it has been widely applied for the cloning of a variety of NP BGCs ranging from 11 to 106 kb from different microbes, including Streptomyces, Sorangium , and Cystobacter ( Binz et al., 2008 ; Lesic and Rahme, 2008 ; Wang et al., 2018 ). In these cases, the reconstitution process was mediated by very short homologous regions (usually 40–50 bp) between a replicative circular vector and a linear DNA molecule, and was therefore termed “linear-circular homologous recombination (LCHR).” However, the approach utilizing Redαβ or the truncated version of RecET is inefficient at mediating homologous recombination between two linear DNA molecules, which hampers its use for direct cloning of target BGCs ( Fu et al., 2012 ).

Fu et al. (2012) discovered that full-length RecE along with RecT considerably increased the efficiency of recombination between two linear DNA molecules (a linearized target DNA fragment and a PCR-amplified linear vector backbone flanked with homology arms to the target DNA). Using this LLHR (linear–linear homologous recombination) approach, ten large NRPS and PKS BGCs (with sizes from 10 to 37 kb) from the genomic DNA of Photorhabdus luminescens were directly cloned into linear expression vectors in a one-step recombination event. However, they failed to direct clone the intact 106 kb salinomycin gene cluster from the genome of Streptomyces albus using LLHR. Finally, the group successfully cloned three fragments of salinomycin BGC using LLHR separately and assembled them into a complete one ( Yin et al., 2015 ).

To improve the performance for direct cloning of large-sized (>50 kb) DNA segments from complex genomes such as mammalian genomes, which are three orders of magnitude larger than bacterial genomes, exonuclease ( in vitro ) combined with RecET recombination ( in vivo ) (ExoCET) was developed ( Wang et al., 2018 ). For the in vitro assembly, several exonucleases including T4 polymerase (T4 pol), T5 exonuclease, T7 exonuclease, DNA polymerase I Klenow fragment, T7 DNA polymerase, λ exonuclease, Exonuclease III, and Phusion DNA polymerase, were tested. The 3’ exonuclease activity of T4 polymerase was selected due to it having the highest efficiency and fidelity ( Wang et al., 2018 ). After exonuclease chew-back, the target DNA fragment and the vector were annealed together via the homology arm (about 80 bp) and were then transformed into E. coli for in vivo HR via Red/ET. This concerted action of T4 pol and Red/ET is believed to be more proficient for the direct cloning of long DNA regions than either T4 pol or Red/ET alone ( Wang et al., 2018 ). ExoCET is generally applicable to a broader range of direct cloning with respect to size (up to 106 kb) and genome complexity ( Wang et al., 2018 ). It should be noted that, in order to ensure a high efficiency for the LLHR-mediated cloning method, genomic DNA must be cleaved by unique restriction enzymes near the 5’ and 3’ ends of target BGCs. However, it is not always easy to find appropriate restriction enzyme cutting sites. With the advent of the programmable CRISPR/Cas9 system, which is able to recognize and cut DNA sequences near target BGCs to easily release linear DNA fragments, this limitation could be overcome ( Lee et al., 2015 ; Wang et al., 2018 ). With improved Red/ET technology and rapidly growing microbial genome sequence data in public databases, a variety of complete NP BGCs have been cloned directly from microbial genomic DNA via LLHR ( Table 1 ).

Examples of BGCs cloned by Red/ET recombination (from 2015 to present).

TAR Cloning of NP BGCs

The assembly of two DNA molecules containing homologous sequences via recombination in yeast was first demonstrated by Kunes et al. (1985) . A couple of years later, a convenient method for plasmid construction using this in vivo bimolecular recombination reaction was developed ( Ma et al., 1987 ). Motivated by this method, a transformation-associated recombination (TAR) strategy in yeast based on this approach was later introduced, allowing for the selective isolation of large genomic regions from complex genomic DNA ( Larionov et al., 1997 ; Noskov et al., 2002 ).

Transformation-associated recombination was initially been used to isolate large regions of mammalian genomic DNA in the 1990s ( Larionov et al., 1997 ). The propagation of TAR-generated DNA constructs depends on ARS-like sequences, which can function as an origin of replication in yeast. The ARS sequences are frequently and randomly distributed throughout all eukaryotic genomes per 20–30 kb on average ( Stinchcomb et al., 1980 ). Chromosomal regions with high G + C content are poor in ARS-like sequences, and ARS frequency might be reduced in prokaryotic genomes, which precludes their isolation via the standard TAR method. Noskov et al. (2003) inserted ARS into the TAR vector, using HIS3 as a positive selection marker and URA3 as a negative marker. The modified TAR cloning system enables the isolation of genomic regions lacking yeast ARS-like sequences (e.g., bacterial genome DNA) and eliminates the high vector recircularization background caused by end-joining during yeast transformation ( Noskov et al., 2003 ). This modified TAR cloning method was further extended to capture microbial NP BGCs by constructing the yeast- E. coli – Streptomyces tri-shuttle vector pTARa. Using pTARa, multiple BGCs were directly cloned or reassembled from environmental DNA (eDNA) libraries ( Kim et al., 2010 ). In contrast to pTARa that harbors oriV , pCAP01, a novel capture vector equipped with a pUC ori , can maintain multiple copies without induction and remained stable even when carrying > 50 kb inserts ( Yamanaka et al., 2012 , 2014 ). Using the pCAP101 vector, a 67 kb silent NRPS BGC responsible for the biosynthesis of taromycin from the marine actinomycete Saccharomonospora sp. CNQ-490 was successfully captured and activated in S. coelicolor M1146 ( Yamanaka et al., 2014 ). However, the construction process of pCAP01-based capture plasmids is tedious and time-consuming. It involves the assembly of a pair of 1-kb capture arms into pCAP01, overlapping with the flanking regions of target BGCs. Larson et al. (2017) streamlined this procedure by employing a fully synthetic 360 bp capture arm, which reduced the duration of the cloning process and opened the door for high-throughput applications. Using this modified TAR method, a 54 kb cosmomycin BGC from Streptomyces sp. CNT-302 was successfully captured ( Larson et al., 2017 ). The range of heterologous hosts compatible with the TAR platform was expanded to the Gram-positive Bacillus subtilis with low G + C content by replacing the high G + C content Streptomyces element in pCAP01 with the Bacillus element ( Bourgouin et al., 1990 ) to yield the yeast- E. coli - B. subtilis tri-shuttle vector pCAPB1. Using pCAPB1, the surfactin BGC was successfully cloned from B. subtilis 1779 ( Li Y. et al., 2015 ). Later, a TAR vector pCAP05 was constructed by introducing an RK2 replicon. It replicates at a low copy number in a wide range of Gram-negative bacteria via the oriV and trfA gene, which determine host range and copy number ( Scott et al., 2003 ; Zhang J. J. et al., 2017 ). Using pCAP05, the violacein BGC (∼8 kb) from marine bacterium Pseudoalteromonas luteoviolacea was cloned and expressed in Pseudomonas putida and Agrobacterium tumefaciens ( Zhang J. J. et al., 2017 ). Overall, TAR has been widely employed for BGC cloning, leading to the identification of many novel NPs ( Alberti et al., 2019 ; Kouprina and Larionov, 2019 ; Table 2 ).

Examples of BGCs cloned by TAR cloning (from 2015 to present).

Although TAR cloning can be used to directly clone NP BGCs of interest, the method exhibits very low cloning efficiency (0.5–2%) due to vector recircularization via end joining in yeast, which leads to time-consuming screening of hundreds of clones. Thus, two different strategies have been introduced to increase the positive rates ( Lee et al., 2015 ; Tang X. et al., 2015 ). The first one is to use a counter-selection marker for colony selection. Tang X. et al. (2015) introduced the URA3 gene under a strong pADH1promoter into pCAP01 in order to generate pCAP03, which allows for convenient screening against recircularization in the presence of 5-FOA. Using pCAP03, a 26 kb thiolactomycin BGC from Salinispora pacifica was captured at a positive rate of 75%, and a 33 kb genome locus containing the thiotetroamide BGC (∼29 kb) was cloned at a positive rate of 20% ( Tang X. et al., 2015 ). The second strategy is to use the RNA-guided Cas9 endonuclease to cleave chromosomal DNA ( Lee et al., 2015 ). Homologous recombination has been reported as more efficient when the linearized capturing vector hooks (homology arms) are located closer to the ends of the target DNA sequences ( Kouprina et al., 2006 ). Although unique restriction enzymes can be theoretically obtained to cleave near the 5’ and 3’ ends of target DNA, it is always challenging to find suitable cutting sites. The programmable CRISPR/Cas9 system was used to precisely cleave both sides of the target DNA, significantly improving TAR cloning efficiency by up to 32% ( Lee et al., 2015 ). Currently, capturing target chromosomal regions requires the screening of less than a dozen transformants. It is conceivable that TAR cloning, combined with a counter-selection marker and the CRISPR/Cas9 system, will further accelerate the direct cloning of microbial NP BGCs. So far, TAR cloning is the only available method for selectively capture chromosomal segments up to 300 kb from complex genomes ( Kouprina and Larionov, 2016 ). Collective examples for the direct cloning of NP BGCs by TAR are summarized in Table 2 .

Site-Specific Integrase-Mediated Cloning

In addition to DNA cloning and assembly methods based on homologous recombination in E. coli or yeast, there are other in vivo cloning systems based on site-specific recombination (SSR), which consist of a specialized recombinase and its target sites. There are two evolutionarily distinct site-specific recombinases with different recombination mechanisms, including tyrosine recombinases (e.g., Cre recombinase) and serine integrases (e.g., ΦC31 and ΦBT1 integrase) ( Fogg et al., 2014 ).

Generally, bacteriophage-derived serine integrases bind to specific 40–60 bp DNA sites (so-called attachment sites derived from the phage attP and cognate bacterial chromosome attB ) and bring these sites together, cut and then rejoin the sites to yield the recombinant product ( Grindley et al., 2006 ). Site-specific serine integration systems have been mainly used to integrate foreign DNA constructs into the attB site of prokaryotes, eukaryotes, or archaea chromosomes for the production of stable engineered strains. Integrases are capable of promoting efficient genomic integration of large NP BGCs (>100 kb) via attP × attB unidirectional recombination ( Myronovskyi and Luzhetskyy, 2013 ). Based on this SSR system, a novel strategy for cloning large BGCs was devised in Streptomyces based on the ΦBT1 integrase ( Du et al., 2015 ). First, the paired ΦBT1 integration sites attB / attP and the replicative plasmid pKC1139 are individually introduced on either side of the target BGC via two single crossover recombination events. Thereafter, the ΦBT1 recombinase is expressed, which mediates the cleavage of the two paired integration sites, resulting in circularization of the target BGC in pKC1139. Recombinant clones containing the target BGC are then extracted and transferred into E. coli for recovery. Using this strategy, the actinorhodin BGC (25 kb) from S. coelicolor , the napsamycin BGC (45 kb), and the daptomycin BGC (157 kb) from Streptomyces roseosporus were successfully isolated with high efficiency greater than 80% ( Du et al., 2015 ). The entire 34 kb neomycin BGC from Streptomyces fradiae CGMCC 4.576 was similarly cloned using the ΦBT1 integration system ( Zheng et al., 2019 ).

