research paper about cloning

Human Cloning: Biology, Ethics, and Social Implications

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• 174 Suppl. 2(6):230-235
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Ethical Issues of Human Cloning

Nasrullah, 1 ; Iqbal, Rana Khalid 1,* ; BiBi, Shahzadi 1 ; Muneer, Sana 1 ; BiBi, Sumaira 1 ; Anwar, Farhana Naureen 2

1 Institute of Molecular Biology and Biotechnology, Bahauddin Zakariya University, Multan, Pakistan

2 Department of Pharmacy, Bahauddin Zakariya University, Multan, Pakistan

Corresponding Author: Dr. Rana Khalid Iqbal, Institute of Molecular Biology and Biotechnology, Bahauddin Zakariya University, Multan-60800, Pakistan. Tel: 00923045326229. E-mail: [email protected]

Received April 03, 2019

Received in revised form December 20, 2019

Accepted January 19, 2020

This is an open access journal, and articles are distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as appropriate credit is given and the new creations are licensed under the identical terms.

Cloning can help us in the research field and medical sciences. But due to ethical and moral values, this idea is not supported. Moreover, it is against ethical values as well. According to modern studies, Human moral values are preferred rather than emotions, but they cannot be ignored. Despite the progress in the stem cell culture, it is still unable to avail the therapeutic benefits. It is said that cloning could be done in the near future, and it is closer to the reality and away from science fiction. Cloning can be carried out by two techniques termed as the somatic cell nuclear transfer and cell mass division. The cloned animal products obtained by the somatic cell nuclear transfer can be used, as they cause no harm and are safe as the noncloned animal products are. Certain harms are related to the twin's growth produced by the cloning procedure that also reinforces on the inhibition of human cloning, as it causes the psychological distress and destroys the universality of an individual, as well as certain ethical and moral values despite which human clones cannot be made. In somatic cell cloning the nucleus (nuclear mass/DNA) can solve many health problems for example organ transplantation, or organ rejection issues. Resulting of all these give rise to a great controversy that either clone of human beings should be produced or not. Although in the near future, the possibility of human clones and their use for different purposes cannot be ignored.

INTRODUCTION

Human cloning can be defined as, the production of individuals, entities or populations, identical or nearly identical to the parent or original organism, from which they were obtained or derived. Herbert Weber, a plant physiologist, termed it as “clon ” (excluded “e ”) and used it for plants in agriculture. However, people tried to get popularity while making the science fiction stories about a human being copied formation through cloning. 1 Haldane went on in his 1963 speech on “biological possibilities for the human species of the next ten thousand years,'' to introduce the scientific community the word “clone ” (including “e ”) to denote the superhumans by genetic cloning. 2

ROLE OF DOLLY SHEEP IN CLONING

Lederberg gained the noble prize for recommended tools for cloning for human beings in 1966 but his two opponents; Paul Ramsey and Joseph Fletcher opposed this idea. 3 Someday, human might be cloned from a single adult somatic cell without sexual reproduction. The idea is opposite to the science fiction and near to the genuine possibility. In Scotland Scientists at the Roslin Institute talked about the cloning of a sheep by a technique that had never succeeded in mammals, technique involves the transplant of genetic material of an adult sheep obtained from a well-differentiated somatic cell inserted into the egg from which nucleus was removed. The sheep born after using this technique was named Dolly on July 5, 1996. 4 Just 20 years after the Dolly sheep cloning, monkeys were cloned. Then, Dolly did not produce identical offspring like prior attempts as Dolly has the genetic material of one of the parents not both. 5 This technique was named “somatic cell nuclear transfer and other.'' Basically, two techniques are available for human cloning, one somatic cell nuclear transfer and other one is cell mass division. Somatic cell nuclear transfer is an artificial technique which produces numerous number of clones as compare to embryo splitting technique which is a natural process produces less or limited number of organisms and cell mass division is a technique which was used for the first time in October 1993. 6 The products of cloned animals can be used, yet there is a controversy on this view that either its use is safe or not never the less, still they are being used as the US has allowed them and said how to use it. 7 Moreover, Science fiction stories and the bioethics as well inspire the human ethics, despite they do not think about the moral values of these sources. 8 The clones are due the fetal cells cloning instead of the adult cells that are well differentiated. 9

ROLE OF CLONING IN RESEARCH AND HUMAN HEALTH

Cloning has a great role in human health, life also in the research's discoveries. Human embryonic cells were separated firstly in 1998. 10 It is being used to cure diseases such as neurological problems, Parkinson's disease, and heart problems. Although cloning has all those benefits still scientists, ethicists, policy-makers, and religious scholars forbid the cloning in human beings, as it has raised many questions such as ethical, emotional, and moral questions as well by the people, it is dangerously prohibited as it is against like the “playing God ” yet according to Islamic point of view the cloning is strongly discouraged, as it is against the lows and against Islamic believes, 11 as Islam say that only Allah almighty can create or destroy anything so cloning equals to the God's creation, as scientists cannot take a place of God and cloning is forbidden by Muslim scholars as it has both types of impacts on human and has many risks on human development and health. Moreover, as cloned organisms have lost the ability to grow into an adult, so medical science has improved and related to the stem cell culturing so it could be used to cure many diseases and improvement of fertility. 12

THE LOW SURVIVAL RATE OF CLONES

There are a lot of risks for the growth and survival of the clones, and most criticism comes from the scientists who knew about the risks and dangers of human cloning as most of the embryos are lost before they reach their birth stage, and the clones would have certain abnormalities as well. The percentage of cloned organisms to reach the period of adulthood is very low at 0.3% for cows and <1% for sheep. Scientists viewed that the clone's production is the wastage of embryos and fetuses. They performed the experiment on the blastocyst of a human. They obtained 242 eggs from 16 women that produced only 30 embryos. 13 Then, they said that it could be abuse for women that gave their eggs but lost the fetuses, and the organisms produced in such a way may suffer certain health issues. The cloning in human may produce certain psychological problems like psychological distress that affects the uniqueness and individuality of an organism. Moreover, it may cause certain issues in earlier or later twin's growth.

CLONE AS AN INDIVIDUALIZED ENTITY AND STAGES OF HUMAN DEVELOPMENT

A human embryo is used for cloning and research purposes, but ethics and moral values oppose it as this destroys the human embryos. Some persons regard the human embryo as an “individualized entity or a person ” that reserve all rights as a human being should have it, as it is the initial stage of human development. 14 15 Some theological perspectives consider the human beings as an individual, but there are different views about what stage of human beings should be considered against the dignity of an individual. 16 From the 1 st day of the human embryo, it has its own existence, and it has a right to live, so we have no right to kill the embryo. However, there is a difference between the different stages of human embryo development. According to the Holy Quran, “We created man of an extraction of clay, then We set him a drop in a safe lodging, then We created of the drop a clot, then We created of the clot a tissue, then We created of the tissue bones, then We covered the bones in flesh; therefore We produced it an another creature. So blessed be God, the Best of Creators. ” According to these verses, we can clone a stem cell at the preensoulment stage, and the elimination of an embryo at this stage is also a sin, but its punishment is less than that of the abortion that is equal to murder.

MORAL ISSUES RELATED TO THE HUMAN CLONING

There are certain moral and ethical values that oppose the cloning in human beings. These moral values are dearest to the people from different religious backgrounds. US President Clinton banned the funding related to being given to the trials on human beings cloning and asked the Advisory Commissions to report about the views of peoples within 90 days, as it was a tough job due to confliction in moral and ethical values. Hence, the National Bioethics Advisory Commission consulted to the scientists, theologians, physicians, etc., to collect the data about ethical and moral values and information about the safety issues of the organism produced by the somatic cell nuclear transfer which was the main issue. 17 However, it raises the questions about the social relations (about families and generations relationships) of the clones, as they may be treated as objects or may disturb the family system.

