Animal cloning: the end of the "Game of Love and Chance"

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1 Animal cloning: the end of the "Game of Love and Chance" Jean-Marc Fraslin, UMR INRA-Agrocampus Rennes, CS 84215, 65, rue de Saint-Brieuc, Rennes, France In 1730, Pierre Carlet de Chamblain de Marivaux published The Game of Love and Chance. Given the knowledge of Genetics at the time, he could never have imagined that the title of his romantic comedy would be an excellent summary of the mechanisms of Reproduction (or more precisely procreation we will come back to this distinction later). In nature, animal reproduction is in fact governed by a subtle game of love and chance. Love, because there is necessarily a harmonious meeting between a male gamete* (a spermatozoon*) and a female gamete (an ovocyte*) during the fertilization stage to create a future new individual. Each gamete, whether male or female, contains a single "stock" of chromosomes (haploid*) in a pronucleus* and the fusion of the two pronuclei produces a double stock of chromosomes to create a diploid* animal. Chance, as this meeting will take place between a few ova (between approximately 1 and 20) and a few million, or even billion, spermatozoa depending on the species. Each of these gametes has a unique genetic heritage, due to the many recombinations that occur during their maturation, during meiosis*. We can therefore see that the individual resulting from this fertilization is the result of a huge lottery, of major genetic mixing. All descendants of the same two parents do in fact have half a paternal genetic heritage and half a maternal one, but none of them are similar (except in the case of identical twins and we will be coming back to this). Now that breeding has been mastered, Humankind has tried to reduce the element of chance by not allowing males and females to mate randomly, and by choosing those that reproduce. This is what we call selection. This method does not however in any way reduce the chance relating to chromosome stocks of gametes and the probability of their meeting. In the second half of the 20 th century, it was the "love" part, and more precisely mating, that zootechnicians decided to reduce in the reproduction of commerical animals, initially through artificial insemination and later using in vitro fertilization methods. So, there is no longer any natural mating but human intervention in the form of inseminators or biologists. These methods have, at the same time, reduced the element of chance, as the gametes involved are taken from a 1

2 few, particularly high-yielding animals. But once again, the meiosis lottery and randomness of fertilization are just as uncertain. The first cloning experiments were intended to purely and simply remove this randomness. But what is a clone exactly? Clones exist in the natural state, in particular in plants. The cutting-taking technique amounts to nothing more than cloning. The cutting, taken from a plant and then planted correctly, has the exact same genetic heritage as the original plant and will produce an identical one. This reproduction technique was, until recently, reserved for the plant world and did not apply to animals. Except, that is, for identical twins! In this case, an ovum which is (naturally or artificially) fertilized by a spermatozoon begins to divide repeatedly, thus beginning normal embryonic growth. But an "accident" will cause the embryo to split and form two half embryos that are alike in every way. If this separation takes place at a very early stage (two cells, for example), each of these two half-embryos will develop normally to form two genetically similar embryos. (Remember that for non-identical twins, two separate ova fertilized by two separate spermatozoa develop at the same time to produce two individuals that have genetic characteristics comparable to any set of siblings). This ability to produce clones of identical twins interested reproduction biologists in the second half of the 20 th century. While in the process of mastering in vitro fertilization, they increased the probability of "accidental" embryo separation by performing a surgical split into two, or even four or eight, cells and then reimplanting these clones into the uteruses of recipient females, to produce as many genetically identical individuals as split cells. However, despite these sophisticated techniques, the starting point remains the fertilization of two gametes, and the results obtained are, despite everything, the result of the "Game of Love and Chance". Cloning technique The cloning idea is nothing new. As early as 1935, i.e. 3 years after the publication of Aldous Huxley's Brave New World, Hans Spemann dreamt up the idea of transplanting diploid cell nuclei into enucleated oocytes. This involved, in what was at the time only a hypothesis, eliminating any element of chance, as the embryo would develop based on the genetic heritage of the diploid adult cell without meiotic change or gamete fusion. Here it is no longer a question of mating but rather reproduction in the "photocopy" sense of the term. Animals created using such a method would therefore be perfect clones of the cell-donor animals. 2