The Cre enzyme, as well as Flp and Dre recombinases, belongs to the tyrosine recombinase family. Cre recombinase can specifically and efficiently catalyze recombination between two specific 34-bp sites called loxP . The Cre/ loxP system is effective in both bacterial and eukaryotic cells. Cre-mediated recombination results in the excision of the intervening DNA segment and produces a circular DNA molecule if two loxP sites in the DNA strand are in the same orientation. Therefore, when a cloning vector backbone is included in the intervening DNA, the circularized DNA molecule can replicate as a plasmid. Using this “Cre/ loxP plus BAC” strategy, the 32 kb T3SS (type 3 secretion system) gene cluster from Photorhabdus luminescens and the 78 kb siderophore BGC from A. tumefaciens were successfully cloned ( Hu et al., 2016 ).

However, as described above, SSR-mediated cloning methods require the initial integration of specific sites into the chromosome in advance. Therefore, they cannot be employed in difficult-to-manipulate organisms. Recently, a robust BGC cloning method named CAPTURE (Cas12a-assisted precise targeted cloning using in vivo Cre- loxP recombination) was developed by combining in vitro Cas12a-based treatment of genome and in vivo Cre- loxp recombination. This method could achieve direct NP BGC cloning with high efficiency ( Enghiad et al., 2021 ). The microbial genome was purified and digested by the Cas12 protein to release the target BGC and then mixed with two PCR-amplified vector elements in a T4 DNA polymerase exo + fill-in DNA assembly reaction to join the three fragments into a linear DNA product. Finally, the linear DNA assembly products were transformed into E. coli expressing Cre recombinase for in vivo Cre- loxp circularization. This method avoids pre-insertion sites at both ends of the BGCs in the genome that are difficult to manipulate genetically. In addition, each PCR amplified vector element only contains one loxP site and does not carry the selection marker and the origin of replication, which could eliminate vector recircularization. Using CAPTURE, 47 NP BGCs ranging from 10 to 113 kb from both Actinomyces and Bacilli were directly cloned with up to 100% efficiency. Heterologous expression of the cloned BGCs led to the discovery of 15 previously uncharacterized NPs ( Enghiad et al., 2021 ).

Concluding Remarks

Exploring new antibiotics to combat against emerging drug resistance as well as the identification of new lead drugs for the treatment of various diseases are of utmost necessity. Thus, mining of NPs will continue to play an indispensable role in the drug discovery field. Traditionally, NP BGCs of interest are often cloned by construction of genomic DNA libraries using cosmids, fosmids, or artificial chromosomes. These methods are sequence-independent and have been proven to be efficient for cloning NP BGCs. However, these conventional methods are not suitable for the large-scale and high-throughput discovery of novel natural agents due to the requirement of extensive screening. With the availability of an increasing number of bacterial genome sequences and progress in genetic manipulation techniques, a variety of approaches for the direct cloning of large-sized BGCs from chromosomes have been developed. After carefully preparing high-quality large DNA fragments harboring putative BGCs and selecting appropriate vectors, these BGCs can be assembled or directly cloned with high efficiency in vitro or in vivo ( Tables 1 – 3 ). Upon cloning, BGCs can be introduced into suitable microbial hosts for heterologous expression and subsequent identification of the corresponding products.

Examples of BGCs cloned via in vitro assembly (from 2015 to present).

The aforementioned methods differ in both mechanism and cloning scale, providing effective means to meet different needs. The development of in vivo , in vitro , or even in vivo/in vitro hybrid strategies, especially those employing Cas9 or Cas12a cleavage, has greatly facilitated the cloning or assembly of microbial NP BGCs. It is therefore expected that these methodologies will greatly improve genome mining efforts that precede the discovery of novel compounds. However, to our knowledge, a universal approach suitable for all experimental situations is still lacking. Therefore, the combination of different cloning approaches, and the establishment of novel, easy-to-use, highly efficient, and accurate cloning methods remain a necessity.

Author Contributions

WW and GZ wrote the draft. YL edited the manuscript. All the authors contributed to the article and approved the submitted version.

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Funding. This work was supported by the National Key Research and Development Program (2019YFA0905400 and 2018YFA0903700) and the National Natural Science Foundation of China (31770088 and 31970083).

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• Methodology
• Open access
• Published: 19 January 2015

Molecular cloning using polymerase chain reaction, an educational guide for cellular engineering

• Sayed Shahabuddin Hoseini 1 , 2 &
• Martin G Sauer 1 , 2 , 3  

Journal of Biological Engineering volume  9 , Article number:  2 ( 2015 ) Cite this article

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Over the last decades, molecular cloning has transformed biological sciences. Having profoundly impacted various areas such as basic science, clinical, pharmaceutical, and environmental fields, the use of recombinant DNA has successfully started to enter the field of cellular engineering. Here, the polymerase chain reaction (PCR) represents one of the most essential tools. Due to the emergence of novel and efficient PCR reagents, cloning kits, and software, there is a need for a concise and comprehensive protocol that explains all steps of PCR cloning starting from the primer design, performing PCR, sequencing PCR products, analysis of the sequencing data, and finally the assessment of gene expression. It is the aim of this methodology paper to provide a comprehensive protocol with a viable example for applying PCR in gene cloning.

Exemplarily the sequence of the tdTomato fluorescent gene was amplified with PCR primers wherein proper restriction enzyme sites were embedded. Practical criteria for the selection of restriction enzymes and the design of PCR primers are explained. Efficient cloning of PCR products into a plasmid for sequencing and free web-based software for the consecutive analysis of sequencing data is introduced. Finally, confirmation of successful cloning is explained using a fluorescent gene of interest and murine target cells.

Conclusions

Using a practical example, comprehensive PCR-based protocol with important tips was introduced. This methodology paper can serve as a roadmap for researchers who want to quickly exploit the power of PCR-cloning but have their main focus on functional in vitro and in vivo aspects of cellular engineering.

Various techniques were introduced for assembling new DNA sequences [ 1 – 3 ], yet the use of restriction endonuclease enzymes is the most widely used technique in molecular cloning. Whenever compatible restriction enzyme sites are available on both, insert and vector DNA sequences, cloning is straightforward; however, if restriction sites are incompatible or if there is even no restriction site available in the vicinity of the insert cassette, cloning might become more complex. The use of PCR primers, in which compatible restriction enzyme sites are embedded, can effectively solve this problem and facilitate multistep cloning procedures.

Although PCR cloning has been vastly used in biological engineering [ 4 – 8 ], practical guides explaining all necessary steps and tips in a consecutive order are scarce. Furthermore, the emergence of new high-fidelity DNA polymerases, kits, and powerful software makes the process of PCR cloning extremely fast and efficient. Here we sequentially explain PCR cloning from the analysis of the respective gene sequence, the design of PCR primers, performing the PCR procedure itself, sequencing the resulting PCR products, analysis of sequencing data, and finally the cloning of the PCR product into the final vector.

Results and discussion

Choosing proper restriction enzymes based on defined criteria.

In order to proceed with a concise example, tdTomato fluorescent protein was cloned into an alpharetroviral vector. Consecutively, a murine leukemia cell line expressing tdTomato was generated. This cell line will be used to track tumor cells upon injection into mice in preclinical immunotherapy studies. However, this cloning method is applicable to any other gene. To begin the cloning project, the gene of interest (GOI) should be analyzed. First, we check whether our annotated sequence has a start codon (ATG, the most common start codon) and one of the three stop codons (TAA, TAG, TGA). In case the gene was previously manipulated or fused to another gene (e.g. via a 2A sequence), it happens that a gene of interest might not have a stop codon [ 9 ]. In such cases, a stop codon needs to be added to the end of your annotated sequence. It is also beneficial to investigate whether your GOI contains an open reading frame (ORF). This is important since frequent manipulation of sequences either by software or via cloning might erroneously add or delete nucleotides. We use Clone Manager software (SciEd) to find ORFs in our plasmid sequences; however, there are several free websites you can use to find ORFs including the NCBI open reading frame finder ( http://www.ncbi.nlm.nih.gov/gorf/gorf.html ).

The tdTomato gene contains ATG start codon and TAA stop codon (Figure  1 ). The size of the tdTomato gene is 716 bp.

Overview of the start and the end of the gene of interest. (A) The nucleotide sequences at the start and the end of the tdTomato gene are shown. The coding strand nucleotides are specified in bold (B) The nucleotide sequences of the forward and reverse primers containing proper restriction enzyme sites and the Kozak sequence are shown.

In a next step, PCR primers that include proper restriction enzyme sites need to be designed for the amplification of the GOI. Several criteria should be considered in order to choose the optimal restriction enzymes. First, binding sites for restriction enzymes should be ideally available at a multiple cloning site within the vector. Alternatively they can be located downstream of the promoter in your vector sequence. Restriction enzymes should be single cutters (single cutters target one restriction site only within a DNA sequence) (Figure  2 A). If they are double or multiple cutters, they should cut within a sequence that is not necessary for proper functioning of the vector plasmid and will finally be removed (Figure  2 B). It is also possible to choose one double cutter or multiple cutter enzymes cutting the vector downstream of the promoter and also not within a vital sequence of the plasmid (Figure  2 C). Double cutter or multiple cutter enzymes have two or more restriction sites on a DNA sequence, respectively. Cutting the vector with double or multiple cutters would give rise to two identical ends. In such a case, the insert cassette should also contain the same restriction enzyme sites on both of its ends. Therefore, when the insert and vector fragments are mixed in a ligation experiment, the insert can fuse to the vector in either the right orientation (from start codon to stop codon) or reversely (from stop codon to start codon). A third scenario can occur, if the vector fragment forms a self-ligating circle omitting the insert at all. Once the DNA has been incubated with restriction enzymes, dephosphorylation of the 5′ and 3′ ends of the vector plasmid using an alkaline phosphatase enzyme will greatly reduce the risk of self-ligation [ 10 ]. It is therefore important to screen a cloning product for those three products (right orientation, reverse orientation, self-ligation) after fragment ligation.

Choosing proper restriction enzymes based on defined criteria for PCR cloning. (A) Two single-cutter restriction enzymes (E1 and E2) are located downstream of the promoter. (B) E1 and E2 restriction enzymes cut the plasmid downstream of the promoter several (here two times for each enzyme) times. (C) The E1 restriction enzyme cuts the plasmid downstream of the promoter more than once. (D) The PCR product, which contains the tdTomato gene and the restriction enzyme sites, was run on a gel before being extracted for downstream applications.

Second, due to higher cloning efficiency using sticky-end DNA fragments, it is desirable that at least one (better both) of the restriction enzymes is a so-called sticky-end cutter. Sticky end cutters cleave DNA asymmetrically generating complementary cohesive ends. In contrast, blunt end cutters cut the sequence symmetrically leaving no overhangs. Cloning blunt-end fragments is more difficult. Nevertheless, choosing a higher insert/vector molar ratio (5 or more) and the use 10% polyethylene glycol (PEG) can improve ligation of blunt-end fragments [ 11 ].

Third, some restriction enzymes do not cut methylated DNA. Most of the strains of E. coli contain Dam or Dcm methylases that methylate DNA sequences. This makes them resistant to methylation-sensitive restriction enzymes [ 12 ]. Since vector DNA is mostly prepared in E. coli , it will be methylated. Therefore avoiding methylation-sensitive restriction enzymes is desirable; however, sometimes the isoschizomer of a methylation-sensitive restriction enzyme is resistant to methylation. For example, the Acc 65I enzyme is sensitive while its isoschizomer kpn I is resistant to methylation [ 13 ]. Isoschizomers are restriction enzymes that recognize the same nucleotide sequences. If there remains no other option than using methylation-sensitive restriction enzymes, the vector DNA needs to be prepared in dam − dcm − E. coli strains. A list of these strains and also common E. coli host strains for molecular cloning is summarized in Table  1 . Information regarding the methylation sensitivity of restriction enzymes is usually provided by the manufacturer.

