cloning

Cloning is a form of asexual reproduction, the creation of genetically identical genes, cells, plants, or animals. Cloning can take many forms, including taking the DNA of a mature organism and introducing it into an egg cell, which then grows up genetically identical to its donor. Cloning also takes place naturally in bacteria and yeast, and when twins occur. In this case, a fertilized egg cell in the course of its many divisions creates two organisms rather than one.

Cloning is done using a technique known as Nuclear Transplantation, stem cells are extracted from blastocysts. for now all the research is concentrated on the uses of stem cells, since stem cells are thought to be able to be converted to other type of cells, I.E: blood cells, muscle cells, bone cells. etc.

An argument for cloning and stem cell research

Tuesday, December 11, 2001, “Stem cells used to heal monkey spines”, so reads a headline in the Japan Times Online, this is just one headline. The debate over the ethical and moral implications of human cloning and stem cell research has raged for years, but one must admit, a headline of that magnitude is hard to ignore. The article goes on to say, “Doctors at Keio University in Tokyo have succeeded in restoring mobility to monkeys crippled with spinal cord injuries, by transplanting neural stem cells obtained from the spinal cords of (dead) fetuses…. the results of the group's experiments may have opened the door to curing injuries of this kind in humans.” Clearly, the issue, once relegated to speculative fiction, indeed in a somewhat ghoulish vein has now become a not-so-horrific reality and as such necessitates observation from a more rational and less emotional perspective.

To examine the subject of cloning objectively, one must first understand some of the science behind it. First, what are stem cells? Stem cells are cells, which can divide for an indefinite amount of time in a laboratory and yield specialized cells. Shortly after an egg is fertilized a single cell capable of producing a complete organism is created, and is called totipotent i.e. it’s potential is total. In just a few hours this cell divides into two identical totipotent cells, both with the potential to develop into a fetus. About four days into the cycle, after several more divisions the cells begin to specialize and form the blastocyst. Inside the blastocyst is a cluster of cells called the inner cell mass. The inner cell mass although capable of forming nearly every type of cell in the human body, cannot form the entire organism without the outer cells which form the placenta and other tissues needed to support the fetus in the womb. These more specialized cells are called pluripotent and are the primary cells that the stem cell lines for research are derived from. It should be noted that since these cell’s potential is not total, they are not totipotent and are therefore not embryos, even if they were to be placed into a woman’s uterus they would not develop into a fetus. ( National Institutes of Health, 2000)

After bearing further specialization these pluripotent cells develop into multipotent cells, cells with specific functions, such as white and red blood cells and various types of skin cells. Multipotent cells are found in children and adults, the most widely understood of which are the blood stem cells that dwell in the bone marrow. Although the results of research in this area are promising the evidence suggests that these cells are found in adults in very small quantities and their amounts may decrease with age. It is for this reason that the bulk of the research now being conducted is with the pluripotent cells.( National Institutes of Health, 2000)

Because these pluripotent cells are central to the genetic decision-making process their research potential is enormous. Medical conditions, such as cancer and birth defects, are caused by abnormal cell division and specialization. A deeper insight into normal cell function could be the key to understanding their root causes and thus the key to the cure. Embryonic stem cells could also be extracted at the blastocyst stage, and with proper stimulation in vitro, coerced to differentiate into different cell lines as a source of potential cell regeneration. Using these embryonic stem cells, scientists could also gain insight into other illnesses such as Parkinson’s disease, Alzheimer’s, and Leukemia, in fact the list of potential benefits is extensive and also includes; treatments for spinal cord injuries, stroke, burns, heart disease, diabetes, osteoarthritis and rheumatoid arthritis.( National Institutes of Health, 2000; John Robertson, 1998)

It is in the creation of human embryos, however, and the subsequent extraction of stem cells thereby destroying the embryo that the current controversy lies. It is for the same reasons that the research has become inextricably linked with the abortion debate. Advocacy groups, governments, and religious groups around the world have reacted to the cloning of the first human embryo in a largely emotional fashion, and are demanding that all stem cell research be stopped.

