A Blast(ocyst) from the Past

Image Credit: HeraldScotland.com

Happy Birthday Dolly

I disposed of the bulk of my lab and lecture notes immediately after dropping out. The boxes took up far too much physical space and I really didn’t need a cupboard packed full of disappointment to remind me of yet another time and place I ran out on. Especially one with no door. I did hold onto a handful of essays though, some of which I haven’t reread until today – ones I’d enjoyed researching and writing at the time and which, maybe, could be construed as having some kind of tenuous philosophical bent.

That horrendous, soul-destroying morning at the growing-finishing pig house signalled the irreversible beginning of the end of my University career; calculating the efficiency of energy conversion in genetically ‘improved’ pigs in terms of the percentage of gross-input-energy retained within their tissues after digestible, indigestible and metabolisable energy had been lost to the environment. Repeated showering to try to rid myself of the stench was, apparently, an unacceptable reason for missing that afternoon’s lab. And every time I see a livestock-in-transit vehicle on the road, my heart breaks all over again.

Dolly the sheep was less than 1-year old at the time. She was never destined for slaughter like those pigs. Instead she was euthanized with an overdose of anaesthetic agent, half a sheep’s life later, on Valentine’s Day; taxidermied and encased in glass for posterity.

Dolly 1C
Image Credit: Sundaypost100.com

Today would be Dolly’s 20th birthday. Today’s Guardian quotes the now Sir Ian Wilmut as saying,

If you’re in research work you’re an optimist, you have to be… Sometimes natural optimism makes us be too hopeful about what can be achieved. I think perhaps we are slowly learning to be more realistic and not make the same mistake again.

My own default factory setting is firmly in the ‘glass-half-empty’ school of hermeneutics.

What follows is the essay I wrote almost two decades ago in response to the announcement of her birth…

Cloning and Transgenics

     Clones may be defined as separate, though identical sequences of nucleic acids, or as genetically identical cells or individuals that have arisen from a single ancestor (Hutchinson, J. S. M. 1993). Some species exist naturally as clones – an example being the shrimp Artemia perthenogenetica which has been reproducing itself asexually for at least 30 million years. Other clones have arisen ‘artificially’ as products of genetic engineering and reproductive technology.

     In 1995 embryologist Dr Ian Wilmut and his team at the Roslin Institute in Edinburgh, with sponsorship from the pharmaceuticals company PPL Therapeutics, created the world’s first cloned lambs: Megan and Morag. The ‘twin’ lambs originated from a single diploid zygote, the genetic complement representing a homologous pairing of maternal and paternal chromosomes. The developing blastocyst (comprising of early inner mass cells surrounded by an extra-embryonic-membrane-forming trophoblast) was bisected to yield two identical embryos which were brought to full term in the wombs of surrogate mothers.

     At progressively advanced stages of embryonic cell differentiation, the same team of scientists went on to successfully clone four lambs from the cells of a 9-day old sheep foetus, and three more lambs from the cells of a foetus that had been aborted after 26 days of pregnancy, via the process of nuclear transference. This involved transplanting the nuclei of the cells cleaved from the embryos, into enucleated oocytes which were likewise fostered in surrogates.

     At the end of last academic year [July 1996] the cloned lamb of an adult, Finn Dorset ewe (aged 6 years) was born. Dolly, as she was subsequently named, is the first animal ever to be cloned from the non-reproductive tissue of a single, fully grown mammal. Her creators, the team led by Dr Wilmut, initiated her genesis by combining material from two sources: i) an unfertilized and enucleated oocyte from the ovary of an unidentified ewe, and ii) a diploid, somatic, donor cell containing a full set of paired chromosomes from the mammary glands of the Finn Dorset ewe. The two cells were forcibly fused into one with an electric current.

     In its original state as a differentiated cell however, the udder cell was restricted in its ability to support the development of the enucleated egg. Before fusion, it had to regain a degree of nuclear potency. This was achieved by starving the cell into a state of dormancy in a minimal salt solution containing only enough growth factor to keep the cell alive at a level of greatly decreased metabolic activity. In such an environment the mature cell ceased all mitotic division and ‘shut down’ all but the most essential of its genes. In effect, it regressed to an earlier, ‘younger’, more primitive and totipotent version of itself.

     After fusing the two manipulated cells into one, Wilmut allowed the resultant cell to grow and divide in the laboratory for one week before implanting it into the uterus of a genetically unrelated, blackface ewe. There, the reconstructed egg developed into a normal, healthy lamb with parturition occurring after a gestation period of 148 days.

     The implication then is that there exist certain proteins and enzymes, intrinsic to the egg, which are so powerful that they are capable of reprogramming a specialized nucleus. It remains to be seen however, whether or not these proteins are capable of reprogramming specialized cells such as those from either muscle tissue or the brain. The degree of reprogramming may also depend on the nature of the organism from which the donor cell was obtained. In sheep, the embryo is slow to activate any of its genes in the first few hours of growth. This period of gene latency may have provided the necessary ‘breathing space’ in which the enzymes contained within the egg could begin reprogramming the udder cell nucleus.

