(idea) by BaronWR Mon Nov 25 2002 at 16:11:31

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 doubt8,11,12). 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

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