In August 2001, President George W. Bush agreed to allow limited government funding of stem cell research on 60 cell lines already in existence “where,” he said, “a life-and-death decision has already been made.” In other words, these existing stem cell lines were derived from embryos left over from fertility treatments or abortions and would be discarded as medical waste. A group of scientists in Virginia complicated matters further by announcing that they had created human embryos for the specific purpose of extracting stem cells. In December 2001, Mr. Bush declared the creation of human embryos by cloning “morally wrong” and said, “We should not as a society grow life to destroy it, and that's exactly what's taking place.” Michael West, President and CEO of Advanced Cell Technology Inc. (ACT), the company in Massachusetts responsible for the cloning of the first human embryo disagreed with the predication that the technology amounted essentially, to the creation of a human being. “We're talking about making human cellular life, not a human life”, he insisted that the research was not meant for human reproduction, and expressed disdain at the idea of it moving toward that end.

Nonetheless the emotionality of the issue can be gauged by the alacrity with which the government, Vatican and pro-life organizations reacted to what many in the scientific community see as “very preliminary and unconvincing evidence.” An article in the Washington Post is quoted, as saying that stopping the research at this point would not be justified, as it was too early in the research process to judge it’s potential. Yet immediately following the publication of ACT’s scientific paper the Vatican made the statement, "Notwithstanding the humanistic intents...this calls for a calm but resolute appraisal which shows the moral gravity of this project and calls for unequivocal condemnation." The statement emphasized the Catholic Churches belief that life begins at conception, thus it believes cloning to be a violation of life. If the reaction from political and religious leaders is such now, before the technology is technically viable, it may be just the tip of the iceberg for what happens when a human being is cloned, and it will happen, it’s only a matter of time.

Due to the governments stance on the matter, as discussed previously, stem cell research is, for the most part privately funded, federal grants amount to a very small part of it. It is this fact that will keep the research going regardless of societal pressures, it’s just too important. The unfortunate fallout from the degradation of public opinion will be the eventual moving ‘underground’ of the research thereby causing a ‘black market’ of cloning if you will, which treads the same ground as the secret abortion clinics of old. It is an attribution of human nature, if there exists a treatment for a particular disease or a unique medical problem that only cloning can solve, people will have it whatever the cost, and wherever they have to go, and whomever they have to deal with to get it. Banning the science completely would create only laws against the practice, not laws to monitor it, in a field that has as much potential for abuse as it does potential benefits this stance is patently dangerous.

Despite the fact that the potential for abuse exists, to a large degree much of the negativity surrounding the issue of human cloning is based on media hysteria, misinformation and science fiction. Huxley’s Brave New World for instance, conveniently neglects the reality that cloning requires a gestating womb and a commitment to rear the child. Although, since the book was written nearly seventy years ago, he can probably be forgiven. That does not however, negate the possibility of total laboratory gestation in the future, but at present most scientists are averse to the idea, and that sentiment seems unlikely to change. Current research shows that it will likely be a very long time before any of the worst of society’s fears are realized, if they are realized at all.Other scenarios talk of slavery and ‘lesser classes’ of people, all of these instances fail to take into account the fact that although a clone would be an exact duplicate genetically, mentally and emotionally it would still be his/her own person. We are all the sum of our experiences and a cloned human would be no different.

The issue of a cloned child being singled out and looked at as less than it’s peers in much the same way as people thought the first IVF or ‘test tube’ baby would be is equally ludicrous. There was a stir initially but eventually the practice became commonplace as will cloning. Nonetheless, with the potential benefits stated above, and others too numerous to mention, human cloning and stem cell research remains one of the greatest hopes of humanity, it would be a crime to see it stopped before those benefits were allowed to come to fruition.