3 In 1962, the British biologist John Gurdon announced that he had successfully performed such a transfer in batrachians using a nucleus from an adult Xenopus* cell. He had to wait until 1970 for the discovery to be proven. Attempts to adapt the technology to mammals continued for thirty years and resulted in the birth of the famous sheep Dolly in July 1996 (Wilmut et al., 1997). Benefits Why produce clones? What are the potential advantages of cloning? When Dolly the sheep was born, Ian Wilmut spoke of the zootechnical advantages of his "creature" when asked about the benefits by journalists. The standardisation of livestock animals would facilitate the work of breeders, and standard veterinary treatment, food rations, product quality and breeding practices would provide breeders with better working conditions. This "ergonomic progress" did however seem slight compared with the cost of producing these animals. Other cloning applications may have some benefits. For example, animals with sought-after zootechnical traits may suffer from sterility and cloning could provide a solution for guaranteeing "descendants". This application seems even more suited to the field of horse racing in which the best performing males are almost systematically castrated to make them gentler. Such champion geldings could, at the end of their career, return to the stud to give their diploid cells rather than their sperm like stallions. Cloning could also be used in the "resurrection" of extinct animals. Several mammoth cloning projects have, for example, been announced based on bodies that have been found frozen. And closer to home, American companies now offer to clone our household pets after the death of the original ones. Similarly, an American company (Transgenics Pets) is hoping to market allergen-free transgenic cats to enable everyone to enjoy these animals (Price: $1,000, i.e. the equivalent of a pedigree cat). Transgenesis is in fact probably the most useful cloning application and not just for our darling pets. Let us go back to Dolly the sheep. Between removing the donor cell and fusing its nucleus with an enucleated oocyte, there is a cell culture phase. We have known for a long, long time now how to change the genetic heritage of cells placed in a culture medium, with considerable efficacy. Here lies the major advantage of cloning. In fact, six months after the birth of Dolly, Polly a new sheep cloned in the same way as Dolly but transgenic was born (Schneike et al., 1997). The Roslin Institute, which has links with the private company PPL Therapeutics, in fact used this cell culture step to insert an additional gene into the sheep genome. This gene, of human origin, led to the production of a protein called factor IX, missing in patients with a certain form of haemophilia. 3

4 This transgene, which was incorporated into the cultured cells, was then transferred to the enucleated oocyte during the fusion stage. Polly the sheep therefore had this additional human gene in all her cells, and expressed the corresponding protein in her milk. She was a real animal "reactor", and all that needed to be done was to purify the human "medicinal" protein in her milk in an attempt to treat certain forms of human haemophilia at a low cost. On 12/01/01, the press announced the birth of the first transgenic monkey (Chan et al., 2001), carrying the gene coding for the fluorescent jellyfish protein GFP (Green fluorescent protein). We cannot help but see this as a step towards human cloning Glossary (source in French: Diploid: a cell is said to be diploid when it has 2n chromosomes, i.e. each chromosome has its homologue. When this is not the case, the cell is haploid or polyploid (one or more of two similar chromosomes for each type of chromosome). Gamete: The gamete is a haploid reproductive cell which has finished meiosis and cytoplasmic differentiation. In animals, female gametes are ova and male gametes, spermatozoa. Haploid: a cell is haploid when it has n chromosomes, i.e. each chromosome is unique. Meiosis: meiosis is a process that takes place during gametogenesis (spermatogenesis or oogenesis), i.e. during the development of gametes. Its aim is to provide haploid cells from diploid cells over two divisions. But in addition to this dividing role, meiosis has an important role to play in interchromosomal and intrachromosomal genetic mixing. Oocyte: female gamete Pronucleus: haploid nucleus of gametes Spermatozoon: male gamete Xenopus: Xenopus laevis, the Southern African clawed frog, is an anura amphibian, used as a model in cell biology and developmental biology. 4

5 Bibliography Chan A. W. S., Chong K. Y., Martinovich C., Simerly C. and Schatten G., Transgenic Monkeys Produced by Retroviral Gene Transfer into Mature Oocytes, Science, 291: Gurdon, J. B., The developmental capacity of nuclei taken from intestinal epithelium cells of feeding tadpoles. Journal of Embryological Experimental Morphology, 34: Huxley A., Brave New World Marivaux P., The Game of Love and Chance Schneike, A.E., Kind, A.J., Ritchie, W.A., Mycock, K., Scott, A.R., Ritchie, M., Wilmut, I., Colman, A. and Campbell, K.H.S., Human factor IX transgenic sheep produced by transfer of nuclei from fetal fibroblasts. Science, 278: Spemann H., Embryonic Development and Induction. New Haven, Yale University Press. Wilmut, I., Schnieke, A.E., McWhir, J., Kind, A.J., and Campbell, K.H.S., Viable offspring derived from fetal and adult mammalian cells, Nature, 385: