research paper about cloning

An official website of the United States government

The .gov means it’s official. Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

The site is secure. The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

• Publications
• Account settings
• My Bibliography
• Collections
• Citation manager

Save citation to file

Email citation, add to collections.

• Create a new collection
• Add to an existing collection

Add to My Bibliography

Your saved search, create a file for external citation management software, your rss feed.

• Search in PubMed
• Search in NLM Catalog
• Add to Search

Human Cloning: Biology, Ethics, and Social Implications

Affiliations.

• 1 MAGI'S LAB, Rovereto (TN), Italy.
• 2 Department of Pharmaceutical Sciences, University of Perugia, Perugia, Italy.
• 3 MAGI EUREGIO, Bolzano, Italy.
• 4 MAGISNAT, Peachtree Corners (GA), USA.
• 5 School of Food Science and Environmental Health, Technological University of Dublin, Dublin, Ireland.
• 6 Department of Psychology and Neuroscience, Dalhousie University, Halifax, Nova Scotia, Ca-nada.
• 7 Department of Ophthalmology, Center for Ocular Regenerative Therapy, School of Medicine, University of California at Davis, Sacramento, CA, USA.
• 8 Centre for Bioethics, Department of Philosophy and Applied Philosophy, University of St. Cyril and Methodius, Trnava, Slovakia.
• 9 Department of Biotechnology Engineering, Ben-Gurion University of the Negev, Beer-Sheva, Israel.
• 10 nstitute of Ophthalmology, Università Cattolica del Sacro Cuore, Fondazione Policlinico Universitario A. Gemelli-IRCCS, Rome, Italy.
• 11 MAGI BALKANS, Tirana, Albania.
• 12 Department of Biotechnology, University of SS. Cyril and Methodius, Trnava, Slovakia.
• 13 International Centre for Applied Research and Sustainable Technology, Bratislava, Slovakia.
• 14 UOC Neurology and Stroke Unit, ASST Lecco, Merate, Italy.
• 15 Center for Preclincal Research and General and Liver Transplant Surgery Unit, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy.
• 16 Department of Pathophysiology and Transplantation, Università degli Studi di Milano, Milan, Italy.
• 17 Department of Biomedical, Surgical and Dental Sciences, Università degli Studi di Milano, Milan, Italy.
• 18 UOC Maxillo-Facial Surgery and Dentistry, Fondazione IRCCS Ca Granda, Ospedale Maggiore Policlinico, Milan, Italy.
• 19 Department of Medical Genetics, Faculty of Medicine, Near East University, Nicosia, Cyprus.
• 20 Department of Medical Genetics, Erciyes University Medical Faculty, Kayseri, Turkey.
• 21 Vascular Diagnostics and Rehabilitation Service, Marino Hospital, ASL Roma 6, Marino, Italy.
• 22 San Francisco Veterans Affairs Health Care System, University of California, San Francisco, CA, USA.
• 23 Univ. Grenoble Alpes, CNRS, Grenoble INP, TIMC-IMAG, SyNaBi, Grenoble, France.
• 24 Department of Biotechnology, University of Tirana, Tirana, Albania.
• 25 Total Lipedema Care, Beverly Hills, California, and Tucson, Arizona, USA.
• 26 Federation of the Jewish Communities of Slovakia.
• 27 Department of Psychological Health and Territorial Sciences, School of Medicine and Health Sciences, "G. d'Annunzio" University of Chieti-Pescara, Chieti, Italy.
• 28 Unit of Molecular Genetics, Center for Advanced Studies and Technology (CAST), "G. d'Annunzio" University of Chieti-Pescara, Chieti, Italy.
• 29 Department of Anatomy and Developmental Biology, University College London, London, UK.
• PMID: 37994769
• DOI: 10.7417/CT.2023.2492

This scholarly article delves into the multifaceted domains of human cloning, encompassing its biological underpinnings, ethical dimensions, and broader societal implications. The exposition commences with a succinct historical and contextual overview of human cloning, segueing into an in-depth exploration of its biological intri-cacies. Central to this biological scrutiny is a comprehensive analysis of somatic cell nuclear transfer (SCNT) and its assorted iterations. The accomplishments and discoveries in cloning technology, such as successful animal cloning operations and advances in the efficiency and viability of cloned embryos, are reviewed. Future improvements, such as reprogramming procedures and gene editing technology, are also discussed. The discourse extends to ethical quandaries intrinsic to human cloning, entailing an extensive contemplation of values such as human dignity, autonomy, and safety. Furthermore, the ramifications of human cloning on a societal plane are subjected to scrutiny, with a dedicated emphasis on ramifications encompassing personal identity, kinship connections, and the fundamental notion of maternity. Culminating the analysis is a reiteration of the imperative to develop and govern human cloning technology judiciously and conscientiously. Finally, it discusses several ethical and practical issues, such as safety concerns, the possibility of exploitation, and the erosion of human dignity, and emphasizes the significance of carefully considering these issues.

Keywords: Human cloning; biology; dignity; ethical considerations; safety; social implications.

PubMed Disclaimer

Similar articles

• Cloning by SCNT: Integrating Technical and Biology-Driven Advances. Moura MT. Moura MT. Methods Mol Biol. 2023;2647:1-35. doi: 10.1007/978-1-0716-3064-8_1. Methods Mol Biol. 2023. PMID: 37041327
• Current Progress and Prospects in Rabbit Cloning. Cao W, Zhao J, Qu P, Liu E. Cao W, et al. Cell Reprogram. 2022 Apr;24(2):63-70. doi: 10.1089/cell.2021.0090. Epub 2022 Feb 15. Cell Reprogram. 2022. PMID: 35167365 Review.
• Cloning of Macaque Monkeys by Somatic Cell Nuclear Transfer. Liu Z, Cai Y, Wang Y, Nie Y, Zhang C, Xu Y, Zhang X, Lu Y, Wang Z, Poo M, Sun Q. Liu Z, et al. Cell. 2018 Feb 8;172(4):881-887.e7. doi: 10.1016/j.cell.2018.01.020. Epub 2018 Feb 1. Cell. 2018. PMID: 29395327
• Human somatic cell nuclear transfer and cloning. Ethics Committee of the American Society for Reproductive Medicine. Ethics Committee of the American Society for Reproductive Medicine. Fertil Steril. 2012 Oct;98(4):804-7. doi: 10.1016/j.fertnstert.2012.06.045. Epub 2012 Jul 12. Fertil Steril. 2012. PMID: 22795681
• 25th ANNIVERSARY OF CLONING BY SOMATIC-CELL NUCLEAR TRANSFER: Cloning, mitochondrial replacement and genome editing: 25 years of ethical debate since Dolly. Greenfield A. Greenfield A. Reproduction. 2021 Jun 11;162(1):F69-F78. doi: 10.1530/REP-20-0635. Reproduction. 2021. PMID: 33878027 Review.

Publication types

• Search in MeSH

Related information

Linkout - more resources, full text sources.

• Societa Editrice Universo, srl
• MedlinePlus Health Information

• Citation Manager

NCBI Literature Resources

MeSH PMC Bookshelf Disclaimer

The PubMed wordmark and PubMed logo are registered trademarks of the U.S. Department of Health and Human Services (HHS). Unauthorized use of these marks is strictly prohibited.

• Search Menu
• Sign in through your institution
• Advance Articles
• Clinical Case Studies
• Journal Club
• Clinical Chemistry Podcasts
• Clinical Trainee Council
• Special Issues
• Clinical Chemistry Guide to Scientific Writing
• Clinical Chemistry Guide to Manuscript Review
• Author Guidelines
• Submission Site
• Self-Archiving Policy
• Call for Papers
• Why Publish?
• About Clinical Chemistry
• Editorial Board
• Advertising & Corporate Services
• Journals on Oxford Academic
• Books on Oxford Academic

Article Contents

• < Previous

The Cloning Debates and Progress in Biotechnology

• Article contents
• Figures & tables
• Supplementary Data

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

• Permissions Icon Permissions

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..

Email alerts

Citing articles via.

• Recommend to Your Librarian
• Advertising and Corporate Services
• Journals Career Network

Affiliations

• Online ISSN 1530-8561
• Print ISSN 0009-9147
• Copyright © 2024 Association for Diagnostics & Laboratory Medicine
• About Oxford Academic
• Publish journals with us
• University press partners
• What we publish
• New features  
• Open access
• Institutional account management
• Rights and permissions
• Get help with access
• Accessibility
• Advertising
• Media enquiries
• Oxford University Press
• Oxford Languages
• University of Oxford

Oxford University Press is a department of the University of Oxford. It furthers the University's objective of excellence in research, scholarship, and education by publishing worldwide

• Copyright © 2024 Oxford University Press
• Cookie settings
• Cookie policy
• Privacy policy
• Legal notice

This Feature Is Available To Subscribers Only

Sign In or Create an Account

This PDF is available to Subscribers Only

For full access to this pdf, sign in to an existing account, or purchase an annual subscription.

• 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

42k Accesses

13 Citations

212 Altmetric

Metrics details

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.

Bryksin AV, Matsumura I: Overlap extension PCR cloning: a simple and reliable way to create recombinant plasmids. Biotechniques 2010, 48: 463. 10.2144/000113418

Article   Google Scholar  

Hughes RA, Miklos AE, Ellington AD: Gene synthesis: methods and applications. Methods Enzymol 2011, 498: 277–309.

Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA, Smith HO: Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 2009, 6: 343-U341. 10.1038/nmeth.1318

Mitchell DB, Ruggli N, Tratschin JD: An improved method for cloning PCR fragments. PCR Methods Appl 1992, 2: 81–2. 10.1101/gr.2.1.81

Radeck J, Kraft K, Bartels J, Cikovic T, Durr F, Emenegger J, et al .: The Bacillus BioBrick Box: generation and evaluation of essential genetic building blocks for standardized work with Bacillus subtilis. J Biol Eng 2013, 7: 29. 10.1186/1754-1611-7-29

Haun RS, Serventi IM, Moss J: Rapid, reliable ligation-independent cloning of PCR products using modified plasmid vectors. Biotechniques 1992, 13: 515–8.

Google Scholar  

Mead DA, Pey NK, Herrnstadt C, Marcil RA, Smith LM: A universal method for the direct cloning of PCR amplified nucleic acid. Biotechnology 1991, 9: 657–63. 10.1038/nbt0791-657

Holton TA, Graham MW: A simple and efficient method for direct cloning of PCR products using ddT-tailed vectors. Nucleic Acids Res 1991, 19: 1156. 10.1093/nar/19.5.1156

Holst J, Szymczak-Workman AL, Vignali KM, Burton AR, Workman CJ, Vignali DA: Generation of T-cell receptor retrogenic mice. Nat Protoc 2006, 1: 406–17. 10.1038/nprot.2006.61

Hoffman LM, Jendrisak J: Heat-labile phosphatase simplifies the preparation of dephosphorylated vector DNA. Gene 1990, 88: 97–9. 10.1016/0378-1119(90)90064-X

Pheiffer BH, Zimmerman SB: Polymer-stimulated ligation: enhanced blunt- or cohesive-end ligation of DNA or deoxyribooligonucleotides by T4 DNA ligase in polymer solutions. Nucleic Acids Res 1983, 11: 7853–71. 10.1093/nar/11.22.7853

Pingoud A, Wilson GG, Wende W: Type II restriction endonucleases-a historical perspective and more. Nucleic Acids Res 2014, 42: 7489–527. 10.1093/nar/gku447

Mural RJ: Cleavage by the restriction endonuclease Asp718, an isoschizomer of KpnI, is sensitive to Escherichia coli Dcm methylation. Nucleic Acids Res 1987, 15: 9085. 10.1093/nar/15.21.9085

Casali N: Escherichia coli host strains. In E coli Plasmid Vectors . Edited by: Casali N, Preston A. Humana Press Inc: Totowa; 2003:27–48.

Chapter   Google Scholar  

Corbett AJ, Brady JL, Lew AM: A single buffer that universally serves both restriction digestion and loading. Mol Biotechnol 2002, 21: 51–5. 10.1385/MB:21:1:051

Wilfinger WW, Mackey K, Chomczynski P: Effect of pH and ionic strength on the spectrophotometric assessment of nucleic acid purity. Biotechniques 1997, 22: 474–6.

Kozak M: An analysis of 5′-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res 1987, 15: 8125–48. 10.1093/nar/15.20.8125

Kozak M: Regulation of translation via mRNA structure in prokaryotes and eukaryotes. Gene 2005, 361: 13–37.

Cline J, Braman JC, Hogrefe HH: PCR fidelity of Pfu DNA polymerase and other thermostable DNA polymerases. Nucleic Acids Res 1996, 24: 3546–51. 10.1093/nar/24.18.3546

Benson LM, Null AP, Muddiman DC: Advantages of Thermococcus kodakaraenis (KOD) DNA polymerase for PCR-mass spectrometry based analyses. J Am Soc Mass Spectr 2003, 14: 601–4. 10.1016/S1044-0305(03)00148-X

Wang Y, Prosen DE, Mei L, Sullivan JC, Finney M, Vander Horn PB: A novel strategy to engineer DNA polymerases for enhanced processivity and improved performance in vitro. Nucleic Acids Res 2004, 32: 1197–207. 10.1093/nar/gkh271

McInerney P, Adams P, Hadi MZ: Error Rate Comparison during Polymerase Chain Reaction by DNA Polymerase. Mol Biol Int 2014, .: .. doi:10.1155/2014/287430

Breslauer KJ, Frank R, Blocker H, Marky LA: Predicting DNA duplex stability from the base sequence. Proc Natl Acad Sci U S A 1986, 83: 3746–50. 10.1073/pnas.83.11.3746

Lodge J, Lund P, Minchin S: Gene cloning principles and applications . New York: Taylor & Francis Group; 2007.

Bolivar F, Rodriguez RL, Greene PJ, Betlach MC, Heyneker HL, Boyer HW, et al .: Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene 1977, 2 (2) : 95–113. 10.1016/0378-1119(77)90000-2

Preston A: Choosing a cloning vector. In E coli Plasmid Vectors . Edited by: Casali N, Preston A. Totowa: Humana Press Inc; 2003:19–26.

Stoker NG, Fairweather NF, Spratt BG: Versatile low-copy-number plasmid vectors for cloning in Escherichia coli. Gene 1982, 18: 335–41. 10.1016/0378-1119(82)90172-X

Bernard P, Gabant P, Bahassi EM, Couturier M: Positive-selection vectors using the F plasmid ccdB killer gene. Gene 1994, 148: 71–4. 10.1016/0378-1119(94)90235-6

Shuman S: Novel approach to molecular cloning and polynucleotide synthesis using vaccinia DNA topoisomerase. J Biological Chem 1994, 269: 32678–84.

Noguchi H, Park J, Takagi T: MetaGene: prokaryotic gene finding from environmental genome shotgun sequences. Nucleic Acids Res 2006, 34: 5623–30. 10.1093/nar/gkl723

Ewing B, Hillier L, Wendl MC, Green P: Base-calling of automated sequencer traces using phred. I. Accuracy assessment. Genome Res 1998, 8: 175–85. 10.1101/gr.8.3.175

Keith CS, Hoang DO, Barrett BM, Feigelman B, Nelson MC, Thai H, et al .: Partial sequence analysis of 130 randomly selected maize cDNA clones. Plant Physiol 1993, 101: 329–32. 10.1104/pp.101.1.329

Richter DC, Ott F, Auch AF, Schmid R, Huson DH: MetaSim: a sequencing simulator for genomics and metagenomics. PLoS One 2008, 3: e3373. 10.1371/journal.pone.0003373

Hoff KJ: The effect of sequencing errors on metagenomic gene prediction. BMC Genomics 2009, 10: 520. 10.1186/1471-2164-10-520

Treangen TJ, Salzberg SL: Repetitive DNA and next-generation sequencing: computational challenges and solutions. Nat Rev Geneti 2012, 13: 36–46.

Prosdocimi F, Ortega JM: Accessing optimal primer distance from insert. In Silico Biol 2005, 5: 469–77.

Engebrecht J, Heilig JS, Brent R: Isolation and purification of DNA. Current Protocols in Immunology 1993.

Bzymek M, Lovett ST: Instability of repetitive DNA sequences: the role of replication in multiple mechanisms. Proc Natl Acad Sci U S A 2001, 98: 8319–25. 10.1073/pnas.111008398

Geisse S, Henke M: Large-scale transient transfection of mammalian cells: a newly emerging attractive option for recombinant protein production. J Struct Funct Genomics 2005, 6: 165–70. 10.1007/s10969-005-2826-4

Szymczak-Workman AL, Vignali KM, Vignali DA: Design and construction of 2A peptide-linked multicistronic vectors. Cold Spring Harbor Protocols 2012, 2012: 199–204.

Szymczak AL, Vignali DA: Development of 2A peptide-based strategies in the design of multicistronic vectors. Expert Opin Biol Ther 2005, 5: 627–38. 10.1517/14712598.5.5.627

Chen X, Zaro JL, Shen WC: Fusion protein linkers: property, design and functionality. Adv Drug Deliv Rev 2013, 65: 1357–69. 10.1016/j.addr.2012.09.039

Yang L, Ukil L, Osmani A, Nahm F, Davies J, De Souza CP, et al .: Rapid production of gene replacement constructs and generation of a green fluorescent protein-tagged centromeric marker in Aspergillus nidulans. Eukaryotic Cell 2004, 3: 1359–62. 10.1128/EC.3.5.1359-1362.2004

Bhide M, Natarajan S, Hresko S, Aguilar C, Bencurova E: Rapid in vitro protein synthesis pipeline: a promising tool for cost-effective protein array design. Mol Biosyst 2014, 10: 1236–45. 10.1039/c4mb00003j

Irian S: Large-scale tag/PCR-based gene expression profiling. World J Microb Biot 2014, 30: 2125–39. 10.1007/s11274-014-1641-0

Schneider BL, Seufert W, Steiner B, Yang QH, Futcher AB: Use of polymerase chain reaction epitope tagging for protein tagging in Saccharomyces cerevisiae. Yeast 1995, 11: 1265–74. 10.1002/yea.320111306

Erster O, Liscovitch M: A modified inverse PCR procedure for insertion, deletion, or replacement of a DNA fragment in a target sequence and its application in the ligand interaction scan method for generation of ligand-regulated proteins. Methods Mol Biol 2010, 634: 157–74. 10.1007/978-1-60761-652-8_12

Li C, Wen A, Shen B, Lu J, Huang Y, Chang Y: FastCloning: a highly simplified, purification-free, sequence- and ligation-independent PCR cloning method. BMC Biotechnol 2011, 11: 92. 10.1186/1472-6750-11-92

Chaudhary VK, Shrivastava N, Verma V, Das S, Kaur C, Grover P, et al .: Rapid restriction enzyme-free cloning of PCR products: a high-throughput method applicable for library construction. PLoS One 2014, 9: e111538. 10.1371/journal.pone.0111538

Mead DA, Pey NK, Herrnstadt C, Marcil RA, Smith LM: A universal method for the direct cloning of PCR amplified nucleic acid. Biotechnology (N Y) 1991, 9: 657–63. 10.1038/nbt0791-657

Stevenson J, Krycer JR, Phan L, Brown AJ: A practical comparison of ligation-independent cloning techniques. PLoS One 2013, 8: e83888. 10.1371/journal.pone.0083888

Ghosh A, Wolenski M, Klein C, Welte K, Blazar BR, Sauer MG: Cytotoxic T cells reactive to an immunodominant leukemia-associated antigen can be specifically primed and expanded by combining a specific priming step with nonspecific large-scale expansion. J Immunother 2008, 31: 121–31. 10.1097/CJI.0b013e31815aaf24

Suerth JD, Maetzig T, Brugman MH, Heinz N, Appelt JU, Kaufmann KB, et al .: Alpharetroviral self-inactivating vectors: long-term transgene expression in murine hematopoietic cells and low genotoxicity. Mol Ther 2012, 20: 1022–32. 10.1038/mt.2011.309

Download references

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.

Author information

Authors and affiliations.

Departments of Pediatric Hematology, Oncology and Blood Stem Cell Transplantation, Hannover, Germany

Sayed Shahabuddin Hoseini & Martin G Sauer

Hannover Center for Transplantation Research, Hannover, Germany

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

Martin G Sauer

You can also search for this author in PubMed   Google Scholar

Corresponding author

Correspondence to Martin G Sauer .

Additional information

Competing interests.

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.

Electronic supplementary material

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)

Rights and permissions

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/4.0 ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/ ) applies to the data made available in this article, unless otherwise stated.

Reprints and permissions

About this article

Cite this article.

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

Download citation

Received : 05 September 2014

Accepted : 08 January 2015

Published : 19 January 2015

DOI : https://doi.org/10.1186/1754-1611-9-2

Share this article

Anyone you share the following link with will be able to read this content:

Sorry, a shareable link is not currently available for this article.

Provided by the Springer Nature SharedIt content-sharing initiative

• Polymerase chain reaction (PCR)
• Recombinant DNA
• Biological engineering
• Educational guide
• Transduction
• Transfection

Journal of Biological Engineering

ISSN: 1754-1611

• General enquiries: [email protected]

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

• View all journals
• Explore content
• About the journal
• Publish with us
• Sign up for alerts
• News and Views Feature
• Published: 16 December 1999

The future of cloning

• J. B. Gurdon 1 , 2 &
• Alan Colman 3  

Nature volume  402 ,  pages 743–746 ( 1999 ) Cite this article

4912 Accesses

95 Citations

9 Altmetric

Metrics details

It is now possible to make clones, or exact genetic copies, of sheep, cows, goats, mice and, probably, humans. This opens the way towards the production of replacement body parts from adult cells.

This is a preview of subscription content, access via your institution

Access options

Subscribe to this journal

Receive 51 print issues and online access

185,98 € per year

only 3,65 € per issue

Buy this article

• Purchase on SpringerLink
• Instant access to full article PDF

Prices may be subject to local taxes which are calculated during checkout

Elsdale, T. R., Gurdon, J. B. & Fischberg, M. J. Embryol. Exp. Morph. 8 , 437–444 (1960).

CAS   PubMed   Google Scholar  

Sun, F. Z. & Moor, R. Curr. Top. Dev. Biol. 30 , 145–173 (1995).

Google Scholar  

Wilmut, I. et al. Nature 385 , 810–813 (1997); erratum, Nature 386 , 200 (1997).

Article   ADS   CAS   Google Scholar  

Wakayama, T. et al. Nature 394 , 369–374 (1998).

Ashworth, D. et al. Nature 394 , 329 (1998).

Signer, E. et al. Nature 394 , 329–330 (1995).

Article   ADS   Google Scholar  

Shiels, P. G. et al. Nature 399 , 316–317 (1999).

Briggs, R. & King, T. J. J. Morphol. 100 , 269–312 (1957).

Article   Google Scholar  

Gurdon, J. B. J. Embryol. Exp. Morphol. 10 , 622–640 (1962).

Gurdon, J. B. & Uehlinger, V. Nature 210 , 1240–1241 (1966).

Gurdon, J. B., Laskey, R. A. & Reeves, O. R. J. Embryol. Exp. Morphol. 34 , 93–112 (1975).

DiBerardino, M. A. & Hoffner, N. J. Science 219 , 862–864 (1983).

Wells, D. N., Misica, P. M. & Tervit, H. R. Biol. Reprod. 60 , 996–1005 (1999).

Article   CAS   Google Scholar  

Campbell, K. H., Ritchie, W. A. & Wilmut, I. Biol. Reprod. 49 , 933–942 (1993).

Blasco, M. A. et al. Cell 91 , 24–34 (1997).

Stennard, F., Ryan, K. & Gurdon, J. B. Curr. Opin. Genet. Dev. 7 , 620–627 (1997).

Chan, A. P. & Gurdon, J. B. Int. J. Dev. Biol. 40 , 441–451 (1996).

Surani, M. A. Cell 93 , 309–312 (1998).

Heard, E., Clerc, P. & Auner, P. Annu. Rev. Genet. 31 , 571–610 (1997).

De Robertis, E. M., Partington, G. A., Longthorne, R. F. & Gurdon, J. B. J. Embryol. Exp. Morphol. 40 , 199–214 (1977).

Bjornson, C. et al. Science 283 , 534–537 (1999).

Azizi, S. A. et al. Proc. Natl Acad. Sci. USA 95 , 3908–3913 (1999).

Thomson, J. A. et al. Science 282 , 1145–1147 (1998).

Shamblott, M. J. et al. Proc. Natl Acad. Sci. USA 95 , 13726–13731 (1998).

Green, J. B. et al. Cell 71 , 731–739 (1992).

Burley, J. & Harris, J. J. Med. Ethics 25 , 108–113 (1999).

Burley, J. Semin. Cell Dev. Biol. 10 , 287–294 (1999).

Lanza, R. P., Cibelli, J. B. & West, M. D. Nature Med. 5 , 975–977 (1999).

Dominko, T. et al. Biol. Reprod. 60 , 1496–1502 (1999).

Bueler, H. et al. Cell 73 , 1339–1347 (1993).

Tearle, R. G. et al. Transplantation 61 , 13–19 (1996).

Snouwaert, J. N. et al. Science 257 , 1083–1088 (1992).

Gurdon, J. B. Proc. R. Soc. Lond. B 198 , 211–247 (1977).

Download references

Author information

Authors and affiliations.

the Wellcome/CRC Institute, Tennis Court Road

J. B. Gurdon

the Department of Zoology, University of Cambridge, Cambridge, CB2 1QR, UK

PPL Therapeutics, Roslin Institute, Edinburgh, EH25 9PP, UK

Alan Colman

You can also search for this author in PubMed   Google Scholar

Corresponding authors

Correspondence to J. B. Gurdon or Alan Colman .

Rights and permissions

Reprints and permissions

About this article

Cite this article.

Gurdon, J., Colman, A. The future of cloning. Nature 402 , 743–746 (1999). https://doi.org/10.1038/45429

Download citation

Issue Date : 16 December 1999

DOI : https://doi.org/10.1038/45429

Share this article

Anyone you share the following link with will be able to read this content:

Sorry, a shareable link is not currently available for this article.

Provided by the Springer Nature SharedIt content-sharing initiative

This article is cited by

Preventing common hereditary disorders through time-separated twinning.

• Alexander Churbanov
• Levon Abrahamyan

BioNanoScience (2018)

Mediators of reprogramming: transcription factors and transitions through mitosis

• Dieter Egli
• Garrett Birkhoff
• Kevin Eggan

Nature Reviews Molecular Cell Biology (2008)

Nuclear transfer cloning and the United Nations

• Anthony CF Perry

Nature Biotechnology (2004)

Quick links

• Explore articles by subject
• Guide to authors
• Editorial policies

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

• Introduction to Genomics
• Educational Resources
• Policy Issues in Genomics
• The Human Genome Project
• Funding Opportunities
• Funded Programs & Projects
• Division and Program Directors
• Scientific Program Analysts
• Contacts by Research Area
• News & Events
• Research Areas
• Research Investigators
• Research Projects
• Clinical Research
• Data Tools & Resources
• Genomics & Medicine
• Family Health History
• For Patients & Families
• For Health Professionals
• Jobs at NHGRI
• Training at NHGRI
• Funding for Research Training
• Professional Development Programs
• NHGRI Culture
• Social Media
• Broadcast Media
• Image Gallery
• Press Resources
• Organization
• NHGRI Director
• Mission and Vision
• Policies and Guidance
• Institute Advisors
• Strategic Vision
• Leadership Initiatives
• Diversity, Equity, and Inclusion
• Partner with NHGRI
• Staff Search

Cloning Fact Sheet

The term cloning describes a number of different processes that can be used to produce genetically identical copies of a biological entity. The copied material, which has the same genetic makeup as the original, is referred to as a clone. Researchers have cloned a wide range of biological materials, including genes, cells, tissues and even entire organisms, such as a sheep.

Do clones ever occur naturally?

Yes. In nature, some plants and single-celled organisms, such as bacteria , produce genetically identical offspring through a process called asexual reproduction. In asexual reproduction, a new individual is generated from a copy of a single cell from the parent organism.

Natural clones, also known as identical twins, occur in humans and other mammals. These twins are produced when a fertilized egg splits, creating two or more embryos that carry almost identical DNA . Identical twins have nearly the same genetic makeup as each other, but they are genetically different from either parent.

What are the types of artificial cloning?

There are three different types of artificial cloning: gene cloning, reproductive cloning and therapeutic cloning.

Gene cloning produces copies of genes or segments of DNA. Reproductive cloning produces copies of whole animals. Therapeutic cloning produces embryonic stem cells for experiments aimed at creating tissues to replace injured or diseased tissues.

Gene cloning, also known as DNA cloning, is a very different process from reproductive and therapeutic cloning. Reproductive and therapeutic cloning share many of the same techniques, but are done for different purposes.

What sort of cloning research is going on at NHGRI?

Gene cloning is the most common type of cloning done by researchers at NHGRI. NHGRI researchers have not cloned any mammals and NHGRI does not clone humans.

How are genes cloned?

Researchers routinely use cloning techniques to make copies of genes that they wish to study. The procedure consists of inserting a gene from one organism, often referred to as "foreign DNA," into the genetic material of a carrier called a vector. Examples of vectors include bacteria, yeast cells, viruses or plasmids, which are small DNA circles carried by bacteria. After the gene is inserted, the vector is placed in laboratory conditions that prompt it to multiply, resulting in the gene being copied many times over.

How are animals cloned?

In reproductive cloning, researchers remove a mature somatic cell , such as a skin cell, from an animal that they wish to copy. They then transfer the DNA of the donor animal's somatic cell into an egg cell, or oocyte, that has had its own DNA-containing nucleus removed.

Researchers can add the DNA from the somatic cell to the empty egg in two different ways. In the first method, they remove the DNA-containing nucleus of the somatic cell with a needle and inject it into the empty egg. In the second approach, they use an electrical current to fuse the entire somatic cell with the empty egg.