RELIGIOUS VIEWS RELATED TO HUMAN CLONING

The technology has many good and bad impacts on human health so is case with the human cloning which has many advantages and disadvantages as well that's why prohibited by most of the religious scholars. It may be accepted before the ensouled stage. Iran is of one of the first countries that had carried out the cloning process according to the values of Islam for therapeutic purposes. They have used this technique for the organ transplantation by following the ethical values at each stage of the embryo by developing the stem cell culture. 18 19 Hence, it is permitted to be carried out for therapeutic and research purposes while taking out of all the possible measurements. 20

ISSUES RELATED TO THE USE OF CLONED ANIMAL PRODUCTS AND THEIR SAFETY

Cloning produces similar individuals that are useful for the animal breeders, as they have good traits in the same to their parents. 21 The cloned animal products faced moral and ethical issues due to controversial views, yet the reports submitted by the US and National Academy of Sciences, National Research Council reveals that the cloned animal products have no side effects and are as safe as are noncloned or commercial animals also no differences in scientific research bases according to the Food and Drug Authority (FDA). The FDA reported the foods such as milk and meat obtained from the cloned organisms is same as noncloned one. 22 That's why FDA has allowed the use of cloned animal products along with their wide range cloning. 23

Cloning is banned due to many ethical and moral values. Moreover, it faces many emotional reactions, psychological, and social issues as well. According to the Islamic point of view, the cloning of an embryo is a sin, and it is against nature due to the hazards that an embryo faces during a cloning process. However, Islam gives its permission to be applied only all the moral and ethical values are followed during the human embryo cloning for therapeutic and research purposes.

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Cell mass division; dolly sheep; human embryo; somatic cell nuclear transfer

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• Methodology
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• Published: 19 January 2015

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

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

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

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

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

Conclusions

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

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

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

Results and discussion

Choosing proper restriction enzymes based on defined criteria.

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

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

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

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

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

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

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

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

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

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

Designing cloning primers based on defined criteria

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

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

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

The Tm of our forward primer is 66°C.

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

http://reverse-complement.com/

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

Performing PCR using proofreading polymerases

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

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

Analysis of sequencing data

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Viral production and transduction of target cells

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

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

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

Cell lines and media

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

Plasmids, primers, PCR and sequencing

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

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

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

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

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

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

Manipulation of DNA fragments

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

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

Production of viral supernatant and transduction of cells

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

Flow cytometry and fluorescence microscope

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

Abbreviations

Polymerase chain reaction

Gene of interest

Open reading frame

Melting temperature

Basic local alignment search tool

Vesicular stomatitis virus G glycoprotein

Luria-Bertani

Dulbecco’s Modified Eagle medium

Roswell Park Memorial Institute

Bovine serum albumin

Ethylenediaminetetraacetic acid

Fluorescence-activated cell sorting

Human embryonic kidney

Phosphate buffered saline

Fetal calf serum

Hydroxyethyl-piperazineethane-sulfonic acid

Ampicillin resistance gene

Posttranscriptional regulatory element

Myeloproliferative sarcoma virus promoter.

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Acknowledgments

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

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Sayed Shahabuddin Hoseini & Martin G Sauer

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

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13036_2014_161_moesm1_esm.xlsx.

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

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

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Human cloning laws, human dignity and the poverty of the policy making dialogue

• Timothy Caulfield 1 , 2 , 3  

BMC Medical Ethics volume  4 , Article number:  3 ( 2003 ) Cite this article

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The regulation of human cloning continues to be a significant national and international policy issue. Despite years of intense academic and public debate, there is little clarity as to the philosophical foundations for many of the emerging policy choices. The notion of "human dignity" is commonly used to justify cloning laws. The basis for this justification is that reproductive human cloning necessarily infringes notions of human dignity.

The author critiques one of the most commonly used ethical justifications for cloning laws – the idea that reproductive cloning necessarily infringes notions of human dignity. He points out that there is, in fact, little consensus on point and that the counter arguments are rarely reflected in formal policy. Rarely do domestic or international instruments provide an operational definition of human dignity and there is rarely an explanation of how, exactly, dignity is infringed in the context reproductive cloning.

It is the author's position that the lack of thoughtful analysis of the role of human dignity hurts the broader public debate about reproductive cloning, trivializes the value of human dignity as a normative principle and makes it nearly impossible to critique the actual justifications behind many of the proposed policies.

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Dolly, the most famous sheep in history, was euthanised on February 14 this year at the age of 6 after being diagnosed with an incurable lung disorder. [ 1 ] Dolly was a famous symbol of both the great possibilities of science and a focal point for public concerns about the social impact of biotechnology. Almost immediately after Dolly's birth, there were calls to introduce regulatory controls of the technology. Though most countries still do not have specific cloning laws [ 2 ], it continues to be a significant national and international policy issue. But despite years of intense academic and public debate, there remains little clarity as to the philosophical foundations for many of the emerging policy choices.

In this paper, I briefly explore one of the most commonly used ethical justifications for cloning laws, the idea that reproductive cloning necessarily infringes notions of human dignity. As we will see, there is, in fact, little consensus on point. Unfortunately, the counter arguments are rarely reflected in formal policy. Few, if any, domestic or international instruments provide an operational definition of human dignity [ 3 , 4 ]and there is rarely an explanation of how, exactly, dignity is infringed in the context reproductive cloning.

Admittedly, I do not provide my own definition of human dignity. I will, however, endeavor to divine the likely definition of human dignity at play in the context of a given social concern. We will see that regardless of the definition that seems to be implied within the social concerns outlined below, there are legitimate counter arguments that weaken the claim that human reproductive cloning necessarily infringes human dignity. Many thoughtful scholars have already done an admirable job attempting to define human dignity and it place in the policy making process. [ 5 – 8 ] The goal of this paper is not to provide a comprehensive review of these possible definitions, and there are many, or to definitively answer the question of whether human reproductive cloning infringes human dignity. Rather, in this paper I argue that the lack of thoughtful policy analysis of the role of human dignity hurts the broader public debate about reproductive cloning, trivializes the potential value of human dignity as a normative principle and makes it nearly impossible to critique the actual justifications behind many of the proposed policies.

Concerns About Human Dignity

Numerous arguments of varying persuasive force have been put forward as justifications for a ban on reproductive cloning. To cite just a few examples, some commentators have suggested that the visceral reaction that many in the public have had to the idea of human reproductive cloning is, from a policy perspective, significant enough to justify, on its own, a regulatory response. [ 9 ] Others have suggested reproductive cloning would have an adverse impact on the social definition of family: "Modernity's assault on the family would thus be complete with the development of cloning. Already stripped of its social function, the family would now be rendered biologically unnecessary, if not irrelevant".[ 10 ] And, of course, there are the clear health and safety issues that are far from being resolved.[ 11 ] Indeed, Dolly's death, while not definitively traceable to the cloning process, again highlighted the possible health risks associated with reproductive cloning. [ 12 ]

However, the broadest concern, and the concern that is often explicitly mentioned in relevant policy statements, is that human reproductive cloning, at some level, infringes notions of human dignity. One of the best known illustrations is UNESCO's Universal Declaration on the Human Genome and Human Rights which recommends a ban on "practices which are contrary to human dignity, such as reproductive cloning". [ 13 ] Similarly, in 1998, the World Health Organization reaffirmed that "cloning for the replication of human individuals is ethically unacceptable and contrary to human dignity and integrity".[ 14 ] The Council of Europe's Convention for the Protection of Human Rights and its Additional Protocol on the Prohibition of Cloning Human Beings states that: "the instrumentalization of human beings through the deliberate creation of genetically identical human beings is contrary to human dignity and thus constitutes a misuse of biology and medicine".[ 15 ]

Despite the existence of such policy statements, and despite almost universal public objection to the idea of reproduction cloning [ 16 ] there is, at least in the academic community, little agreement about the role of human dignity in this context. Indeed, it has been suggested that "aside from the moral debate on whether the embryo is a human being arguments about human dignity do not hold up well under rational reflection".[ 17 ]

Below I briefly consider some of the reasons commentators remain skeptical of the claim that reproductive cloning infringes human dignity. The goal is not to provide a comprehensive analysis of all the relevant critiques, but to simply highlight a few of the counter arguments and substantive considerations that remain largely absent from a consideration of human dignity in the context of formal policy development.

Autonomy and Uniqueness

At the heart of many of the human dignity arguments, often implicitly, is the idea that copying someone's genome is a morally problematic action. From the perspective of human dignity, the concern is founded on the assumption that a clone's autonomy will be compromised and that a person's genome is singularly important to human uniqueness.[ 18 ] For those who espouse this view, dignity is obviously closely related to autonomy (likely to some version of the classic Kantian view of dignity) and the ability to make autonomous choices. Moreover, dignity is connected to human "uniqueness," though it is rarely explained why this is so. As Donald Bruce argues: "Willfully to copy the human genetic identity seems to go beyond something inherent in human dignity and individuality". [ 19 ] Many policy statements, such as the few noted above, seem to adopt this view and specifically link genetic identity with the concept of human dignity. Other statements simply assert that "the production of identical human individuals" [ 20 ] or the creation of a "genetic 'copy"' [ 21 ] should be banned.

The ethos that underlies these positions is, of course, both scientifically inaccurate and philosophically problematic. Without resolving the point, let us assume that, somehow, uniqueness is central to an individual's dignity. We must ask, then, what role our genome has in our uniqueness and, more to the point, why copying it infringes human dignity. Our genome plays a key role in how we develop, but it is hardly determinative of who we are as individuals. Is an identical twin's dignity compromised because of the mere existence of a sibling with an identical genome? More importantly, our genes do not, on their own, bind our future life to a particular course. Absent other external factors (such as social or parental expectations), an individual's autonomy is not compromised solely because he/she does not have a unique genome. To believe otherwise is to adopt a deterministic view of the role of genes that is simply wrong. [ 22 , 23 ] There are very few human traits that are controlled solely by genetic factors, and this is particularly true of the infinitely complex characteristics that make us who we are as individuals. [ 24 ] A human clone would be wholly unique and, as such, it is difficult to maintain that even a "uniqueness" view of human dignity is dependant on having a unique genome.

From a policy perspective, it is worth noting that a variety of commentators have long questioned the deterministic argument that underlies the autonomy/uniqueness concern about reproductive cloning. For example, shortly after the birth of Dolly Sir John Polkinghorne noted that " [o]ne of the by-products of the furor about Dolly has been to remind thoughtful people of the poverty and implausibility of a genetic reductionist account of human nature". [ 25 ] George Wright takes this idea to an extreme length by suggesting that reproductive cloning would actually promote human dignity by proving the inaccuracy of genetic determinism. "Human cloning may well serve to highlight, to emphasize, and to set off with greater clarity, quite apart from anyone's intentions, the mysterious capacities that comprise and express our human dignity".[ 26 ]