Fourth, it makes cloning easier if the buffer necessary for the full functionality of restriction enzymes is the same because one can perform double restriction digest. This saves time and reduces the DNA loss during purification. It may happen that one of the restriction enzymes is active in one buffer and the second enzyme is active in twice the concentration of the same buffer. For example the Nhe I enzyme from Thermo Scientific is active in Tango 1X buffer (Thermo Scientific) and Eco R1 enzyme is active in Tango 2X buffer (Thermo Scientific). In such cases, the plasmid DNA needs to be first digested by the enzyme requiring the higher buffer concentration (here Eco R1). This will be followed by diluting the buffer for the next enzyme (requiring a lower concentration (here Nhe I)) in the same buffer. However, the emergence of universal buffers has simplified the double digest of DNA sequences [ 15 ]. In our example the vector contains the Age I and Sal I restriction sites. These enzyme sites were used for designing PCR primers (Figure  1 ). It is essential for proper restriction enzyme digestion that the plasmid purity is high. DNA absorbance as measured by a spectrophotometer can be used to determine the purity after purification. DNA, proteins, and solvents absorb at 260 nm, 280 nm, and 230 nm, respectively. An OD 260/280 ratio of >1.8 and an OD 260/230 ratio of 2 to 2.2 is considered to be pure for DNA samples [ 16 ]. The OD 260/280 and 260/230 ratios of our exemplary plasmid preparations were 1.89 and 2.22, respectively. We observed that the purity of the gel-extracted vector and insert DNA fragments were lower after restriction digest; ligation works even in such cases, however, better results can be expected using high-purity fragments.

The following plasmid repository website can be useful for the selection of different vectors (viral expression and packaging, empty backbones, fluorescent proteins, inducible vectors, epitope tags, fusion proteins, reporter genes, species-specific expression systems, selection markers, promoters, shRNA expression and genome engineering): http://www.addgene.org/browse/ .

A collection of cloning vectors of E. coli is available under the following website: http://www.shigen.nig.ac.jp/ecoli/strain/cvector/cvectorExplanation.jsp .

Designing cloning primers based on defined criteria

For PCR primer design, check the start and stop codons of your GOI. Find the sequence of the desired restriction enzymes (available on the manufacturers’ websites) for the forward primer (Figure  3 A). It needs to be located before the GOI (Figure  1 B). The so-called Kozak sequence is found in eukaryotic mRNAs and improves the initiation of translation [ 17 ]. It is beneficial to add the Kozak sequence (GCCACC) before the ATG start codon since it increases translation and expression of the protein of interest in eukaryotes [ 18 ]. Therefore, we inserted GCCACC immediately after the restriction enzyme sequence Age I and before the ATG start codon. Then, the first 18 to 30 nucleotides of the GOI starting from the ATG start codon are added to the forward primer sequence. These overlapping nucleotides binding to the template DNA determine the annealing temperature (Tm). The latter is usually higher than 60°C. Here, we use Phusion high-fidelity DNA polymerase (Thermo Scientific). You can use the following websites for determination of the optimal Tm: http://www.thermoscientificbio.com/webtools/tmc/ .

Designing primers based on defined criteria for PCR cloning. (A-B) Sequences of the forward and the reverse primer are depicted. The end of the coding strand is to be converted into the reverse complement format for the reverse primer design. For more information, please see the text.

https://www.neb.com/tools-and-resources/interactive-tools/tm-calculator .

The Tm of our forward primer is 66°C.

Choose the last 18 to 30 nucleotides including the stop codon of your GOI for designing the reverse primer (Figure  3 B). Then calculate the Tm for this sequence which should be above 60°C and close to the Tm of the forward primer. Tm of the overlapping sequence of our reverse primer was 68°C. Then, add the target sequence of the second restriction enzyme site (in this case Sal I) immediately after the stop codon. Finally, convert this assembled sequence to a reverse-complement sequence. The following websites can be used to determine the sequence of the reverse primer:

http://reverse-complement.com/

http://www.bioinformatics.org/sms/rev_comp.html This is important since the reverse primer binds the coding strand and therefore its sequence (5′ → 3′) must be reverse-complementary to the sequence of the coding strand (Figure  1 A).

Performing PCR using proofreading polymerases

Since the PCR reaction follows logarithmic amplification of the target sequence, any replication error during this process will be amplified. The error rate of non-proofreading DNA polymerases, such as the Taq polymerase, is about 8 × 10 −6 errors/bp/PCR cycle [ 19 ]; however, proofreading enzymes such as Phusion polymerase have a reported error rate of 4.4 × 10 −7 errors/bp/PCR cycle. Due to its superior fidelity and processivity [ 20 – 22 ], the Phusion DNA polymerase was used in this example. It should be noted that Phusion has different temperature requirements than other DNA polymerases. The primer Tm for Phusion is calculated based on the Breslauer method [ 23 ] and is higher than the Tm using Taq or pfu polymerases. To have optimal results, the Tm should be calculated based on information found on the website of the enzyme providers. Furthermore, due to the higher speed of Phusion, 15 to 30 seconds are usually enough for the amplification of each kb of the sequence of interest.

After the PCR, the product needs to be loaded on a gel (Figure  2 D). The corresponding band needs to be cut and the DNA extracted. It is essential to sequence the PCR product since the PCR product might include mutations. There are several PCR cloning kits available some of which are shown in Table  2 . We used the pJET1.2/blunt cloning vector (Thermo Scientific, patent publication: US 2009/0042249 A1, Genbank accession number EF694056.1) and cloned the PCR product into the linearized vector. This vector contains a lethal gene ( eco47IR ) that is activated in case the vector becomes circularized. However, if the PCR product is cloned into the cloning site within the lethal gene, the latter is disrupted allowing bacteria to grow colonies upon transformation. Circularized vectors not containing the PCR product express the toxic gene, which therefore kills bacteria precluding the formation of colonies. Bacterial clones are then to be cultured, plasmid DNA consecutively isolated and sequenced. The quality of isolated plasmid is essential for optimal sequencing results. We isolated the plasmid DNA from a total of 1.5 ml cultured bacteria (yield 6 μg DNA; OD 260/280 = 1.86; OD 260/230 = 2.17) using a plasmid mini-preparation kit (QIAGEN). The whole process of PCR, including cloning of the PCR product into the sequencing vector and transfection of bacteria with the sequencing vector can be done in one day. The next day, bacterial clones will be cultured overnight before being sent for sequencing.

Analysis of sequencing data

Sequencing companies normally report sequencing data as a FASTA file and also as ready nucleotide sequences via email. For sequence analysis, the following websites can be used:

http://blast.ncbi.nlm.nih.gov/Blast.cgi

http://xylian.igh.cnrs.fr/bin/align-guess.cgi

Here we will focus on the first website. On this website page, click on the “nucleotide blast” option (Figure  4 A). A new window opens. By default, the “blastn” (blast nucleotide sequences) option is marked (Figure  4 B). Then check the box behind “Align two or more sequences”. Now two boxes will appear. In the “Enter Query Sequence” box (the upper box), insert the desired sequence of your gene of interest, which is flanked by the restriction sites you have already designed for your PCR primers. In the “Enter Subject Sequence” box (the lower box), enter the sequence or upload the FASTA file you have received from the sequencing company. Then click the “BLAST” button at the bottom of the page. After a couple of seconds, the results will be shown on another page. A part of the alignment data is shown in Figure  4 C. For interpretation, the following points should be considered: 1) the number of identical nucleotides (shown under the “Identities” item) must be equal to the nucleotide number of your gene of interest. In our example, the number of nucleotides of the tdTomato gene together with those of the restriction enzyme sites and the Kozak sequence was 735. This equals the reported number (Figure  4 C). 2) The sequence identity (under the “Identities” item) should be 100%. Occasionally, the sequence identity is 100% but the number of identical nucleotides is lower than expected. This can happen if one or more of the initial nucleotides are absent. Remember, all sequencing technologies have an error rate. For Sanger sequencing, this error rate is reported to range from 0.001% to 1% [ 30 – 33 ]. Nucleotide substitution, deletion or insertion can be identified by analyzing the sequencing results [ 34 ]. Therefore, if the sequence identity does not reach 100%, the plasmid should be resequenced in order to differentiate errors of the PCR from simple sequencing errors. 3) Gaps (under the “Gaps” item) should not be present. If gaps occur, the plasmid should be resequenced.

Sequence analysis of the PCR product using the NCBI BLAST platform. (A) On the NCBI BLAST webpage, the “nucleotide blast” option is chosen (marked by the oval line). (B) The “blastn” option appears by default (marked by the circle). The sequence of the gene of interest (flanked by the restriction sites as previously designed for the PCR primers) and the PCR product are to be inserted to the “Enter Query Sequence” and “Enter Subject Sequence” boxes. Sequences can also be uploaded as FASTA files. (C) Nucleotide alignment of the first 60 nucleotides is shown. Two important items for sequence analysis are marked by oval lines.

The average length of a read, or read length, is at least 800 to 900 nucleotides for Sanger sequencing [ 35 ]. For the pJET vector one forward and one reverse primer need to be used for sequencing the complete gene. These primers can normally cover a gene size ranging up to 1800 bp. If the size of a gene is larger than 1800, an extra primer should be designed for each 800 extra nucleotides. Since reliable base calling does not start immediately after the primer, but about 45 to 55 nucleotides downstream of the primer [ 36 ], the next forward primer should be designed to start after about 700 nucleotides from the beginning of the gene. Different websites, including the following, can be used to design these primers:

http://www.ncbi.nlm.nih.gov/tools/primer-blast/

http://www.yeastgenome.org/cgi-bin/web-primer

http://www.genscript.com/cgi-bin/tools/sequencing_primer_design

Being 735 bp in length, the size of the PCR product in this example was well within the range of the pJET sequencing primers.

After choosing the sequence-verified clone, vector and insert plasmids were digested by the Age I and Sal I restriction enzymes (Figure  5 ). This was followed by gel purification and ligation of the fragments. Transformation of competent E. coli with the ligation mixture yielded several clones that were screened by restriction enzymes. We assessed eight clones, all of which contained the tdTomato insert (Figure  6 ). It is important to pick clones that are large. Satellite clones might not have the right construct. We used a fast plasmid mini-preparation kit (Zymo Research) to extract the plasmid from 0.6 ml bacterial suspension. The yield and purity were satisfying for restriction enzyme-based screening (2.3 μg DNA; OD 260/280 = 1.82; OD 260/230 = 1.41). For large-scale plasmid purification, a maxi-preparation kit (QIAGEN) was used to extract the plasmid from 450 ml of bacterial culture (yield 787 μg DNA; OD 260/280 = 1.89; OD 260/230 = 2.22). The expected yield of a pBR322-derived plasmid isolation from 1.5 ml and 500 ml bacterial culture is about 2-5 μg and 500-4000 μg of DNA, respectively [ 37 ].

Vector and insert plasmid maps A) Illustration of the CloneJET plasmid containing the PCR product. Insertion of the PCR product in the cloning site of the plasmid disrupts the integrity of the toxic gene eco47IR and allows the growth of transgene positive clones. The plasmid was cut with the Age I and Sal I enzymes generating two fragments of 3 kb and 0.7 kb in size. The 0.7 kb fragment (tdTomato gene) was used as the insert for cloning. (B) Illustration of the vector plasmid. The plasmid was cut with the Age I and Sal I enzymes generating two fragments of 4.9 kb and 0.7 kb in size. The 4.9 kb fragment was used as the vector for cloning. AMP: Ampicillin resistance gene; PRE: posttranscriptional regulatory element; MPSV: myeloproliferative sarcoma virus promoter.

Screening of the final plasmid with restriction enzymes. Illustration of the final plasmid is shown. For screening, the plasmid was cut with the Bsiw I enzyme generating two fragments of 4.8 kb and 0.8 kb in size. AMP: Ampicillin resistance gene; PRE: posttranscriptional regulatory element; MPSV: myeloproliferative sarcoma virus promoter.

Some plasmids tend to recombine inside the bacterial host creating insertions, deletions and recombinations [ 38 ]. In these cases, using a recA-deficient E. coli can be useful (Table  1 ). Furthermore, if the GOI is toxic, incubation of bacteria at lower temperatures (25-30°C) and using ABLE C or ABLE K strains might circumvent the problem.

Viral production and transduction of target cells

To investigate the in vitro expression of the cloned gene, HEK293T cells were transfected with plasmids encoding the tdTomato gene, alpharetroviral Gag/Pol, and the vesicular stomatitis virus glycoprotein (VSVG) envelope. These cells, which are derived from human embryonic kidney, are easily cultured and readily transfected [ 39 ]. Therefore they are extensively used in biotechnology and gene therapy to generate viral particles. HEK293T cells require splitting every other day using warm medium. They should not reach 100% confluency for optimal results. To have good transfection efficiency, these cells need to be cultured for at least one week to have them in log phase. Transfection efficiency was 22%, as determined based on the expression of tdTomato by fluorescence microscopy 24 hours later (Figure  7 A-B). To generate a murine leukemia cell line expressing the tdTomato gene for immunotherapy studies, C1498 leukemic cells were transduced with freshly harvested virus (36 hours of transfection). Imaging studies (Figure  7 C) and flow cytometric analysis (Figure  7 D) four days after transduction confirmed the expression of tdTomato in the majority of the cells.

Assessing in vitro expression of the cloned gene. (A, B) HEK293T cells were transfected with Gag/Pol, VSVG, and tdTomato plasmids. The expression of the tdTomato gene was assessed using a fluorescence microscope. Fluorescent images were superimposed on a bright-field image for the differentiation of positively transduced cells. Transfection efficiency was determined based on the expression of tdTomato after 24 hours. Non-transfected HEK293T cells were used as controls (blue histogram). (C, D) The murine leukemia cell line C1498 was transduced with fresh virus. Four days later, transgene expression was assessed by fluorescence microscopy (C) and flow cytometry (D) . Non-transduced C1498 cells were used as controls (blue histogram). Scale bars represent 30 μm.

In this manuscript, we describe a simple and step-by-step protocol explaining how to exploit the power of PCR to clone a GOI into a vector for genetic engineering. Several PCR-based creative methods have been developed being extremely helpful for the generation of new nucleotide sequences. This includes equimolar expression of several proteins by linking their genes via a self-cleaving 2A sequence [ 40 , 41 ], engineering fusion proteins, as well as the use of linkers for the design of chimeric proteins [ 42 – 44 ]. Furthermore, protein tags [ 45 , 46 ] and mutagenesis (site-directed, deletions, insertions) [ 47 ] have widened the applications of biological engineering. The protocol explained in this manuscript covers for most situations of PCR-assisted cloning; however, alternative PCR-based methods are available being restriction enzyme and ligation independent [ 6 , 48 – 51 ]. They are of special interest in applications where restriction enzyme sites are lacking; nevertheless, these methods might need several rounds of PCR or occasionally a whole plasmid needs to be amplified. In such cases, the chance of PCR errors increases and necessitates sequencing of multiple clones. In conclusion, this guideline assembles a simple and straightforward protocol using resources that are tedious to collect on an individual basis thereby trying to minimize errors and pitfalls from the beginning.

Cell lines and media

The E. coli HB101 was used for the preparation of plasmid DNA. The bacteria were cultured in Luria-Bertani (LB) media. Human embryonic kidney (HEK) 293 T cells were cultured in Dulbecco’s Modified Eagle medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 2 mM L-glutamine, 100 mg/ml streptomycin, and 100 units/ml penicillin. A myeloid leukemia cell line C1498 [ 52 ], was cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with the same reagents used for DMEM. Cells were split every other day to keep them on log phase.

Plasmids, primers, PCR and sequencing

A plasmid containing the coding sequence of the tdTomato gene, plasmid containing an alpha-retroviral vector, and plasmids containing codon-optimized alpharetroviral gag/pol [ 53 ] were kindly provided by Axel Schambach (MHH Hannover, Germany). A forward (5′- ACCGGTGCCACCATGGCCACAACCATGGTG-3′) and a reverse (5′-GTCGACTTACTTGTACAGCTCGTCCATGCC-3′) primer used for the amplification of the tdTomato gene were synthesized by Eurofins Genomics (Ebersberg, Germany).

The optimal buffers for enzymes or other reagents were provided by the manufacturers along with the corresponding enzymes or inside the kits. If available by the manufacturers, the pH and ingredients of buffers are mentioned. Primers were dissolved in ultrapure water at a stock concentration of 20 pmol/μl. The template plasmid was diluted in water at a stock concentration of 50 ng/μl. For PCR, the following reagents were mixed and filled up with water to a total volume of 50 μl: 1 μl plasmid DNA (1 ng/μl final concentration), 1.25 μl of each primer (0.5 pmol/μl final concentration for each primer), 1 μL dNTP (10 mM each), 10 μl of 5X Phusion HF buffer (1X buffer provides 1.5 mM MgCl2), and 0.5 μl Phusion DNA polymerase (2U/μl, Thermo Scientific).

PCR was performed using a peqSTAR thermocycler (PEQLAB Biotechnologie) at: 98°C for 3 minutes; 25 cycles at 98°C for 10 seconds, 66°C for 30 seconds, 72°C for 30 seconds; and 72°C for 10 minutes. To prepare a 0.8% agarose gel, 0.96 g agarose (CARL ROTH) was dissolved in 120 ml 1X TAE buffer (40 mM Tris, 20 mM acetic acid, 1 mM EDTA, pH of 50X TAE: 8.4) and boiled for 4 minutes. Then 3 μl SafeView nucleic acid stain (NBS Biologicals) was added to the solution and the mixture was poured into a gel-casting tray.

DNA was mixed with 10 μl loading dye (6X) (Thermo Scientific) and loaded on the agarose gel (CARL ROTH) using 80 V for one hour in TAE buffer. The separated DNA fragments were visualized using an UV transilluminator (365 nm) and quickly cut to minimize the UV exposure. DNA was extracted from the gel slice using Zymoclean™ Gel DNA Recovery Kit (Zymo Research). The concentration of DNA was determined using a NanoDrop 2000 spectrophotometer (Thermo Scientific).

For sequence validation, the PCR product was subcloned using CloneJET PCR cloning kit (Thermo Scientific). 1 μl of blunt vector (50 ng/μl), 50 ng/μl of the PCR product, and 10 μl of 2X reaction buffer (provided in the kit) were mixed and filled with water to a total volume of 20 μl. 1 μl of T4 DNA ligase (5 U/μl) was added to the mixture, mixed and incubated at room temperature for 30 minutes. For bacterial transfection, 10 μl of the mixture was mixed with 100 μl of HB101 E. coli competent cells and incubated on ice for 45 minutes. Then the mixture was heat-shocked (42°C/2 minutes), put on ice again (5 minutes), filled up with 1 ml LB medium and incubated in a thermomixer (Eppendorf) for 45 minutes/37°C/450RPM. Then the bacteria were spun down for 4 minutes. The pellet was cultured overnight at 37°C on an agarose Petri dish containing 100 μg/mL of Ampicillin. The day after, colonies were picked and cultured overnight in 3 ml LB containing 100 μg/mL of ampicillin.

After 16 hours (overnight), the plasmid was isolated from the cultured bacteria using the QIAprep spin miniprep kit (QIAGEN) according to the manufacturer’s instructions. 720 to 1200 ng of plasmid DNA in a total of 12 μl water were sent for sequencing (Seqlab) in Eppendorf tubes. The sequencing primers pJET1.2-forward (5′-CGACTCACTATAGGGAG-3′), and pJET1.2-reverse (5′-ATCGATTTTCCATGGCAG-3′), were generated by the Seqlab Company (Göttingen, Germany). An ABI 3730XL DNA analyzer was used by the Seqlab Company to sequence the plasmids applying the Sanger method. Sequence results were analyzed using NCBI Blast as explained in the Results and discussion section.

Manipulation of DNA fragments

For viewing plasmid maps, Clone Manager suite 6 software (SciEd) was used. Restriction endonuclease enzymes (Thermo Scientific) were used to cut plasmid DNA. 5 μg plasmid DNA, 2 μl buffer O (50 mM Tris–HCl (pH 7.5 at 37°C), 10 mM MgCl2, 100 mM NaCl, 0.1 mg/mL BSA, Thermo Scientific), 1 μl Sal I (10 U), and 1 μl AgeI (10 U) were mixed in a total of 20 μl water and incubated (37°C) overnight in an incubator to prevent evaporation and condensation of water under the tube lid. The next day, DNA was mixed with 4 μl loading dye (6X) (Thermo Scientific) and run on a 0.8% agarose gel at 80 V for one hour in TAE buffer. The agarose gel (120 ml) contained 3 μl SafeView nucleic acid stain (NBS Biologicals). The bands were visualized on a UV transilluminator (PEQLAB), using a wavelength of 365 nm, and quickly cut to minimize the UV damage. DNA was extracted from the gel slices using the Zymoclean™ gel DNA recovery kit (Zymo Research). The concentration of DNA was determined using a NanoDrop 2000 spectrophotometer (Thermo Scientific).

For the ligation of vector and insert fragments, a ligation calculator was designed (the Excel file available in the Additional file 1 ) for easy calculation of the required insert and vector volumes. The mathematical basis of the calculator is inserted into the excel spreadsheet. The size and concentration of the vector and insert fragments and the molar ratio of vector/insert (normally 1:3) must be provided for the calculation. Calculated amounts of insert (tdTomato) and vector (alpha-retroviral backbone) were mixed with 2 μl of 10X T4 ligase buffer (400 mM Tris–HCl, 100 mM MgCl2, 100 mM DTT, 5 mM ATP (pH 7.8 at 25°C), Thermo Scientific), 1 μl of T4 ligase (5 U/μl, Thermo Scientific), filled up to 20 μl using ultrapure water and incubated overnight at 16°C. The day after, HB101 E. coli was transfected with the ligation mixture as mentioned above. The clones were picked and consecutively cultured for one day in LB medium containing ampicillin. Plasmid DNA was isolated using Zyppy™ plasmid miniprep kit (Zymo Research) and digested with proper restriction enzymes for screening. Digested plasmids were mixed with the loading dye and run on an agarose gel as mentioned above. The separated DNA fragments were visualized using a Gel Doc™ XR+ System (BIO-RAD) and analyzed by the Image Lab™ software (BIO-RAD). The positive clone was cultured overnight in 450 ml LB medium containing ampicillin. Plasmid DNA was isolated using QIAGEN plasmid maxi kit (QIAGEN), diluted in ultrapure water and stored at −20°C for later use.