In both processes, the egg is allowed to develop into an early-stage embryo in the test-tube and then is implanted into the womb of an adult female animal.

Ultimately, the adult female gives birth to an animal that has the same genetic make up as the animal that donated the somatic cell. This young animal is referred to as a clone. Reproductive cloning may require the use of a surrogate mother to allow development of the cloned embryo, as was the case for the most famous cloned organism, Dolly the sheep.

What animals have been cloned?

Over the last 50 years, scientists have conducted cloning experiments in a wide range of animals using a variety of techniques. In 1979, researchers produced the first genetically identical mice by splitting mouse embryos in the test tube and then implanting the resulting embryos into the wombs of adult female mice. Shortly after that, researchers produced the first genetically identical cows, sheep and chickens by transferring the nucleus of a cell taken from an early embryo into an egg that had been emptied of its nucleus.

It was not until 1996, however, that researchers succeeded in cloning the first mammal from a mature (somatic) cell taken from an adult animal. After 276 attempts, Scottish researchers finally produced Dolly, the lamb from the udder cell of a 6-year-old sheep. Two years later, researchers in Japan cloned eight calves from a single cow, but only four survived.

Besides cattle and sheep, other mammals that have been cloned from somatic cells include: cat, deer, dog, horse, mule, ox, rabbit and rat. In addition, a rhesus monkey has been cloned by embryo splitting.

Have humans been cloned?

Despite several highly publicized claims, human cloning still appears to be fiction. There currently is no solid scientific evidence that anyone has cloned human embryos.

In 1998, scientists in South Korea claimed to have successfully cloned a human embryo, but said the experiment was interrupted very early when the clone was just a group of four cells. In 2002, Clonaid, part of a religious group that believes humans were created by extraterrestrials, held a news conference to announce the birth of what it claimed to be the first cloned human, a girl named Eve. However, despite repeated requests by the research community and the news media, Clonaid never provided any evidence to confirm the existence of this clone or the other 12 human clones it purportedly created.

In 2004, a group led by Woo-Suk Hwang of Seoul National University in South Korea published a paper in the journal Science in which it claimed to have created a cloned human embryo in a test tube. However, an independent scientific committee later found no proof to support the claim and, in January 2006, Science announced that Hwang's paper had been retracted.

From a technical perspective, cloning humans and other primates is more difficult than in other mammals. One reason is that two proteins essential to cell division, known as spindle proteins, are located very close to the chromosomes in primate eggs. Consequently, removal of the egg's nucleus to make room for the donor nucleus also removes the spindle proteins, interfering with cell division. In other mammals, such as cats, rabbits and mice, the two spindle proteins are spread throughout the egg. So, removal of the egg's nucleus does not result in loss of spindle proteins. In addition, some dyes and the ultraviolet light used to remove the egg's nucleus can damage the primate cell and prevent it from growing.

Do cloned animals always look identical?

No. Clones do not always look identical. Although clones share the same genetic material, the environment also plays a big role in how an organism turns out.

For example, the first cat to be cloned, named Cc, is a female calico cat that looks very different from her mother. The explanation for the difference is that the color and pattern of the coats of cats cannot be attributed exclusively to genes. A biological phenomenon involving inactivation of the X chromosome (See sex chromosome ) in every cell of the female cat (which has two X chromosomes) determines which coat color genes are switched off and which are switched on. The distribution of X inactivation, which seems to occur randomly, determines the appearance of the cat's coat.

What are the potential applications of cloned animals?

Reproductive cloning may enable researchers to make copies of animals with the potential benefits for the fields of medicine and agriculture.

For instance, the same Scottish researchers who cloned Dolly have cloned other sheep that have been genetically modified to produce milk that contains a human protein essential for blood clotting. The hope is that someday this protein can be purified from the milk and given to humans whose blood does not clot properly. Another possible use of cloned animals is for testing new drugs and treatment strategies. The great advantage of using cloned animals for drug testing is that they are all genetically identical, which means their responses to the drugs should be uniform rather than variable as seen in animals with different genetic make-ups.

After consulting with many independent scientists and experts in cloning, the U.S. Food and Drug Administration (FDA) decided in January 2008 that meat and milk from cloned animals, such as cattle, pigs and goats, are as safe as those from non-cloned animals. The FDA action means that researchers are now free to using cloning methods to make copies of animals with desirable agricultural traits, such as high milk production or lean meat. However, because cloning is still very expensive, it will likely take many years until food products from cloned animals actually appear in supermarkets.

Another application is to create clones to build populations of endangered, or possibly even extinct, species of animals. In 2001, researchers produced the first clone of an endangered species: a type of Asian ox known as a guar. Sadly, the baby guar, which had developed inside a surrogate cow mother, died just a few days after its birth. In 2003, another endangered type of ox, called the Banteg, was successfully cloned. Soon after, three African wildcats were cloned using frozen embryos as a source of DNA. Although some experts think cloning can save many species that would otherwise disappear, others argue that cloning produces a population of genetically identical individuals that lack the genetic variability necessary for species survival.

Some people also have expressed interest in having their deceased pets cloned in the hope of getting a similar animal to replace the dead one. But as shown by Cc the cloned cat, a clone may not turn out exactly like the original pet whose DNA was used to make the clone.

What are the potential drawbacks of cloning animals?

Reproductive cloning is a very inefficient technique and most cloned animal embryos cannot develop into healthy individuals. For instance, Dolly was the only clone to be born live out of a total of 277 cloned embryos. This very low efficiency, combined with safety concerns, presents a serious obstacle to the application of reproductive cloning.

Researchers have observed some adverse health effects in sheep and other mammals that have been cloned. These include an increase in birth size and a variety of defects in vital organs, such as the liver, brain and heart. Other consequences include premature aging and problems with the immune system. Another potential problem centers on the relative age of the cloned cell's chromosomes. As cells go through their normal rounds of division, the tips of the chromosomes, called telomeres, shrink. Over time, the telomeres become so short that the cell can no longer divide and, consequently, the cell dies. This is part of the natural aging process that seems to happen in all cell types. As a consequence, clones created from a cell taken from an adult might have chromosomes that are already shorter than normal, which may condemn the clones' cells to a shorter life span. Indeed, Dolly, who was cloned from the cell of a 6-year-old sheep, had chromosomes that were shorter than those of other sheep her age. Dolly died when she was six years old, about half the average sheep's 12-year lifespan.

What is therapeutic cloning?

Therapeutic cloning involves creating a cloned embryo for the sole purpose of producing embryonic stem cells with the same DNA as the donor cell. These stem cells can be used in experiments aimed at understanding disease and developing new treatments for disease. To date, there is no evidence that human embryos have been produced for therapeutic cloning.

The richest source of embryonic stem cells is tissue formed during the first five days after the egg has started to divide. At this stage of development, called the blastocyst, the embryo consists of a cluster of about 100 cells that can become any cell type. Stem cells are harvested from cloned embryos at this stage of development, resulting in destruction of the embryo while it is still in the test tube.

What are the potential applications of therapeutic cloning?

Researchers hope to use embryonic stem cells, which have the unique ability to generate virtually all types of cells in an organism, to grow healthy tissues in the laboratory that can be used replace injured or diseased tissues. In addition, it may be possible to learn more about the molecular causes of disease by studying embryonic stem cell lines from cloned embryos derived from the cells of animals or humans with different diseases. Finally, differentiated tissues derived from ES cells are excellent tools to test new therapeutic drugs.

What are the potential drawbacks of therapeutic cloning?