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References

National Institutes of Health.(2000, May). Stem Cells: A Primer Available: http://www.nih.gov/news/stemcell/primer.htm

John Robertson Texas Law Review 76: 1371. (1998). Liberty, Identity, and Human Cloning Available: http://www.humancloning.org/liberty.htm

Simon Smith .The benefits of human cloning. Available: http://www.humancloning.org/benefits.htm

CNN.com/HEALTH. (26 November 2001). Bush: Human cloning 'morally wrong'Available: http://www.cnn.com/2001/HEALTH/11/26/human.cloning/index.html

CNN.com/allpolitics.com with Time. (10 August 2001). Bush to allow limited stem cell funding Available: http://www.cnn.com/2001/ALLPOLITICS/08/09/stem.cell.bush/

CNN.com/HEALTH. (24 August 2000). Stem cells: When politics and science collide Available: http://www.cnn.com/2000/HEALTH/08/24/embryo8_23.a.tm.tm/

News.bbc.co.uk. (26 November 2001). Embryo clone prompts debate Available: http://news.bbc.co.uk/hi/english/sci/tech/newsid_1677000/1677327.stm

Shasta Darlington. (2001). Vatican Slams U.S. Human Embryo Cloning Available: http://abcnews.go.com/wire/SciTech/reuters20011126_247.html

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The Issues and Benefits of Whole Organism Cloning

Introduction

The cloning of Dolly the sheep from an adult cell in 1997 proved beyond doubt that the creation of healthy organisms using the technique of nuclear transfer was possible.¹ Since then a large number of other mammals have been cloned, including mice, goats and cows.² Several scientists have attracted media attention by claiming that they are planning to clone a human being, but the science of cloning is still not well understood, and, ethically, cloning is controversial because of the high mortality rate involved due to oocyte (egg cell)/donor nucleus cell cycle incompatibility and epigenetic differences between the donor nucleus and a normal oocyte nucleus. However the potential benefits from whole organism cloning are great.

Nuclear Transfer

The process of cloning by nuclear transfer is a comparatively simple procedure: the nucleus of an oocyte is removed using a very fine syringe, and replaced with the nucleus of a different cell, and the combined cell is activated by an electric shock. A successfully developing embryo is then implanted in a surrogate mother.^1,3 Although this method has had some notable successes, and managed to clone a whole range of animals, including sheep, mice, cows and goats, the success rate of the procedure is very low, and doubts still remain about the long term prospects for even apparently healthy cloned animals.

Ethical Issues

This high mortality rate is the major ethical objection to whole organism cloning. The mortality rate of the recombined oocytes created by nuclear transfer, is usually about 99%², each of these failure destroys an oocyte, which had the potential to form a new organism, or if the failure happens later in the pregnancy, causes considerable pain to an organism. Even successful births produce far more abnormal offspring than is normal, and the long term health of clones is still relatively unknown. At current success rates this is considered an unethical loss, particularly if the method were to be used in human cloning, as even if the initial failed recombined oocytes are considered ethically justifiable, a position similar to the UK"s position on embryo research which allows embryos to be kept until they are 14 days old, at which a nervous system starts to form, and the embryo becomes capable of feeling pain, a significant number of embryos over 14 days old will also be lost.⁵ It is generally believed in the scientific community that while clones are genetically identical (with the exception of mitochondrial DNA, which is inherited from the oocyte donor) the effects of external conditions on the organism are likely to make it impossible to create identical copies of an organism, but the popular media has spread the idea that clones are completely identical⁶. This is particularly important with human cloning, where various misunderstood beliefs still exist, such as the possibility the people can be 'resurrected' by creating clones of them, or that clones will be completely indistinguishable from the cloned person⁷. A successful human clone would be a unique individual and as such should have identical rights to a human produced conventionally.

Problems

The high failure rate of cloning by nuclear transfer is due to two main factors: Cell cycle synchronisation and epigenetic differences in the genome structure.⁸

Cell Cycle Synchronisation

In order for the nucleus and enucleated (cell with the nucleus removed) oocyte to fuse correctly, they must be at a compatible stage of their cell cycles, so that the ploidy (amount of DNA) is maintained, both cells are at compatible stages of mitosis and meiosis and the optimum amount of time is allowed for genetic reprogramming. This is usually done by using nuclei from cells before DNA replication, known as the G1 (cell growth before DNA replication) or G0 (G1 but cell cycle is artificially arrested or cell is not dividing) stages and inserting them into oocytes during metaphase II. The success of this process depends on the cell cycle of the animal involved, as those with longer periods between division and the beginning of embryonic development, one of the reasons sheep were cloned before more conventional laboratory animals such as mice, which have a shorter gap for genetic reprogramming between activation and the beginning of major transcription.^8,9,10 However the increased understanding of the cell cycle has made this less of a problem.