Production of viral supernatant and transduction of cells

HEK293T cells were thawed, split every other day for one week and grown in log phase. The day before transfection, 3.5 × 10 6 cells were seeded into tissue culture dishes (60.1 cm 2 growth surface, TPP). The day after, the cells use to reach about 80% confluence. If over confluent, transfection efficiency decreases. The following plasmids were mixed in a total volume of 450 μl ultrapure water: codon-optimized alpharetroviral gag/pol (2.5 μg), VSVG envelope (1.5 μg), and the alpharetroviral vector containing the tdTomato gene (5 μg). Transfection was performed using calcium phosphate transfection kit (Sigma-Aldrich). 50 μl of 2.5 M CaCl 2 was added to the plasmid DNA and the mixture was briefly vortexed. Then, 0.5 ml of 2X HEPES buffered saline (provided in the kit) was added to a 15 ml conical tube and the calcium-DNA mixture was added dropwise via air bubbling and incubated for 20 minutes at room temperature. The medium of the HEK293T cells was first replaced with 8 ml fresh medium (DMEM containing FCS and supplement as mentioned above) containing 25 μM chloroquine. Consecutively the transfection mixture was added. Plates were gently swirled and incubated at 37°C. After 12 hours, the medium was replaced with 6 ml of fresh RPMI containing 10% FCS and supplements. Virus was harvested 36 hours after transfection, passed through a Millex-GP filter with 0.22 μm pore size (Millipore), and used freshly to transduce C1498 cells. Before transduction, 24 well plates were coated with retronectin (Takara, 280 μl/well) for 2 hours at room temperature. Then, retronectin was removed and frozen for later use (it can be re-used at least five times) and 300 μl of PBS containing 2.5% bovine serum albumin (BSA) was added to the wells for 30 minutes at room temperature. To transduce C1498 cells, 5 × 10 4 of cells were spun down and resuspended with 1 ml of fresh virus supernatant containing 4 μg/ml protamine sulfate. The BSA solution was removed from the prepared plates and plates were washed two times with 0.5 ml PBS. Then cells were added to the wells. Plates were centrifuged at 2000RPM/32°C/90 minutes. Fresh medium was added to the cells the day after.

Flow cytometry and fluorescence microscope

For flow cytometry assessment, cells were resuspended in PBS containing 0.5% BSA and 2 mM EDTA and were acquired by a BD FACSCanto™ (BD Biosciences) flow cytometer. Flow cytometry data were analyzed using FlowJo software (Tree Star). Imaging was performed with an Olympus IX71 fluorescent microscope equipped with a DP71 camera (Olympus). Images were analyzed with AxioVision software (Zeiss). Fluorescent images were superimposed on bright-field images using adobe Photoshop CS4 software (Adobe).

Abbreviations

Polymerase chain reaction

Gene of interest

Open reading frame

Melting temperature

Basic local alignment search tool

Vesicular stomatitis virus G glycoprotein

Luria-Bertani

Dulbecco’s Modified Eagle medium

Roswell Park Memorial Institute

Bovine serum albumin

Ethylenediaminetetraacetic acid

Fluorescence-activated cell sorting

Human embryonic kidney

Phosphate buffered saline

Fetal calf serum

Hydroxyethyl-piperazineethane-sulfonic acid

Ampicillin resistance gene

Posttranscriptional regulatory element

Myeloproliferative sarcoma virus promoter.

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Acknowledgments

The authors would like to thank Jessica Herbst, Abbas Behpajooh, Christian Kardinal and Juwita hübner for their fruitful discussions. We also thank Gang Xu for helping to design the cover page. This work was supported by the Deutsche Forschungsgemeinschaft, the Bundesministerium für Bildung und Forschung, the Deutsche Jose-Carreras Leukämiestiftung (grants SFB-738, IFB-TX CBT_6, DJCLS R 14/10 to M.G.S.) and the Ph.D. program Molecular Medicine of the Hannover Medical School.

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Sayed Shahabuddin Hoseini & Martin G Sauer

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Department of Pediatric Hematology and Oncology, Medizinische Hochschule Hannover, OE 6780, Carl-Neuberg-Strasse 1, 30625, Hannover, Germany

Martin G Sauer

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The authors declare that they have no competing interests.

Authors’ contributions

SSH conceived the study subject, carried out experiments and drafted the initial manuscript. MGS participated in study design and coordination and edited the manuscript. Both authors have read and approved the final manuscript.

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13036_2014_161_moesm1_esm.xlsx.

Additional file 1: Ligation calculator. To calculate the amounts of the vector and insert fragments for a ligation reaction, you need to provide the size of the vector and insert (in base pairs), the molar ration of insert/vector (normally 3 to 5), vector amount (normally 50 to 100 ng), and vector and insert fragment concentrations (ng/μl). The computational basis of this ligation calculator is mentioned in the lower box. (XLSX 50 KB)

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Hoseini, S.S., Sauer, M.G. Molecular cloning using polymerase chain reaction, an educational guide for cellular engineering. J Biol Eng 9 , 2 (2015). https://doi.org/10.1186/1754-1611-9-2

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About Cloning

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Cloning refers to the process of producing multiple individual organisms with identical genes, which may occur naturally or artificially. A gene is the basic biological unit that determines an organism's characteristics. Genes are made up of even smaller molecules called deoxyribonucleic acid (DNA) that carry and replicate genetic information.

Cloning occurs as a natural outcome of biological reproduction. Some bacteria and plants reproduce asexually—that is, there is no mixing of genetic material from multiple parent organisms. Instead, these organisms reproduce by creating genetically identical copies, or clones, of themselves. Identical twins, or two separate offspring resulting from a single fertilized egg, are considered clones of one another but not of their parent organisms.

Clones can also be created artificially...    ( Opposing Viewpoints )

• Should cloning of humans be permitted?
• What are the potential benefits and risks of cloning?
• What are the ethical controversies surrounding cloning?
• What are the social and cultural impacts of cloning?
• Will clones really be physically and behaviorally identical?
• If human cloning is allowed, what type of legislation might be enacted to control it?
• Do any laws currently exist which address the issue of cloning?
• Next: Library Resources >>
• Last Updated: Aug 29, 2024 3:56 PM
• URL: https://libguides.broward.edu/Cloning

40+ Cloning Essay Topics: From Sheep to Identity

Today, cloning is not only limited to the realm of science fiction anymore. It’s a real industry and scientific area of research that may significantly change our lives. Perhaps, it’s one of the most controversial investigations in biology. Active debates in the media have led to a widespread belief that cloning may lead to extreme danger. Science fiction books and movies have contributed a lot to this state of affairs as well.

Generally speaking, cloning allows for the reproduction of genetically similar organisms (also known as clean lines). For example, plant cloning is a common process in nature and farming. Usually, the plant is propagated by shoots, cuttings, tendrils, etc. As you can see, nature has cloned organisms for billions of years. So, why can’t we clone other species?

Since the invention of the term “clone” in 1963, genetic engineering has significantly developed. Scientists have learned how to extract genes, develop a polymerase chain reaction method, decode the human genome, and clone a number of mammals. The next step is obvious – human cloning, but it’s connected with various religious, ethical, and technological issues. Should we be afraid of human cloning? Is it possible to clone Hitler or Jesus Christ?

In the list below we want to share with you a list of cloning debate topics connected to animal and human cloning. Enjoy!

Human cloning essay topics

Human cloning has become an extremely popular theme of science fiction, and people have already despaired over the idea of when this technology will step over from the pages and screens to real life. Here we want to share with you some issues and questions related to human cloning.

• Persuade your audience whether human cloning should be allowed.
• Describe the disease-associated gene. How can cloning overcome such a disease? How will it affect diagnosis and treatment?
• Think about whether the potential benefits of human cloning outweigh the ethical side of the creation of artificial life.
• Is it physically safe to clone humans?
• Describe the current findings and future perspectives of human reproductive cloning.
• What are the ethical issues connected to human cloning?
• Discuss the main findings of the Human Genome Project. What are the implications? How would it affect you personally?
• Analyze the issues connected to human cloning in the context of organ transplantation.
• Describe the potential of therapeutic cloning in regenerative medicine. Is it a viable technology?
• Analyze the book “Frankenstein” by Mary Shelley from the perspective of stem cell cloning.
• Analyze the book “Our Posthuman Future” by F. Fukuyama in regards to the advantages and disadvantages of cloning.
• Would you allow the creation of your clone? Why or why not?
• How would human cloning affect us on a global scale?
• Analyze “Never Let Me Go,” a book by Ishiguro, in terms of scientific human cloning.
• Explore reproductive cloning in terms of medical ethics.
• How can human cloning affect our relationships?
• Analyze the reproductive cloning from the perspective of Kant’s theory and Leon Kass’s arguments.
• Approve or disapprove the following statement: “The cloning technology is not perfect; it can lead to the death of the fetus.”
• Analyze cloning from the perspective that it may be used by businessmen to sell organs for transplantation.
• What governmental regulations should be used to control cloning?
• Would it be ethically and morally right to clone dead people?
• Do you think a clone has a soul?
• How is human cloning represented in the media?
• Analyze the situation where the clone is killed by the original from a legislative and ethical side.
• What are the main problems with reproductive cloning? Why haven’t we cloned a human yet?

Interesting cloning debate topics

There are many issues related to cloning that have begun to grow since the 2000s. Today, the number of provocative questions has only increased, and opinions have polarized, dividing people into two opposing camps.

• Describe the positive and negative sides of cloning.
• Is cloning the right path for science? What dangers does it have? Is it worth the money for the research?
• Analyze the characteristics of artificial cloning.
• What is the future of cloning? What are the most prospective researches?
• What are the current methods of using cloning? Describe the latest technologies.
• Describe the positive and negative sides of animal cloning.
• Discuss cloning in the context of bioethics.
• Does cloning relate to responsible citizenship? How?
• Analyze the cloning of bacteria and yeast, and its applications.
• Discuss cloning from a religious perspective. Can we say that cloning is like playing as God?
• Analyze the animal cloning business in South Korea. What insights can it give other countries?
• What are the major ethical dilemmas of genetic cloning?
• Do you think it is bad to clone endangered species? In what situations can cloning be justified?
• Analyze George Bush’s speech on cloning.
• Should scientists be obliged to share both benefits and burdens of cloning research?
• Do you think the US government should invest in cloning?
• Explore the philosophical issues of cloning.
• Analyze the current status of cloning in the US. Is there any governmental law or regulation on cloning in the US? Are there any scientific programs related to cloning?
• Explore the peculiarities of the Dolly sheep cloning.
• Is it ethically right to clone pets?
• Explore the issues connected to cloning animals for food. What cloned animals are already used for food? Should we use cloned animals for food?
• Will cloning limit genetic diversity?

Obviously, cloning has significant potential advantages and several possible negative consequences. As with many scientific advancements of the past, such as airplanes and computers, the only threat is our narrow mindset.

We hope that our list of cloning essay topics will give you some food for thought. If you can’t manage to write an essay by yourself, our writers will eagerly help you! Fill in the order form and enjoy your life!

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In 1997 Dolly the Sheep was the first mammal ever to be cloned. Since that time, the discussion has turned towards the possibilities of cloning human beings either for research (“therapeutic”) or reproductive purposes, and even as a potential means for organ farming. Cloning is also known as “somatic cell nuclear transfer” (SCNT), the technical process by which cloning is performed. Cloning is a dominant topic under the broader category of biotechnology. Ethical issues specific to human cloning include: the safety and efficacy of the procedure, cloning for destructive embryonic stem cell research, the effects of reproductive cloning on the child/parent relationship, and the commodification of human life as a research product.