Many researchers think it is worthwhile to explore the use of embryonic stem cells as a path for treating human diseases. However, some experts are concerned about the striking similarities between stem cells and cancer cells. Both cell types have the ability to proliferate indefinitely and some studies show that after 60 cycles of cell division, stem cells can accumulate mutations that could lead to cancer. Therefore, the relationship between stem cells and cancer cells needs to be more clearly understood if stem cells are to be used to treat human disease.

What are some of the ethical issues related to cloning?

Gene cloning is a carefully regulated technique that is largely accepted today and used routinely in many labs worldwide. However, both reproductive and therapeutic cloning raise important ethical issues, especially as related to the potential use of these techniques in humans.

Reproductive cloning would present the potential of creating a human that is genetically identical to another person who has previously existed or who still exists. This may conflict with long-standing religious and societal values about human dignity, possibly infringing upon principles of individual freedom, identity and autonomy. However, some argue that reproductive cloning could help sterile couples fulfill their dream of parenthood. Others see human cloning as a way to avoid passing on a deleterious gene that runs in the family without having to undergo embryo screening or embryo selection.

Therapeutic cloning, while offering the potential for treating humans suffering from disease or injury, would require the destruction of human embryos in the test tube. Consequently, opponents argue that using this technique to collect embryonic stem cells is wrong, regardless of whether such cells are used to benefit sick or injured people.

Last updated: August 15, 2020

Warning: The NCBI web site requires JavaScript to function. more...

An official website of the United States government

The .gov means it's official. Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you're on a federal government site.

The site is secure. The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

• Publications
• Account settings
• Browse Titles

NCBI Bookshelf. A service of the National Library of Medicine, National Institutes of Health.

National Academy of Sciences (US), National Academy of Engineering (US), Institute of Medicine (US) and National Research Council (US) Committee on Science, Engineering, and Public Policy. Scientific and Medical Aspects of Human Reproductive Cloning. Washington (DC): National Academies Press (US); 2002.

Scientific and Medical Aspects of Human Reproductive Cloning.

• Hardcopy Version at National Academies Press

2 Cloning: Definitions And Applications

In this chapter, we address the following questions in our task statement:

What does cloning of animals including humans mean? What are its purposes? How does it differ from stem cell research?

To organize its response to those questions, the panel developed a series of subquestions, which appear as the section headings in the following text.

• WHAT IS MEANT BY REPRODUCTIVE CLONING OF ANIMALS INCLUDING HUMANS?

Reproductive cloning is defined as the deliberate production of genetically identical individuals. Each newly produced individual is a clone of the original. Monozygotic (identical) twins are natural clones. Clones contain identical sets of genetic material in the nucleus—the compartment that contains the chromosomes—of every cell in their bodies. Thus, cells from two clones have the same DNA and the same genes in their nuclei.

All cells, including eggs, also contain some DNA in the energy-generating “factories” called mitochondria. These structures are in the cytoplasm, the region of a cell outside the nucleus. Mitochondria contain their own DNA and reproduce independently. True clones have identical DNA in both the nuclei and mitochondria, although the term clones is also used to refer to individuals that have identical nuclear DNA but different mitochondrial DNA.

• HOW IS REPRODUCTIVE CLONING DONE?

Two methods are used to make live-born mammalian clones. Both require implantation of an embryo in a uterus and then a normal period of gestation and birth. However, reproductive human or animal cloning is not defined by the method used to derive the genetically identical embryos suitable for implantation. Techniques not yet developed or described here would nonetheless constitute cloning if they resulted in genetically identical individuals of which at least one were an embryo destined for implantation and birth.

The two methods used for reproductive cloning thus far are as follows:

• Cloning using somatic cell nuclear transfer ( SCNT ) [ 1 ]. This procedure starts with the removal of the chromosomes from an egg to create an enucleated egg. The chromosomes are replaced with a nucleus taken from a somatic (body) cell of the individual or embryo to be cloned. This cell could be obtained directly from the individual, from cells grown in culture, or from frozen tissue. The egg is then stimulated, and in some cases it starts to divide. If that happens, a series of sequential cell divisions leads to the formation of a blastocyst, or preimplantation embryo. The blastocyst is then transferred to the uterus of an animal. The successful implantation of the blastocyst in a uterus can result in its further development, culminating sometimes in the birth of an animal. This animal will be a clone of the individual that was the donor of the nucleus. Its nuclear DNA has been inherited from only one genetic parent.

The number of times that a given individual can be cloned is limited theoretically only by the number of eggs that can be obtained to accept the somatic cell nuclei and the number of females available to receive developing embryos. If the egg used in this procedure is derived from the same individual that donates the transferred somatic nucleus, the result will be an embryo that receives all its genetic material—nuclear and mitochondrial—from a single individual. That will also be true if the egg comes from the nucleus donor's mother, because mitochondria are inherited maternally. Multiple clones might also be produced by transferring identical nuclei to eggs from a single donor. If the somatic cell nucleus and the egg come from different individuals, they will not be identical to the nuclear donor because the clones will have somewhat different mitochondrial genes [ 2 ; 3 ]

• Cloning by embryo splitting. This procedure begins with in vitro fertilization ( IVF ): the union outside the woman's body of a sperm and an egg to generate a zygote. The zygote (from here onwards also called an embryo) divides into two and then four identical cells. At this stage, the cells can be separated and allowed to develop into separate but identical blastocysts, which can then be implanted in a uterus. The limited developmental potential of the cells means that the procedure cannot be repeated, so embryo splitting can yield only two identical mice and probably no more than four identical humans.

The DNA in embryo splitting is contributed by germ cells from two individuals—the mother who contributed the egg and the father who contributed the sperm. Thus, the embryos, like those formed naturally or by standard IVF , have two parents. Their mitochondrial DNA is identical. Because this method of cloning is identical with the natural formation of monozygotic twins and, in rare cases, even quadruplets, it is not discussed in detail in this report.

• WILL CLONES LOOK AND BEHAVE EXACTLY THE SAME?

Even if clones are genetically identical with one another, they will not be identical in physical or behavioral characteristics, because DNA is not the only determinant of these characteristics. A pair of clones will experience different environments and nutritional inputs while in the uterus, and they would be expected to be subject to different inputs from their parents, society, and life experience as they grow up. If clones derived from identical nuclear donors and identical mitocondrial donors are born at different times, as is the case when an adult is the donor of the somatic cell nucleus, the environmental and nutritional differences would be expected to be more pronounced than for monozygotic (identical) twins. And even monozygotic twins are not fully identical genetically or epigenetically because mutations, stochastic developmental variations, and varied imprinting effects (parent-specific chemical marks on the DNA) make different contributions to each twin [ 3 ; 4 ].

Additional differences may occur in clones that do not have identical mitochondria. Such clones arise if one individual contributes the nucleus and another the egg—or if nuclei from a single individual are transferred to eggs from multiple donors. The differences might be expected to show up in parts of the body that have high demands for energy—such as muscle, heart, eye, and brain—or in body systems that use mitochondrial control over cell death to determine cell numbers [ 5 ; 6 ].

• WHAT ARE THE PURPOSES OF REPRODUCTIVE CLONING?

Cloning of livestock [ 1 ] is a means of replicating an existing favorable combination of traits, such as efficient growth and high milk production, without the genetic “lottery” and mixing that occur in sexual reproduction. It allows an animal with a particular genetic modification, such as the ability to produce a pharmaceutical in milk, to be replicated more rapidly than does natural mating [ 7 ; 8 ]. Moreover, a genetic modification can be made more easily in cultured cells than in an intact animal, and the modified cell nucleus can be transferred to an enucleated egg to make a clone of the required type. Mammals used in scientific experiments, such as mice, are cloned as part of research aimed at increasing our understanding of fundamental biological mechanisms.