Instrumentalism

For some, it is not the technical copying of a genome that gives rise to concerns about reproductive cloning, but the possibility that cloning will be used in a way that instrumentalizes the clone. Again, this issue is likely tied to the concern that reproductive cloning would infringe the basic Kantian tenet to treat every human being as an end, not as a means. [ 27 ] It is certainly possible that the use of reproductive cloning for the purpose of creating an individual for a particular life role could infringe the resultant clone's dignity. However, it is the pressure or social expectations (expectations that are necessarily informed by an inaccurate view of the role of genes) placed on the individual clone that challenge the clone's human dignity, not the process of reproductive cloning. As noted by Pattinson, the act of cloning could be implicated in an intention to "violate the rights of the clone in the future." He goes on to note, however, that in such circumstances, "it is not the cloning as such that violates the clone's rights, but the intention to make the clone worse off (relative to its alternatives) in the future". [ 28 ]

That said, some argue that the mere act of cloning instrumentalizes the clone, "because the clone is created for the primary benefit not of the individual but of some third party as a means to an end". [ 29 ] This argument is problematic for a number of reasons. First, it raises the interesting question of whether an act done prior to the birth of an individual can infringe the dignity of that individual. Even if an individual is created with instrumental intentions, if, after the birth of the individual, he/she is treated as an equal member of the community, as an autonomous individual and with respect, is the individual's dignity still being infringed?

Second, if one accepts that our genes do not determine our life course or who we are as individuals, it is unclear how the technical act of cloning is more problematic, in relation to instrumentalism, than having children through IVF or, for that matter, making children the natural way for the sole purpose of producing an heir, labour or a means of old age support. Of course, one could argue that, for the sake of consistency, these latter activities should also be banned. However, monitoring and assessing the motives of perspective parents would not, quite obviously, be a practical or appropriate state policy.

Finally, these kind of instrumentalist concerns assume that cloning would always be done for instrumentalist purposes, which may not be the case (e.g., individuals may simply wish to use cloning for the same reason people use IVF, for the purpose of having biologically related offspring). As noted by Steven Malby: "From the point of view of dignity, the desire to treat infertility clearly does not violate any of the parameters associated with an objective perspective of dignity". [ 30 ] At a minimum, it is hard to support the argument that all forms of reproductive cloning will inevitably infringe human dignity. "We should distinguish among the different forms, uses, and contexts of human cloning in assessing the relationship between cloning and human dignity".[ 31 ]

Replication

Closely tied to the concerns regarding instrumentalism and the copying of an individual's genome, are the claims that the asexual nature of the process is "unnatural," that cloning is "replication" and not "reproduction" and that, therefore, by implication, cloning degrades human dignity. Gilbert Meilaender notes that we "find asexual reproduction only in the lowest forms of life. ... Children conceived sexually are 'begotten, not made.' When a man and a woman beget a child, that child is formed out of what they are. What we beget is like ourselves, equal to us in dignity and not at our disposal". [ 32 ]

Though individuals may not feel comfortable with the process (just as many did not feel comfortable with cadaveric research, in vitro fertilization and sperm donation), there must be something about the "replication" process that infringes human dignity. It is unclear how, exactly, the asexual nature of the process, on its own, is problematic from the perspective of human dignity. Again, people may have nefarious motivations for using cloning – just as they may have questionable reasons for using IVF or having children the natural way – but aside from religious arguments regarding the moral status of the embryo and the significance of sexual union, there seems to be little to support the notion that "replication" infringes human dignity.

Meilaender's claim that being created by a sexual union that is beyond "reason or will" is central to our dignity seems to suggest that the thousands of children born as a result of reproductive technologies are, somehow, less worthy of dignity. [ 33 ] Surely the process used to produce an individual is completely irrelevant to the respect and dignity the individual deserves once born. In fact, if we lived in a society that allowed individuals created by cloning, or any other process, to be treated as less than human, reproductive cloning would be far from our most pressing policy concern.