Epigenetic Differences

While the genetic code of a cell is the same throughout the organism and remains unchanged throughout its life, the DNA itself will have a number of epigenetic differences depending on what type of cell it is. This is particularly true for the differences between an oocyte and a fully differentiated somatic cell (cell that will not be passed on to the next generation). The four main differences are differentiation, imprinting, the inactive X-chromosome (in female animals) and telomere lengths.

Cell Differentiation

As an oocyte develops into an organism the cells it is composed from become more and more specialised. At a molecular level this is due to the switching on and off of genes by alterations to the genetic material and the proteins (most importantly histones and chromatins) round it by mechanisms such as methylation and structural changes in the proteins that govern access to the DNA by the cell transcriptional machinery. These control which genes are expressed, and which are not. Many genes are only expressed in certain cells or at certain phases in the life cycle, and remain permanently inactivated otherwise This means that as a cell becomes more differentiated the genes that are activate and inactive on the genome are very different from the genes that are active and inactive in the oocyte. In the nuclear transfer process therefore, the gene still carry these epigenetic modifications, which will change which genes are expressed at which time. This is one of the reasons why so many failed clones show the signs of inappropriate gene expression, and the high rate at which the success of nuclear transfer decreases with the differentiation of the cells (in mouse studies, a nucleus taken from a two cell embryo has only a 22% chance of developing offspring and a nucleus from a four cell embryo has a 14% chance)⁸.

Imprinting

A fertilised oocyte contains a set of DNA from each parent, however certain genes from each parent are expressed differently depending on whether they were inherited from the father or the mother, in a similar way to the effects of differentiation on the genome, a phenomenon known as imprinting. It is not known how much of the maternal and paternal imprinting survives in fully differentiated cells, but the fact that many imprinted genes are involved with early development leads to the conclusion that lack of imprinting is one of the reasons for the high failure rate and inappropriate gene expression seen in many failed experiments.⁸

Inactive X-chromosome

Female cells have two copies of the X-chromosome but one of the copies is randomly inactivated. Female embryos have two active X-chromosome, but only the paternal copy is inactivated in the placenta. It is thought that if the inactive X-chromosome is not reactivated, then the paternal copy of the X-chromosome may be activated, and expressed in the placenta, although it is not known what problems this will cause.⁸

Telomeres

Telomeres have been found to be shorter in cloned animals than in normal animals, by a factor proportional to the age of the differentiated cells from which were the nuclear donors¹¹. This could be detrimental to clones as it is believed that telomeres protect the genome from damage, and telomere loss, which occurs when a cell divides, is believed to be a cause of ageing. However, this appears to have little effect on the health of clones: there is a high mortality rate before and shortly after birth, but this appears to be more due to inappropriate gene expression than premature ageing or genetic damage and all the healthy cloned animals produced so far appear to have aged normally, and another study involving several generations of cloned mice shows no sign of telomere shortening, in fact the become longer, although they admit the possibility of selection for telomere length as one of the reasons for a decreased success rate with each successive generation¹². It appears that the telomere problem can be solved simply be selected cells that are likely to have gone through the fewest divisions and minimising the time in which they are cultured.

Genetic Reprogramming

These alterations to the genome would seem to make cloning an impossibility, but it appears that enzymes and structural proteins in the cytoplasm of the oocyte can genetically reprogram the genome, reversing many of these problems on it and making it totipotent again, as well as reactivating the inactive X-chromosome and restoring telomeres, (although this is in doubt^8,11,12). This makes it possible for the simple process of nuclear transfer to create viable embryos, although the success rate for adult cells is very low and even foetuses that survive to term are likely to suffer birth defects from inappropriate gene expression. It appears that these effects occur in the interval between the transfer and the onset of major transcription, so animals with a longer interval are easier to clone than animals with a short interval. In order to improve the success rate, a much greater knowledge of the events to occur in cell reprogramming is required and it the transferred nuclei will probably have to be chemically pre-reprogrammed to reach a reliable success rate.⁸

Benefits

There are several important benefits of a reliable cloning method for cloning whole organisms: the most important one is the flexibility it will allow in the genetic engineering of mammals. Other organisms, such as plants and prokaryotes can be engineered, then cultured from a single cell, however, up till now genetic engineers wishing to genetically engineer an entire organism have had to engineer fertilised oocytes. The difficulty of getting oocytes and the low success rate, of much genetic engineering coupled with the difficulty of non-destructively testing for gene expression mean that genetically engineering an entire organism is wasteful and unethical, because of the number of viable oocytes that will be destroyed. An efficient cloning system, would enable geneticists to genetically engineer a culture of somatic cells, then transfer the recombinant nuclear material to an oocyte, dramatically reducing the number of oocytes that would be wasted. Several steps have already been made in this direction, including the successful production of genetically engineered sheep.¹³ Cloning has also been proposed as a solution for a number of other problems, including infertility treatment and an aid to animal conservation. The investigation of the problems of nuclear transfer will also yield valuable data about the process of cell differentiation, development and gene activation.

Conclusion

The creation of viable offspring from somatic cells is an important biological step, but several problems still remain which are most likely to be solved be an increased understanding of the chemical processes of early development and improved techniques, most likely in the form of pre-reprogramming the somatic nucleus to return to totipotency. Once the high failure rate of cloning is reduced, though, whole organism cloning will become a very useful tool for the biologist⁸.

References:

1. I. Wilmut, A.E. Scnieke, J. McWHir, A.J. Kind & K.H.S. Campbell. Viable offspring derived from fetal and adult mammalian cells. Nature 385, 810-813 (1997)
2. I. Wilmut & Peterson L. A. Somatic cell nuclear transfer (cloning) efficiency. (http://www.roslin.ac.uk/public/webtablesGR.pdf) (2002)
3. Nuclear transfer chart: http://www.infigen.com/pages/processes/transfer/Nuclear_chart.html
4. K. H. S. Campbell, J. McWhir, W.A. Ritchie & I. Wilmut. Sheep cloned by nuclear transfer from a cultured cell line. Nature 380, 64-66 (1996)
5. Human Embryo research guide from Human Embryology and Fertilisation Authority http://www.hfea.gov.uk/Downloads/Leaflets/embresh.pdf
6. Star Wars II: Attack of the Clones. (2002)
7. The Boys from Brazil (1978)
8. I. Wilmut, N.Beaujean, P.A.de Sousa, A. Dinnyes, T.J. King, L.A.Paterson, D.N. Wells & L.E. Young. Somatic cell nuclear transfer. Nature 419, 583-585 (2002)
9. D. Solter. Lambing by nuclear transfer. Nature 380, 24-25 (1996)
10. C. Stewart. An udder way of making lambs. Nature 385, 769-771 (1997)
11. P. G. Sheils, A.J. Kind, K.H.S. Campbell, D. Waddington, I. Wilmut, A. Colman, A.E. Schneik. Analysis of telomere lengths in cloned sheep. Nature 399, 316-317 (1999)
12. T. Wakayama, Y. Shinkai, K.L.K. Tamashiro, H. Niida, D.C. Blanchard, R.J. Blanchard, A. Ogura, K. Tanemura, M. Tachibama, A.C.F. Perry, D.F. Colgan, P. Mombearts, & R. Yanagimachi. Cloning of mice to six generations. Nature 407, 318-319
13. K. J. McCreath, J. Howgraft, K.H.S. Campbell, A. Colman, A.E. Schneleke & A.J. Kind Production of gene targeted sheep by nuclear transfer from cultured somatic cells. Nature 405, 1066-1069