In principle, those people who might wish to produce children through human reproductive cloning [ 9 ] include:

• Infertile couples who wish to have a child that is genetically identical with one of them, or with another nucleus donor
• Other individuals who wish to have a child that is genetically identical with them, or with another nucleus donor
• Parents who have lost a child and wish to have another, genetically identical child
• People who need a transplant (for example, of cord blood) to treat their own or their child's disease and who therefore wish to collect genetically identical tissue from a cloned fetus or newborn.

Possible reasons for undertaking human reproductive cloning have been analyzed according to their degree of justification. For example, in reference 10 it is proposed that human reproductive cloning aimed at establishing a genetic link to a gametically infertile parent would be more justifiable than an attempt by a sexually fertile person aimed at choosing a specific genome.

Transplantable tissue may be available without the need for the birth of a child produced by cloning. For example, embryos produced by in vitro fertilization ( IVF ) can be typed for transplant suitability, and in the future stem cells produced by nuclear transplantation may allow the production of transplantable tissue.

The alternatives open to infertile individuals are discussed in Chapter 4 .

• HOW DOES REPRODUCTIVE CLONING DIFFER FROM STEM CELL RESEARCH?

The recent and current work on stem cells that is briefly summarized below and discussed more fully in a recent report from the National Academies entitled Stem Cells and the Future of Regenerative Medicine [ 11 ] is not directly related to human reproductive cloning. However, the use of a common initial step—called either nuclear transplantation or somatic cell nuclear transfer ( SCNT )—has led Congress to consider bills that ban not only human reproductive cloning but also certain areas of stem cell research. Stem cells are cells that have the ability to divide repeatedly and give rise to both specialized cells and more stem cells. Some, such as some blood and brain stem cells, can be derived directly from adults [ 12 - 19 ] and others can be obtained from preimplantation embryos. Stem cells derived from embryos are called embryonic stem cells ( ES cells ). The above-mentioned report from the National Academies provides a detailed account of the current state of stem cell research [ 11 ].

ES cells are also called pluripotent stem cells because their progeny include all cell types that can be found in a postimplantation embryo, a fetus, and a fully developed organism. They are derived from the inner cell mass of early embryos (blastocysts) [ 20 - 23 ]. The cells in the inner cell mass of a given blastocyst are genetically identical, and each blastocyst yields only a single ES cell line. Stem cells are rarer [ 24 ] and more difficult to find in adults than in preimplantation embryos, and it has proved harder to grow some kinds of adult stem cells into cell lines after isolation [ 25 ; 26 ].

Production of different cells and tissues from ES cells or other stem cells is a subject of current research [ 11 ; 27 - 31 ]. Production of whole organs other than bone marrow (to be used in bone marrow transplantation) from such cells has not yet been achieved, and its eventual success is uncertain.

Current interest in stem cells arises from their potential for the therapeutic transplantation of particular healthy cells, tissues, and organs into people suffering from a variety of diseases and debilitating disorders. Research with adult stem cells indicates that they may be useful for such purposes, including for tissues other than those from which the cells were derived [ 12 ; 14 ; 17 ; 18 ; 25 - 27 ; 32 - 43 ]. On the basis of current knowledge, it appears unlikely that adults will prove to be a sufficient source of stem cells for all kinds of tissues [ 11 ; 44 - 47 ]. ES cell lines are of potential interest for transplantation because one cell line can multiply indefinitely and can generate not just one type of specialized cell, but many different types of specialized cells (brain, muscle, and so on) that might be needed for transplants [ 20 ; 28 ; 45 ; 48 ; 49 ]. However, much more research will be needed before the magnitude of the therapeutic potential of either adult stem cells or ES cells will be well understood.

One of the most important questions concerning the therapeutic potential of stem cells is whether the cells, tissues, and perhaps organs derived from them can be transplanted with minimal risk of transplant rejection. Ideally, adult stem cells advantageous for transplantation might be derived from patients themselves. Such cells, or tissues derived from them, would be genetically identical with the patient's own and not be rejected by the immune system. However, as previously described, the availability of sufficient adult stem cells and their potential to give rise to a full range of cell and tissue types are uncertain. Moreover, in the case of a disorder that has a genetic origin, a patient's own adult stem cells would carry the same defect and would have to be grown and genetically modified before they could be used for therapeutic transplantation.

The application of somatic cell nuclear transfer or nuclear transplantation offers an alternative route to obtaining stem cells that could be used for transplantation therapies with a minimal risk of transplant rejection. This procedure—sometimes called therapeutic cloning, research cloning, or nonreproductive cloning, and referred to here as nuclear transplantation to produce stem cells —would be used to generate pluripotent ES cells that are genetically identical with the cells of a transplant recipient [ 50 ]. Thus, like adult stem cells, such ES cells should ameliorate the rejection seen with unmatched transplants.

Two types of adult stem cells—stem cells in the blood forming bone marrow and skin stem cells—are the only two stem cell therapies currently in use. But, as noted in the National Academies' report entitled Stem Cells and the Future of Regenerative Medicine , many questions remain before the potential of other adult stem cells can be accurately assessed [ 11 ]. Few studies on adult stem cells have sufficiently defined the stem cell's potential by starting from a single, isolated cell, or defined the necessary cellular environment for correct differentiation or the factors controlling the efficiency with which the cells repopulate an organ. There is a need to show that the cells derived from introduced adult stem cells are contributing directly to tissue function, and to improve the ability to maintain adult stem cells in culture without the cells differentiating. Finally, most of the studies that have garnered so much attention have used mouse rather than human adult stem cells.

ES cells are not without their own potential problems as a source of cells for transplantation. The growth of human ES cells in culture requires a “feeder” layer of mouse cells that may contain viruses, and when allowed to differentiate the ES cells can form a mixture of cell types at once. Human ES cells can form benign tumors when introduced into mice [ 20 ], although this potential seems to disappear if the cells are allowed to differentiate before introduction into a recipient [ 51 ]. Studies with mouse ES cells have shown promise for treating diabetes [ 30 ], Parkinson's disease [ 52 ], and spinal cord injury [ 53 ].

The ES cells made with nuclear transplantation would have the advantage over adult stem cells of being able to provide virtually all cell types and of being able to be maintained in culture for long periods of time. Current knowledge is, however, uncertain, and research on both adult stem cells and stem cells made with nuclear transplantation is required to understand their therapeutic potentials. (This point is stated clearly in Finding and Recommendation 2 of Stem Cells and the Future of Regenerative Medicine [ 11 ] which states, in part, that “studies of both embryonic and adult human stem cells will be required to most efficiently advance the scientific and therapeutic potential of regenerative medicine.”) It is likely that the ES cells will initially be used to generate single cell types for transplantation, such as nerve cells or muscle cells. In the future, because of their ability to give rise to many cell types, they might be used to generate tissues and, theoretically, complex organs for transplantation. But this will require the perfection of techniques for directing their specialization into each of the component cell types and then the assembly of these cells in the correct proportion and spatial organization for an organ. That might be reasonably straightforward for a simple structure, such as a pancreatic islet that produces insulin, but it is more challenging for tissues as complex as that from lung, kidney, or liver [ 54 ; 55 ].

The experimental procedures required to produce stem cells through nuclear transplantation would consist of the transfer of a somatic cell nucleus from a patient into an enucleated egg, the in vitro culture of the embryo to the blastocyst stage, and the derivation of a pluripotent ES cell line from the inner cell mass of this blastocyst. Such stem cell lines would then be used to derive specialized cells (and, if possible, tissues and organs) in laboratory culture for therapeutic transplantation. Such a procedure, if successful, can avoid a major cause of transplant rejection. However, there are several possible drawbacks to this proposal. Experiments with animal models suggest that the presence of divergent mitochondrial proteins in cells may create “minor” transplantation antigens [ 56 ; 57 ] that can cause rejection [ 58 - 63 ]; this would not be a problem if the egg were donated by the mother of the transplant recipient or the recipient herself. For some autoimmune diseases, transplantation of cells cloned from the patient's own cells may be inappropriate, in that these cells can be targets for the ongoing destructive process. And, as with the use of adult stem cells, in the case of a disorder that has a genetic origin, ES cells derived by nuclear transplantation from the patient's own cells would carry the same defect and would have to be grown and genetically modified before they could be used for therapeutic transplantation. Using another source of stem cells is more likely to be feasible (although immunosuppression would be required) than the challenging task of correcting the one or more genes that are involved in the disease in adult stem cells or in a nuclear transplantation-derived stem cell line initiated with a nucleus from the patient.

In addition to nuclear transplantation, there are two other methods by which researchers might be able to derive ES cells with reduced likeli hood for rejection. A bank of ES cell lines covering many possible genetic makeups is one possibility, although the National Academies report entitled Stem Cells and the Future of Regenerative Medicine rated this as “difficult to conceive” [ 11 ]. Alternatively, embryonic stem cells might be engineered to eliminate or introduce certain cell-surface proteins, thus making the cells invisible to the recipient's immune system. As with the proposed use of many types of adult stem cells in transplantation, neither of these approaches carries anything close to a promise of success at the moment.

The preparation of embryonic stem cells by nuclear transplantation differs from reproductive cloning in that nothing is implanted in a uterus. The issue of whether ES cells alone can give rise to a complete embryo can easily be misinterpreted. The titles of some reports suggest that mouse embryos can be derived from ES cells alone [ 64 - 72 ]. In all cases, however, the ES cells need to be surrounded by cells derived from a host embryo, in particular trophoblast and primitive endoderm. In addition to forming part of the placenta, trophoblast cells of the blastocyst provide essential patterning cues or signals to the embryo that are required to determine the orientation of its future head and rump (anterior-posterior) axis. This positional information is not genetically determined but is acquired by the trophoblast cells from events initiated soon after fertilization or egg activation. Moreover, it is critical that the positional cues be imparted to the inner cells of the blastocyst during a specific time window of development [ 73 - 76 ]. Isolated inner cell masses of mouse blastocysts do not implant by themselves, but will do so if combined with trophoblast vesicles from another embryo [ 77 ]. By contrast, isolated clumps of mouse ES cells introduced into trophoblast vesicles never give rise to anything remotely resembling a postimplantation embryo, as opposed to a disorganized mass of trophoblast. In other words, the only way to get mouse ES cells to participate in normal development is to provide them with host embryonic cells, even if these cells do not remain viable throughout gestation (Richard Gardner, personal communication). It has been reported that human [ 20 ] and primate [ 78 - 79 ] ES cells can give rise to trophoblast cells in culture. However, these trophoblast cells would presumably lack the positional cues normally acquired during the development of a blastocyst from an egg. In the light of the experimental results with mouse ES cells described above, it is very unlikely that clumps of human ES cells placed in a uterus would implant and develop into a fetus. It has been reported that clumps of human ES cells in culture, like clumps of mouse ES cells, give rise to disorganized aggregates known as embryoid bodies [ 80 ].

Besides their uses for therapeutic transplantation, ES cells obtained by nuclear transplantation could be used in laboratories for several types of studies that are important for clinical medicine and for fundamental research in human developmental biology. Such studies could not be carried out with mouse or monkey ES cells and are not likely to be feasible with ES cells prepared from normally fertilized blastocysts. For example, ES cells derived from humans with genetic diseases could be prepared through nuclear transplantation and would permit analysis of the role of the mutated genes in both cell and tissue development and in adult cells difficult to study otherwise, such as nerve cells of the brain. This work has the disadvantage that it would require the use of donor eggs. But for the study of many cell types there may be no alternative to the use of ES cells; for these cell types the derivation of primary cell lines from human tissues is not yet possible.

If the differentiation of ES cells into specialized cell types can be understood and controlled, the use of nuclear transplantation to obtain genetically defined human ES cell lines would allow the generation of genetically diverse cell lines that are not readily obtainable from embryos that have been frozen or that are in excess of clinical need in IVF clinics. The latter do not reflect the diversity of the general population and are skewed toward genomes from couples in which the female is older than the period of maximal fertility or one partner is infertile. In addition, it might be important to produce stem cells by nuclear transplantation from individuals who have diseases associated with both simple [81] and complex (multiple-gene) heritable genetic predilections. For example, some people have mutations that predispose them to “Lou Gehrig's disease” (amyotrophic lateral sclerosis, or ALS); however, only some of these individuals become ill, presumably because of the influence of additional genes. Many common genetic predilections to diseases have similarly complex etiologies; it is likely that more such diseases will become apparent as the information generated by the Human Genome Project is applied. It would be possible, by using ES cells prepared with nuclear transplantation from patients and healthy people, to compare the development of such cells and to study the fundamental processes that modulate predilections to diseases.

Neither the work with ES cells , nor the work leading to the formation of cells and tissues for transplantation, involves the placement of blastocysts in a uterus. Thus, there is no embryonic development beyond the 64 to 200 cell stage, and no fetal development.

2-1. Reproductive cloning involves the creation of individuals that contain identical sets of nuclear genetic material ( DNA ). To have complete genetic identity, clones must have not only the same nuclear genes, but also the same mitochondrial genes.

2-2. Cloned mammalian animals can be made by replacing the chromosomes of an egg cell with a nucleus from the individual to be cloned, followed by stimulation of cell division and implantation of the resulting embryo.

2-3. Cloned individuals, whether born at the same or different times, will not be physically or behaviorally identical with each other at comparable ages.

2-4. Stem cells are cells that have an extensive ability to self-renew and differentiate, and they are therefore important as a potential source of cells for therapeutic transplantation. Embryonic stem cells derived through nuclear transplantation into eggs are a potential source of pluripotent (embryonic) stem cell lines that are immunologically similar to a patient's cells. Research with such cells has the goal of producing cells and tissues for therapeutic transplantation with minimal chance of rejection.

2-5. Embryonic stem cells and cell lines derived through nuclear transplantation could be valuable for uses other than organ transplantation. Such cell lines could be used to study the heritable genetic components associated with predilections to a variety of complex genetic diseases and test therapies for such diseases when they affect cells that are hard to study in isolation in adults.

2-6. The process of obtaining embryonic stem cells through nuclear transplantation does not involve the placement of an embryo in a uterus, and it cannot produce a new individual.

• Cite this Page National Academy of Sciences (US), National Academy of Engineering (US), Institute of Medicine (US) and National Research Council (US) Committee on Science, Engineering, and Public Policy. Scientific and Medical Aspects of Human Reproductive Cloning. Washington (DC): National Academies Press (US); 2002. 2, Cloning: Definitions And Applications.
• PDF version of this title (5.5M)
• Disable Glossary Links

In this Page

Related information.

• PMC PubMed Central citations
• PubMed Links to PubMed

Recent Activity

• Cloning: Definitions And Applications - Scientific and Medical Aspects of Human ... Cloning: Definitions And Applications - Scientific and Medical Aspects of Human Reproductive Cloning

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

Connect with NLM

National Library of Medicine 8600 Rockville Pike Bethesda, MD 20894

Web Policies FOIA HHS Vulnerability Disclosure

Help Accessibility Careers