Community Dignity

It has also been suggested that reproductive cloning may adversely impact "communal dignity" or "the dignity of humankind". [ 34 ] While a detailed discussion of this issues is beyond the scope of this paper, it should be remembered that not all agree that "communities" have dignity in the same way that individuals have dignity. Indeed, most traditional legal applications of human dignity emphasize not the community but the protection of individual rights, often in an effort to guard against state imposed incursion upon individual autonomy. [ 35 , 36 ] As summarized by Deirk Ullrich in relation to law in Canada and Germany: "human dignity is an indispensable compass in our continuing journey to promote and protect the rights and freedoms of the individual". [ 37 ] That said, there are those who take a more expansive, less Western centric, view of dignity, suggesting, for instance, that dignity is also relevant to the way in "which groups visualize and constitute themselves." [ 38 ] This type of reference to "communal dignity" can be found in documents such as the UNESCO Declaration: "no research or its applications concerning the human genome, in particular in the fields of biology, genetics and medicine, should prevail over the respect for human rights, fundamental freedoms and human dignity of individuals or, where applicable, of groups of people" [ 39 ]

However, even if one accepts a community view of human dignity, we see that in the context of reproductive cloning much of the concerns remain closely associated with individual autonomy. For example, Malby poses the question thus: "Does dignity impose a responsibility to protect a key feature of humanity (our 'genetic heritage'), from which (to an undetermined extent) we acquire key capacities such as autonomy and the capacity for moral thought?".[ 40 ] But if one's genetic make up is not a key feature to our autonomy and moral thought, and few could genuinely claim that it is, then a central plank of this concern is lost.

The Policy Response

Early in the cloning debate, many of the above points were noted by well-known scholars from a wide range of philosophical perspectives. [ 41 – 43 ] Nevertheless, there are few policy making entities that have, at least on the surface, engaged the human dignity debate in any meaningful manner. [ 44 ]

In Canada, for example, the government has recommended a ban on all forms of human cloning. The Health Canada information document that accompanied the publication of the proposed law simply claims, without any explanation of how or why, that human cloning "would be banned because it treats human beings as though they were objects and does not respect the individuality of human beings". [ 45 ] A later report by the Parliamentary Standing Committee on Health also recommends a ban on human cloning. The Committee noted that the recommendation is based on a number of core principles, including human dignity, but the Committee makes no attempt to relate the recommendation to the notion of human dignity. [ 46 ]

The two US reports, the 2002 US President's Council on Bioethics [ 47 ] and the 1997 Report of the National Bioethics Advisory Commission [ 48 ], do, at least, discuss the fallacy of genetic determinism. Nevertheless, they do not connect this analysis to the issue of human dignity and both conclude that reproductive cloning still creates problems in relation to individual autonomy. For example, the President's Council concludes that " [w]hat matters is the cloned individual's perception of the significance of the 'precedent life' and the way that perception cramps and limits a sense of self and independence". [ 49 ] Because this concern is based on the psychological harm associated with deterministic expectations, and not on the actual impact of cloning technology, they do little to support the argument that cloning, as a technology, infringes human dignity. In fact, as I have noted elsewhere, cloning laws that are not accompanied by thoughtful policy analysis may have the unintended effect of legitimizes perceptions of genetic determinism.[ 50 ]

Why Human Dignity?

If one were to take a skeptical view of the policy making process, it would not be hard to conclude that concern for human dignity is used as a justification for cloning laws precisely because the notion of human dignity is both so revered and so ill-defined. This fits well with the broad, generalized concerns that the public seems to have about reproductive cloning. As noted by Ronald Dworkin, the public isn't terribly worried about safety or research ethics, but have "some deeper, less articulate ground for that revulsion, even if they have not or perhaps cannot fully articulate that ground, but can express it only in heated and logically inappropriate language, like [a] bizarre reference to 'fundamental human rights..."' [ 51 ]

This view of public attitudes is supported by survey data. Risk and safety are not the issues driving public reaction. When asked, the public often lists morality and/or religion as the basis for their objection to human cloning. [ 52 ] As such, policy makers can safely use the concept of human dignity to reflect general unspecified condemnation. For a good percentage of the public, human reproductive cloning simply seems immoral and, for lack of a better philosophical argument, it is declared that it infringes human dignity. Dworkin puts it in less secular terms: "It is wrong, people say, particularly after more familiar objections have been found wanting, to play God". [ 53 ]

Another reason concerns for human dignity may be used so frequently as a justification for cloning bans is that they allow policy makers to avoid more socially controversial and politically charged rationales, such as those based on a particular religious perspective or abortion politics. It is far easier, at least politically, to say that a given law is based on concern for human dignity than on, for example, a Christian view of the moral status of the embryo – though there seems little doubt that religious perspectives have played an important role in the policy process. [ 54 ]

In addition, the use of human dignity allows policy makers to avoid the appearance that they are seeking to regulate morality. For many legal scholars, moral belief or repugnance "is not sufficient to outlaw conduct engaged in by consenting adults". [ 55 ]

Finally, I suspect that much of the debate remains scientifically ill-informed. Media images of reproductive cloning, which are everywhere, often portray clones as "carbon copies". [ 56 ] These representations undoubtedly impact the public's "intuitive" response to the technology and the public's desire to ban the technology.

In fact, I too have intuitive concerns regarding the appropriateness of human reproductive cloning. I believe that reproductive cloning will have little practical use, the health and safety concerns will likely endure for decades, and it may create some challenging genetic enhancement issues. There are, no doubt, sound reasons to consider the tight regulation of reproductive cloning.

Why, then, is the ad hoc use of the notion of human dignity in the context of reproductive cloning a problem? It hurts public debate. Though I am tremendously skeptical of the worth of intuitive reactions as a justification for a given law, particularly criminal prohibitions [ 57 ] if general cultural anxiety is one of the rationales for a proposed ban, then this should be explicitly stated. Policy makers should not dress up the argument as a concern for human dignity in order to create the perception of legitimacy. By doing so, transparency in policy making is obscured or even lost. As noted by Shaun Pattinson in his critique of the Canadian government's use of human dignity as a justification for a ban: "Once again we are left with the feeling that other arguments are in play but remain unsure as to what those arguments are". [ 58 ] But without knowing that these "other arguments" are, it is impossible to have an informed policy discussion.

If the concerns about cloning are based on the fear that we live in a world increasingly governed by inaccurate views of genetic determinism and, therefore, people may have inappropriate ideas of what cloning can do, [ 59 ] then this too should be stated. Indeed, it could be argued that we should be focussing our policy making energy not on the technology but on the possible causes of the deterministic sentiments that may motivate the desire to use reproductive cloning. Unfortunately, "genetic determinism" is a much more challenging and amorphous policy target as compared with human cloning technology.

In addition, using human dignity as a blanket argument against all forms of human cloning makes it much more difficult to reflect rationally on the true risks and benefits of the technology. Such claims can have powerful rhetorical force (no one is against the idea of human dignity!). [ 60 ] But, as noted by Beyleveld and Brownsword, "from any perspective that values rational debate about human genetics, it is an abuse of the concept of human dignity to operate it as a veto on any practice that is intuitively disliked".[ 61 ]

Finally, we are in danger of trivializing and degrading the potential normative value of human dignity. There seems little doubt that the rapid advances that are occurring in the field of science, and biotechnology in particular, will continue to create new social and regulatory challenges, many of which may also raise issues associated with notions of human dignity. The way we handle current science policy issues stands as a precedent for future analysis. The ad hoc application of human dignity in relation to human cloning will undoubtedly impact how it is applied to future technologies. We should strive to apply the principle in a logical and coherent fashion otherwise the notion of human dignity is in danger of being eroded to the point where it stands as nothing more than a symbol of amorphous cultural anxiety.

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Thanks to Lori Sheremeta, Nola Ries, Angela Long, Jai Shah, Jason Robert, the peer reviewers and to Genome Prairie, the Stem Cell Network and the AHFMR for their funding support.

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The future of cloning

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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.
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