     In similar experiments performed on amphibians in the 1970s by John Gurdon at the University of Cambridge, nuclei from the skin cells of adult frogs were injected into enucleated frog eggs. Gurdon introduced an intermediate transplantation step into his experiment by transferring the nuclei of developing embryos into a second enucleated egg. This was for the specific purpose of affording the transplanted genome sufficient time to readjust to the embryonic environment in order that a normal blastula, capable of developing into a tadpole, could be formed. But, whilst many of his embryos grew into apparently normal tadpoles, none reached adulthood.

     Wilmut encountered the same problem with numbers in his experiments. Out of 277 fusions of enucleated oocytes with starved udder cells, Dolly proved to be the only viable live birth. According to Wilmut, this is because it is extremely difficult to synchronize the cycles of division in the initial stages of fusing the two cells.

     The principal purpose of cloning mammals is to advance the development of drug therapies for the treatment of certain life-limiting human diseases, and to develop cell culture systems from which it is possible to engineer genetically ‘elite’ agricultural livestock (Wilmut et al. 1997). Cloning, therefore, provides a powerful new tool for research into areas of both therapeutic and economic importance. Growing such transplanted cells in cultures in the laboratory provides a way of producing, in principle, unlimited numbers of genetically identical transgenic animals. Genetic uniformity within laboratory species would eradicate the anomalies of experimental results caused by the varying responses of the phenotypes to trial drugs or test diets – because of the genetic variation between the individuals. Cloning via nuclear transfer coupled with gene targeting, (disrupting or mutating a designated gene by homologous recombination, that is the genetic exchange between identical or almost identical DNA sequences), will allow researchers to utilize animals other than mice as models to test treatments for human diseases (Hartl, D. L. 1994).

     There are several possible applications for cloning larger mammals in medical research. Pigs are already being used as ‘bioreceptors’ to produce in their milk the human blood clotting protein – factor VIII – in order to treat human haemophilia; transgenic sheep are currently being used to produce the therapeutic protein α-1-antitrypsin for the treatment of cystic fibrosis in phase-2 clinical trials; and a California-based biotechnology company called GenPharm have engineered ‘Herman the wonder bull’ to possess the gene that encodes for human lactoferrin (HLF), which confers antibacterial and iron transport properties to people. Some, though not all of Herman’s female progeny are capable of producing milk which contains HLF (Raven, P. H. & Johnson, G. B. 1996); other herds of cows are presently being engineered to produce special human proteins in the place of some normal bovine proteins, in order that cows milk may become digestible by human infants. Using clones of such animals would increase the speed and efficiency with which the gene mutations are incorporated into the germ line.

     Pigs have also been developed as donors of hearts and kidneys for surgical xenotransplantation but, so far, with limited success. Though the pig tissues have been engineered to be coated with certain human proteins, such xenografts are still very often rejected by the human body due to the presence of remaining cell surface pig histocompatibility molecules. Nuclear transfer and gene targeting would improve the chances of success of xenografts by replacing the pig proteins that are responsible for organ rejection with much less antigenic human proteins. It is conceivable that a patient’s own cells may at some stage in the future be utilized for such therapies in the treatment of Leukaemia and Parkinson’s disease, by the cloning of specific body parts as well as of whole organisms.

     With respect to Agriculture, the most obvious advantage of cloning farm animals is in the production of a central, elite genetic stock for purchase and use by the commercial farmer; where banks of engineered embryos would complement the already existing banks of semen collected from high merit bulls. Gene targeting could be employed to produce phenotypic traits in beef bulls and dairy cows that are of high economic importance, such as growth rate, feed conversion efficiency, muscle:fat ratio, total lactation and longevity. In a single generation a farmer could significantly raise the performance of his herd, before returning to the more traditional methods of selective breeding. Genetic diversity could be protected in such a scenario by employing systems whereby limited numbers of each clone of each genotype would be made available for retail, and each buyer would be limited to a certain number of purchases.

     The first transgenic salmon have been engineered by Canadian fisheries scientists to have shortened production cycles and heavier adult weights by recombining growth hormone genes into the developing embryos. Large scale cloning of animals such as these would go some way to increasing world-wide food production.

     The benefits of cloning for endangered species are less clear. Genomes could be cryostatically preserved in liquid nitrogen from skin biopsies and blood samples to serve as emergency stocks should the wild populations deteriorate to dangerously low levels but it is conceptually difficult to reconcile the need for genetic diversity with the act of preserving a single instance or limited versions of a genome.

     Dr Wilmut is confident that new therapies for diseases such as cystic fibrosis, developed as a direct result of animal cloning, will be marketable within the next couple of decades. Beyond that he maintains that predictions regarding the direction of science and technology are futile. In an interview with Andrew Ross he admitted that, due to the nature of his research, he may have contributed towards a scientific movement that he does not wish to see happen. He is most concerned that strict and unequivocal legislative laws should be enforced to prevent any kind of manipulation with human embryos. The notion that human cloning is the next obvious step may be fanciful, and it opens up an ethical minefield which is beyond the scope of this essay, suffice it to say that the right of a human being to live out an autonomous and dignified existence does not depend upon the fusion of sperm and egg (or lack thereof) as representing the starting point of any particular generation.