SUGGESTED RESOURCES

• Michael Sleasman, “Bioethics Past, Present, and Future: Important Signposts in Human Dignity” (An overview of bioethics and the breadth of issues it encompasses)
• John Kilner, “An Overview of Human Cloning"
• Dean Clancy, “To Clone or Not to Clone? Reflections from the Executive Director of the President’s Council on Bioethics”
• John Kilner, “Human Cloning"
• John Kilner and Robert George, “Human Cloning: What’s at Stake”

Bibliography

Position statement.

Human cloning is the creation of a human being whose genetic make-up is nearly identical 1 to that of a currently or previously existing individual. Recent developments in animal cloning coupled with advances in human embryonic stem cell research have heightened the need for legislation on this issue. Despite their nearly identical titles, the two bills currently being considered by Congress call for markedly different policies on this critical issue. Though both seek a ban on what is being called "reproductive" cloning--in which a clonal human embryo is implanted in a woman with the intent that a cloned human being will be born--they differ dramatically with respect to what is being termed "therapeutic" cloning. This latter type of cloning involves the creation and subsequent destruction of a clonal human embryo for the purposes of scientific or medical research. 2 Such embryonic destruction is usually carried out as a means of obtaining the embryo's "stem cells"--cells which some believe have the potential to revolutionize medicine by restoring the health of persons suffering from a variety of debilitating conditions. 3 Because the prospect of human cloning carries great potential to impact humanity in ways previously only imagined, it is exceedingly important that Congress adopt legislation that will protect society and the citizens who live in it--both now and for generations to come. To achieve this end, we believe that a comprehensive ban prohibiting both "reproductive" and "therapeutic" cloning is needed. In support of this assertion, we offer the following:

I. The overwhelming consensus in this country that human reproductive cloning should not be permitted necessitates a ban on both reproductive and "therapeutic" cloning.

An overwhelming majority of scientists, lawyers, health care professionals, ethicists and the general public has spoken out strongly against creating a human baby via what is being termed "reproductive cloning." 4 While most U.S. citizens support a ban on the reproductive cloning of human beings, they may or may not support a ban on "therapeutic" cloning. Yet, to enact a ban on the former while simultaneously permitting the latter would almost certainly result in instances of both reproductive and "therapeutic" cloning. Support for this premise is as follows:

First, if a ban only on reproductive cloning were adopted, enforcement would require the legally mandated destruction of human embryos created via cloning. That is, if it were legal to create clonal embryos for "therapeutic"--but not for reproductive--purposes, the demise of these embryos would be required in order to prevent the illegal practice of reproductive cloning from occurring. A non-comprehensive ban would thereby establish "for the first time in American history a class of embryos that it is a crime not to destroy, a felony not to treat as anything except disposable tissue ."5 Although abortion is currently legal in this country, the majority of U.S. citizens would surely react strongly against and refuse to adhere to a governmental policy that mandated the destruction of human life (or the punishment/ incarceration of women known to have defied the law by giving birth to human clones). 6

Indeed, if clonal human embryos were created in the laboratory for "therapeutic" purposes, the mandate that they not be implanted or otherwise allowed to progress toward birth would prove very difficult to defend. Therefore, the birth of clonal human beings--the very thing such a ban would intend to prohibit--would likely result. 7 Consider the following scenarios:

• A clonal embryo is produced with the "therapeutic" intent of producing tissue needed to save the life of a seriously ill child. Before the tissue can be obtained, the child dies. Her grieving parents, distraught over their tragic loss, request that the embryo be implanted so that they may have another child.
• A man agrees to be cloned with the intent of donating the resultant embryo to research. Subsequent to creation of the clonal embryo, he learns that both he and his wife are infertile. Realizing that their prospect for having a genetically related child suddenly appears to be compromised, the man changes his mind and requests that his clone be implanted in his wife instead.

In cases like these, authorities would be hard pressed to deny the wishes of those desiring to implant a clonal embryo. As Leon Kass, Addie Clark Harding Professor in The College and the Committee on Social Thought at the University of Chicago, has aptly remarked, "Once the genies put the cloned embryos into the bottles, who can strictly control where they go?" 8 Currently, the parents of embryos created via in vitro fertilization (IVF) are given absolute decision-making power as to whether their embryos are implanted, cryopreserved, donated to another couple or to research, or destroyed. Fertility clinics often go to great lengths to determine parents' wishes regarding embryos who have been stored for a long period of time and honor decisions both for and against implantation. 9 Certainly, the fertility industry would have a difficult time denying people the same choice simply because their embryos resulted from cloning. The implantation of human life--regardless of how that life originated--should not be regarded as a prohibitable act.

Second, if the laboratory creation of clonal embryos was permitted but the implantation of such embryos was banned, logistical problems regarding enforcement of such a system would undoubtedly arise. To prevent a cloned embryo from being implanted within the private context of a doctor/patient relationship would prove to be impossible. Policies that would require genetic testing of every baby upon birth to ensure that he or she is not a clone would likely be regarded as a violation of privacy. Furthermore, such testing would itself fail to ensure that human cloning had not occurred, as the baby could be a clone of an unknown or unrevealed person, rather than being a near genetic duplicate of one of the parents. As a result, the threat to levy fines on or otherwise punish those who clone often would not serve as a deterrent. Also, those who could be proven to have cloned (e.g., parents who cloned an ill son with the hope that his clone could provide healthy tissue for transplant) would likely not be stopped from engaging in such action if they believed it could save the life of their child. Therefore, policies that would prohibit the implantation of clonal human embryos would often be unenforceable and would fail to deter human reproductive cloning.

II. To mandate the destruction of clonal human embryos created for research purposes would constitute a break with our nation's longstanding legal tradition and the majority of public sentiment.

While some proponents of "therapeutic" cloning have characterized the cloning controversy as primarily a dispute between those who are "pro-life" and those who are "pro-choice," extracorporeal human embryos historically have been accorded the right to certain protections by those on both sides of the abortion debate. The following excerpt from Richard Doerflinger's June 20, 2001 testimony before the U.S. House Subcommittee on Health illustrates this point in convincing fashion:

...the one practice in human embryo research that is widely condemned even by supporters of abortion rights is the special creation of human embryos solely for the purpose of research that will kill them. In 1994 the National Institutes of Health did propose funding such [a practice], as part of a larger proposal for funding human embryo research generally. The moral outcry against this aspect of the proposal, however, was almost universal. Opinion polls showed massive opposition and the NIH panel making the recommendation was inundated with over 50,000 letters of protest. The Washington Post, while reaffirming its support for legalized abortion, attacked the Panel's recommendation as follows:

The creation of human embryos specifically for research that will destroy them is unconscionable... [I]t is not necessary to be against abortion rights, or to believe human life literally begins at conception, to be deeply alarmed by the notion of scientists purposely causing conceptions in a context entirely divorced from even the potential of reproduction. 10

The Chicago Sun-Times likewise editorialized:

We can debate all day whether an embryo is or isn't a person. But it is unquestionably human life, complete with its own unique set of human genes that inform and drive its own development. The idea of the manufacture of such a magnificent thing as a human life purely for the purpose of conducting research is grotesque, at best .11

In the end, President Clinton set aside the recommendation for creation of research embryos. Every year since then, Congress has prohibited funding for all harmful embryo research at the National Institutes of Health, through the Dickey amendment to the annual Labor/HHS appropriations bills. .12 However, even members of Congress who have led the opposition to the Dickey amendment agree with its rejection of special creation of human embryos for research. On the only occasion when an amendment was offered on the House floor to weaken the Dickey amendment, the sponsors emphasized that it would leave intact the clause rejecting the creation of embryos for research. .13 Similarly, the recent NIH guidelines for embryonic stem cell research, as well as Senator Specter's Stem Cell Research Act of 2001, explicitly reject the idea of using embryos specially created for research purposes." .14

Also among those who have rejected the creation of human embryos for use in destructive research is the National Bioethics Advisory Commission (NBAC) appointed by President Clinton. .15 Much of the public also opposes the creation and subsequent destruction of clonal embryos for research purposes. 16

III. The United States should promote ethical scientific and medical research, and not merely the progress of research, as "good ends" do not justify any and all means to achieve those ends.

Some have suggested recently that "America is likely to be [the] most important battleground" in the debate over human cloning. 17 This is largely due to the fact that the U.S. has the most highly developed biotechnology industry in the world. Indeed, pressure from scientists seeking to close in on medical breakthroughs is immense, and they are consistently among the most vocal advocates of "therapeutic" cloning. 18 In his June 20, 2001 testimony before the U.S. House Subcommittee on Health, Thomas Okarma, President of Geron Corporation, issued the following statement:

Our nation is on the cusp of reaping the long dreamed-of rewards from our significant investment in biomedical research. The U.S. biotech industry is the envy of much of the world, especially our ability to turn basic research at NIH and universities into applied research at biotech companies and, in turn, into new therapies and cures for individual patients. Using somatic cell nuclear transfer and other cloning technologies, biotech researchers will continue to learn about cell differentiation, re-programming and other areas of cell and molecular biology. Armed with this information, they can eventually crack the codes of diseases and conditions that have plagued us for hundreds of years, indeed, for millennia.

Also offering testimony before the Subcommittee on Health, George Mason University Professor of Public Policy Francis Fukuyama noted that many have fallen prey to the fear that the U.S. "will risk falling behind technologically if we hobble ourselves by restricting either research into or the actual procedure of cloning." 19 Similar reasoning was endorsed over a decade ago when James Watson urged Congress to fund the Human Genome Project on the premise that "what is good for U.S. business is good for the nation." 20 Today, those employed in science and public policy may indeed stand to gain both professionally and economically from engaging in cloning research; 21 however, even if such vocational and material benefits are not the primary motives behind the research--but merely accompany the central goal of improving human health--this praiseworthy pursuit should not be achieved by whatever means are available. 22

In illustrating this point, Kevin FitzGerald, a molecular geneticist and bioethicist who is himself engaged in cancer research at Georgetown University, has eloquently argued that the potential for obtaining benefits from scientific and medical research--regardless of how significant such benefits may be or who may stand to be helped by them--does not in itself translate into a license to engage in that particular research. For example, scientists would likely learn some very valuable information about the environmental contributions to cancer by administering known carcinogens to a group of people and then varying factors such as diet and sun exposure. To do so, however, would certainly be unethical and almost no one would advocate going forward with such experimentation. 23

With regard to the cloning debate, it may indeed be helpful to keep in mind that a hallmark of scientific research is to do no harm until it has been absolutely determined that no alternative means for obtaining a desired good exist. The recent succession of advances in non-embryonic stem cell research indicate that we have not yet reached that point of determination. 24 A corollary to this principle is that even when no other means exist, there are still restrictions against inflicting harm. 25 The importance of adhering to this principle should be enshrined in every scientist and citizen alike, as horrific examples of failure to ascribe to this cardinal rule abound in all too recent history.

Tragically, the last century and a half has been marred by numerous atrocities against vulnerable human beings in the name of progress and medical benefit. In the 19th century, vulnerable human beings were bought and sold in the town square as slaves and bred as though they were animals. 26 In this century, the vulnerable were executed mercilessly and subjected to demeaning experimentation at Dachau and Auschwitz. 27 At mid-century, the vulnerable were subjects of our own government's radiation experiments without their knowledge or consent. 28 Likewise, vulnerable African-Americans in Tuskegee, Alabama were victimized as subjects of a government-sponsored research project to study the effects of syphilis. 29 Currently, we are witness to the gross abuse of mental patients used as subjects in purely experimental research. 30 These experiments were and are driven by a crass utilitarian ethos which results in the creation of a "sub-class" of human beings, allowing the rights of the few to be sacrificed for the sake of potential benefit to the many. These unspeakably cruel and inherently wrong acts against human beings have resulted in the enactment of laws and policies which require the protection of human rights and liberties, including the right to be protected from the tyranny of the quest for scientific progress.

We are aware that "therapeutic" cloning research has been endorsed by many on the basis of its alleged potential to relieve the suffering of those afflicted by debilitating disease or disability. While we acknowledge that the desire to heal people is certainly a laudable goal and understand that many have invested their lives in realizing this goal, we also recognize that we simply are not free to pursue good ends via unethical means. As Fukuyama perceptively noted in his testimony:

The United States, as an economically, politically and culturally dominant force in the world, will have an enormous impact on other societies. The Council of Europe has already passed a ban on cloning; to date, 24 countries (including Germany, France, Italy and Japan) have already enacted national bans on cloning, while 16 have banned creation of embryos for research purposes. The United States can do a great deal to either reinforce (or else undermine) [what constitutes acceptable scientific and medical research]. 31

Precisely because our nation is a global power, it possesses a momentous opportunity to set a standard on both reproductive and "therapeutic" human cloning. Failure to set standards which are ethical will cause this country--and perhaps others--to reap once again the tragic consequences of unethical scientific and medical research.

IV. The pursuit of therapies for human disease and disability via "therapeutic" cloning would likely leave many Americans without acceptable means to relieve their suffering.

Some proponents of "therapeutic" cloning have alleged that if a comprehensive ban is enacted, those who advocated such legislation should be held responsible for the continued suffering of patients who might have benefitted from therapies derived from embryonic stem cells. However, given that many Americans have indicated they would resist treatments derived from embryonic stem cells due to their personal moral convictions, 32 serious consideration to the manner in which therapies are derived would seem warranted. That is to say, concern for the suffering should extend equally to all who suffer, and therapies should be developed which will not discriminate on the basis of moral convictions. Given that many Americans afflicted with debilitating disease and/or disability would likely refuse treatments derived from destructive embryo research, would the fervent commitment to helping the suffering that is often voiced by proponents of "therapeutic" cloning really best be served by research on embryonic, as opposed to non-embryonic, 33 stem cells? Indeed, if a treatment or cure for a particular disease was developed from embryonic stem cells, researchers most likely would not seek to develop an alternative therapy from non-embryonic stem cells but would instead move on to pursue the development of therapies for other human afflictions.  Thus, if "therapeutic" cloning were to be legally accepted, the suffering of many patients might actually be extended--rather than ended or lessened--as they might be forced to continue in their suffering unless they are willing to abandon their moral commitments.

V. Human beings have a right not to be created for purposes of experimentation.

A bill permitting "therapeutic" cloning while prohibiting reproductive cloning would constitute the legalization of a wholly unethical practice in that it would legally condone and even legally require the demise of human embryos created for research purposes. The destruction of human embryos is profoundly disturbing, and research which necessitates such an act should be proscribed--regardless of the potential for scientific and medical gain. That some individuals would be destroyed in the name of medical science constitutes a threat to us all.

Regardless of one's views on abortion or personhood of the human embryo, human embryos are unequivocally human beings and therefore should not be subjected to destructive research. An international scientific consensus now recognizes that human embryos are biologically human beings beginning at fertilization and acknowledges the physical continuity of human growth and development from the one-cell stage forward. 34 In the 1970s and 1980s, some frog and mouse embryologists referred to human embryos in the first week or two of development as "pre-embryos," claiming that they deserved less respect than embryos in later stages of development. 35 Today, however, some embryology textbooks openly refer to the term "pre-embryo" as a scientifically invalid and "inaccurate" term that has been "discarded," and others who once used the term have quietly dropped it from new editions. 36 Both the Human Embryo Research Panel 37 and the National Bioethics Advisory Commission 38 have also rejected the term, describing the human embryo from his or her earliest stages of development as a living organism and a "developing form of human life." 39 The claim that an early human embryo becomes a human being only after he or she has reached certain stages of development (e.g., after 14 days or following implantation in the womb) is therefore a scientific myth. Finally, the historic and well-respected 1995 Ramsey Colloquium statement on embryo research acknowledges that:

The [embryo] is human; it will not articulate itself into some other kind of animal. Any being that is human is a human being. If it is objected that, at five days or fifteen days, the embryo does not look like a human being, it must be pointed out that this is precisely what a human being looks like--and what each of us looked like--at five or fifteen days of development. 40

The term "pre-embryo," and all that it implies, is therefore scientifically invalid. Human embryos are not mere biological tissues or clusters of cells; they are the tiniest of human beings. 41 Thus, we have a moral responsibility not to deliberately harm them. To ignore this responsibility would be to engage in morally unacceptable age discrimination, resulting in the disregard for and destruction of human life based solely on its developmental stage. 42

In addition to the fact that "therapeutic" cloning runs counter to our nation's longstanding legal tradition and much of public sentiment, 43 the creation of embryos for research via "therapeutic" cloning is for some even more insidious than embryo research in general. As George Annas points out:

To create human embryos solely for research--or to sell them, or to use them in toxicity testing--seems morally wrong because it seems to cheapen the act of procreation and turn embryos into commodities . . . . The moral problem with making embryos for research is that as a society we do not want to see embryos treated as products or mere objects for fear that we will cheapen the value of parenting, risk commercializing procreation and trivialize the act of procreation. 44

While embryonic stem cell research necessitates the destruction of already existing human embryos, "therapeutic" cloning "goes one step further and entails the deliberate creation--as well as the sacrifice--of human embryos for the alleged good of others." 45 Never before has the creation of embryos with the intent of destroying them for research purposes been legally accepted, and many of the most prominent advocates of human embryonic stem cell research have been adamant about holding the line here. 46 Leon Kass notes that:

The prospect of creating new human life solely to be exploited in this way has been condemned on moral grounds by many people [sic]--including The Washington Post, President Clinton and many other supporters of a woman's right to abortion--as displaying a profound disrespect for life. Even those who are willing to scavenge so-called "spare embryos"--those products of in vitro fertilization made in excess of people's reproductive needs, and otherwise likely to be discarded--draw back from creating human embryos explicitly and solely for research purposes. They reject outright what they regard as the exploitation and the instrumentalization of nascent human life. In addition, others who are agnostic about the moral status of the embryo see the wisdom of not . . . offending the sensibilities of their fellow citizens who are opposed to such practices. 47

Indeed, it would seem that the manufacture of embryos for research purposes would cause embryos increasingly to be regarded as expendable commodities who have their value rooted solely in what they are able to offer others. Such a mentality might especially prevail with regard to clonal embryos, whose genetic blueprint would be easily reproducible. If we begin creating and subsequently destroying human embryos in the name of research, we will inevitably move into ever more bleak territory, which, had we been wise, we would have shuddered to enter in the first place.

Finally, it is important to recognize that although research on human embryos is widely accepted in the event that it may afford therapeutic benefit to the embryo, so-called "therapeutic" cloning is in no way beneficial to the embryo. In his May 2, 2001 testimony before the Senate Commerce Subcommittee on Science, Technology and Space, Richard Doerflinger offered the following:

...the experiments contemplated [in therapeutic cloning] are universally called " non therapeutic" experimentation in law and medical ethics--that is, the experiments harm or kill the research subject (in this case the cloned embryo) without any prospect of benefitting that subject. This standard meaning of "nontherapeutic" research is found, for example, in state laws forbidding such research on human embryos as a crime. Experiments performed on one subject solely for possible benefit to others are never called "therapeutic research" in any other context, and there is no reason to change that in this context.

Thus, the term "therapeutic cloning" is actually a misnomer. Such cloning holds no therapeutic value whatsoever for the clonal embryo as, far from benefitting from the research, the embryo is destroyed in it. The painful lessons of the past should have taught us that human beings must not be conscripted for research without their permission--no matter what the alleged justification--especially when that research means the forfeiture of their health or lives. Even if an individual's death is believed to be otherwise imminent, we still do not have a license to engage in lethal experimentation--just as we may not experiment on death row prisoners or harvest their organs without their consent. Of all human beings, embryos are the most defenseless against abuse. A policy advocating the use of clonal human embryos in destructive research would violate the rights of human beings not to be experimented upon.

VI. Conclusion.

Because the prospect of human cloning carries great potential to impact humanity in ways previously only imagined, it is exceedingly important that Congress adopt legislation that will protect society and the citizens who live in it--both now and for generations to come. We believe that the following points are of primary significance to the current legislative debate on this issue:

• The overwhelming consensus in this country that human reproductive cloning should not be permitted necessitates a ban on both reproductive and "therapeutic" cloning.
• To mandate the destruction of clonal human embryos created for research purposes would constitute a break with our nation's longstanding legal tradition and much of public sentiment.
• The United States should promote ethical scientific and medical research, and not merely the progress of research, as "good ends" do not justify any and all means to achieve those ends.
• The pursuit of therapies for human disease and disability via "therapeutic" cloning would likely leave many Americans without acceptable means to relieve their suffering.
• Human beings have a right not to be created for purposes of experimentation.

It is our contention that careful consideration of these points leads to support for a comprehensive ban prohibiting both the reproductive and "therapeutic" cloning of human beings. Failure to adopt such a ban will result in scientific, ethical, and legal failures--the scope and consequences of which will be of great magnitude.

1 In the cloning process, DNA from an existing individual is transferred into an egg cell devoid of genetic material. Components of the egg cell called mitochondria contain their own DNA; thus, provided that the egg cell donor and the donor of DNA are different individuals, a human clone would not be wholly identical in the genetic sense to his or her progenitor. Cloning Human Beings: Report and Recommendations of the National Bioethics Advisory Commission (Rockville, MD), June 1997, pp. 17-18.

2 The term "therapeutic cloning" is actually a misnomer. See Section V (paragraph #5) of this document for discussion of this poinnt.

3 Davor Solter and John Gearhart, "Enhanced: Putting Stem Cells to Work," Science 283 (March 5, 1999):1468-1470; Robert Lanza, et al., "Human Therapeutic Cloning," Nature Medicine 5 (1999):975-977; Robert Lanza, et al., "Prospects for the Use of Nuclear Transfer in Human Transplantation," Nature Biotechnology 17 (1999):1171-1174.

4 Rudolf Jaenisch and Ian Wilmut, "Don't Clone Humans!" Science 291 (March 30, 2001):2552; "Americans Overwhelmingly Oppose Human Cloning," United States Conference of Catholic Bishops ICR poll, June 7, 2001 (In this survey of 1013 adults, the question "Should scientists be allowed to use human cloning to try to create children for infertile couples?" was posed. 84.6% of respondents answered No, 12.4% of respondents answered Yes, 2.6% of respondents answered that they didn't know, and 0.4% of respondents refused to answer the question); ABC News Nightline poll, February 24, 1997.

5 J. Bottum and William Kristol, "For a Total Ban on Human Cloning," The Weekly Standard 6:40 (July 2/July 9), (editorial).

6 In his article "Preventing a Brave New World" (The New Republic, May 21, 2001), Leon Kass refers to the demand that clonal embryos be destroyed as "a bitter pill to swallow even for pro-choice advocates" (p. 36).

7 Lori Andrews, professor at Chicago-Kent College of Law and national/international advisor on genetic and reproductive technologies, has pointed out that even though the United Kingdom has passed a law banning reproductive cloning but permitting "therapeutic" cloning, it is important to note that the UK fertility industry is much more centrally regulated than is that of the United States B which has almost no formal guidelines regarding the use of reproductive technologies. Andrews has remarked that, "In the United States, there is no way a law based on the British model requiring termination of embryos would pass. Quite to the contrary, laws forbidding embryo termination . . . are much more likely" (emphasis added). The Clone Age: Adventures in the New World of Reproductive Technology (New York: Henry Holt and Company, 1999), p. 74.

8 Leon R. Kass and James Q. Wilson. The Ethics of Human Cloning (Washington, D.C.: American Enterprise Institute, 1998), p. 51.

9 American Society of Reproductive Medicine, "Disposition of Abandoned Embryos," Ethics Committee Report, 1997.

10 Editorial, "Embryos: Drawing the Line," The Washington Post, October 2, 1994, p. C6.

11 Editorial, "Embryo Research Is Inhuman," Chicago Sun-Times, October 10, 1994, p. 25.

12 The current version is Section 510 of the Labor/HHS appropriations bill for Fiscal Year 2001, H.R. 5656 (enacted through Section 1(a)(1) of H.R. 4577, the FY >01 Consolidated Appropriations Act, Public Law 106-554). It bans funding any creation of human embryos (by cloning or other means) for research purposes and any research in which human embryos are harmed or destroyed.

13 "Let me say that I agree with our colleagues who say that we should not be involved in the creation of embryos for research. I completely agree with my colleagues on that score," said Rep. Nancy Pelosi, arguing in favor of research on "spare" embryos originally created for fertility treatment. The sponsor of the weakening amendment, Rep. Nita Lowey, said: "I want to make it very clear: We are not talking about creating embryos . . . President Clinton again has made it very clear that early-stage embryo research may be permitted but that the use of federal funds to create embryos solely for research purposes would be prohibited. We can all be assured that the research at the National Institutes of Health will be conducted with the highest level of integrity. No embryos will be created for research purposes...." 142 Cong. Record at H7343 (July 11, 1996). The weakening amendment failed nonetheless, 167 to 256. Id. at H7364. While this debate concerned federal funding, supporters of the Lowey amendment said it was "very hard to understand" why standards for ethical research should be different for publicly funded and privately funded research. See remarks of Rep. Fazio at H7341-2.

14 The NIH guidelines deny funding for "research utilizing pluripotent stem cells that were derived from human embryos created for research purposes," and "research in which human pluripotent stem cells are derived using somatic cell nuclear transfer, i.e., the transfer of a human somatic cell nucleus into a human or animal egg." National Institutes of Health Guidelines for Research Using Human Pluripotent Stem Cells, 65 Fed. Reg. 51976-81 (August 25, 2000) at 51981. Senator Specter's bill supports embryonic stem cell research but insists that "the research involved shall not result in the creation of human embryos." 107th Congress, S. 723, Sec. 2.

15 In 1997 NBAC considered the prospect of cloning human embryos to create "customized stem cell lines" but described this as "a rather expensive and far-fetched scenario" which was also fraught with moral concerns. The NBAC declared that, "Because of ethical and moral concerns raised by the use of embryos for research purposes it would be far more desirable to explore the direct use of human cells of adult origin to produce specialized cells or tissues for transplantation into patients." Cloning Human Beings: Report and Recommendations of the National Bioethics Advisory Commission (Rockville, MD: June 1997), pp. 30-31. The Commission outlined three alternative avenues of stem cell research, two of which seemed not to involve creating human embryos at all.

16 "Americans Overwhelmingly Oppose Human Cloning," United States Conference of Catholic Bishops ICR poll, June 7, 2001.

17 J. Bottum and William Kristol, "For a Total Ban on Human Cloning," The Weekly Standard 6:40 (July 2/July 9), (editorial).

18 Davor Solter and John Gearhart, "Enhanced: Putting Stem Cells to Work," Science 283 (March 5, 1999):1468-1470; Robert Lanza, et al., "Human Therapeutic Cloning," Nature Medicine 5 (1999):975-977; Robert Lanza, et al., "Prospects for the Use of Nuclear Transfer in Human Transplantation," Nature Biotechnology 17 (1999):1171-1174, 1999.

19 June 20, 2001 testimony before the U.S. House Subcommittee on Health.

20 Testimony before the Subcommittee on Science, Research, and Technology of the House Committee on Science, Space and Technology, Coordination of Genome Projects in Committee Report on H.R. 4502 and S. 1966, the Biotechnology Competitiveness Act (Comm. Print 138, 1988).

21 Ronald Cole-Turner, Beyond Cloning: Religion and the Remaking of Humanity. (Harrisburg, PA: Trinity Press International, 2001), p. 99.

22 For a critique of the utilitarian justification for human cloning, see Kilner, John F., et al. (ed.), The Reproduction Revolution: A Christian Appraisal of Sexuality, Reproductive Technology and the Family (Grand Rapids, MI: Eerdmans; and United Kingdom: Paternoster, 2000), pp. 128-132. See also chapter 15 of this volume for a legal assessment of why human cloning should be legally prohibited.

23 Bioethics Colloquium lecture, Trinity International University, Deerfield, Illinois, March 22, 2001.

24 Recent advances in non-embryonic stem cell research suggest that it may not even be necessary to obtain stem cells by destroying human embryos in order to treat disease. A growing number of researchers believe that non-embryonic stem cells may soon be used to develop treatments for afflictions such as Parkinson's disease, Alzheimer's disease, immune disorders, congestive heart failure, degenerative diseases, and other debilitating conditions. Such researchers are working to further research on "adult," rather than embryonic, stem cells. In light of these promising new scientific advances, we promote the development of methods to repair and regenerate human tissue which do not require the destruction of embryonic human life. However, even if such methods do not prove to be as valuable in treating disease as are human embryonic stem cells, use of the latter in the name of medical progress is still not justifiable for the reasons stated in this document. (For an ongoing update of advances in non-embryonic stem cell research, please access www.stemcellresearch.org .)

25 Bioethics Colloquium lecture, Trinity International University, Deerfield, Illinois, March 22, 2001.

26 David Brion Davis, The Problem of Slavery in Western Culture (Ithaca, NY: Cornell Univ. Press), 1966.

27 George J. Annas and Michael A. Grodin (eds.), The Nazi Doctors and the Nuremberg Code: Human Rights in Human Experimentation(New York: Oxford University Press), 1992.

28 Ronald Munson, "Medical Experimentation and Informed Consent," in Intervention and Reflection: Basic Issues in Medical Ethics, 5th ed. (New York: Wadsworth Publishing Company, 1996), pp. 323-25.

29 James Jones, Bad Blood: The Tuskegee Syphilis Experiment (New York: Free Press), 1981.

30 The Center for Mental Health Services: Protection and Advocacy Program for Individuals with Mental Illness (Rockville, MD); web site: www.mentalhealth.org .

31 June 20, 2001 testimony before the U.S. House Subcommittee on Health.

32 Do No Harm: The Coalition of Americans for Research Ethics; web site: www.stemcellresearch.org .

33 Please see endnote #24 for a discussion of promising alternatives to embryonic stem cell research.

34 R. Warwick, Nomina Anatomica, 3rd ed. (Edinburgh: Churchill Livingstone), 1989 [the 6th ed. of Nomina Anatomica includes the international standard for scientifically correct terminology in human embryology]; Ronan O'Rahilly and Fabiola Muller, Human Embryology and Teratology (New York: Wiley-Liss), 1992; William J. Larsen, Human Embryology (New York: Churchill Livingstone), 1993; Bruce M. Carlson, Human Embryology and Developmental Biology (St. Louis: Mosby), 1994; Keith L. Moore and T.V.N. Persaud, The Developing Human: Clinically Oriented Embryology, 6th ed. (Philadelphia: W.B. Saunders Co.), 1998; Bradley Patten, Human Embryology, 3rd ed. (New York: McGraw-Hill), 1968. Stedman's Medical Dictionary (Baltimore: Williams and Wilkens), 1990.

35 Clifford Grobstein, "External Human Fertilization," Scientific American 240 (1979):57-67; Clifford Grobstein, Science and the Unborn: Choosing Human Futures (New York: Basic Books), 1988.

36 Ronan O'Rahilly and Fabiola Muller, Human Embryology and Teratology, 2nd ed. (New York: Wiley-Liss), 1992 (op.cit.): "The ill-defined and inaccurate term preembryo . . . is not used in this book," (p.55). In the 1996 2nd edition of this text, O'Rahilly and Muller repeat this rejection based on the fact that the term is "ill-defined," "inaccurate," "unjustified," and "equivocal" (p.81). See also C. Ward Kischer, "The Big Lie in Human Embryology: The Case of the Preembryo," Linacre Quarterly 64(1997):53-61.

37 National Institutes of Health: Report of the Human Embryo Research Panel (Bethesda, MD: NIH), November 1994.

38 National Bioethics Advisory Commission, Cloning Human Beings (Rockville, MD), June 1997.

40 The Ramsey Colloquium, which is sponsored by the Institute on Religion and Public Life, is a group of Jewish and Christian theologians, philosophers and scholars that meets periodically to consider questions of ethics, religion and public life. It is named after Paul Ramsey (1913-1988), the distinguished ethicist.

41 See, for example, Bruce M. Carlson, "Introduction to the Developing Human" in Human Embryology and Developmental Biology (St. Louis: Mosby), 1994.

42 Personal conversation with John Kilner, PhD, Director of The Center for Bioethics and Human Dignity, July 10, 2001.

43 Please see Section II of this document for discussion of this point.

44 George J. Annas, "The Politics of Human-Embryo Research--Avoiding Ethical Gridlock" (editorial) New England Journal of Medicine 334 (May 16, 1996).

45 Dónal O'Mathúna, "Cloning and Stem Cell Research: Wrong Motives on Both Sides of the Atlantic," Dignity newsletter (The Center for Bioethics and Human Dignity, Fall 2000).

46 In the National Institutes of Health Guidelines for Research Using Human Pluripotent Stem Cells (accessible at www.nih.gov/news/stemcell/stemcellguidelines.htm ), the following statement appears: "Investigators seeking NIH funds for research using hPSCs [human pluripotent (embryonic) stem cells] are required to provide documentation, prior to the award of any NIH funds, that embryos were created for the purposes of fertility treatment. President Clinton, many members of Congress, the NIH Human Embryo Research Panel and the NBAC have all embraced the distinction between embryos created for research purposes and those created for reproductive purposes." The NBAC report Ethical Issues in Human Stem Cell Research Executive Summary (September 1999) may be accessed at www.bioethics.gov/stemcell_exec_intro.htm .

47 Leon R. Kass, "Preventing a Brave New World," in The New Republic, May 21, 2001, p. 36.

Life with Borders

Dolly’s clone was not easily conceived, for it took researchers 277 attempts before they produced 29 embryos that survived longer than six days. Even then, only one lamb was born as a result! And here is where the ethical implications begin to appear, for if a similar ratio of human embryos were used in an attempted human cloning, the loss of human life would be morally unconscionable.

Cloning Humans: Leon Kass and Kevin FitzGerald on the "Post-Human Future"

Why human cloning must be banned now, to clone or not to clone reflections from the executive director of the president's council on bioethics, the interface between science and ethics: probing the deeper questions, human cloning: what's at stake, the body and the quest for control: appreciating the complex nature of human embodiment.

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