The Brave New World of Mammalian Cloning
The demonstration by Wilmut et al. that a nucleus from an adult mammary gland cell from a sheep can be reprogrammed by the oocyte cytoplasm to direct the development of a new individual has caused developmental biologists to re-examine the process of cell differentiation. This process does not, as we have thought until quite recently, irreversibly preclude totipotency. Clearly, cytoplasmic factors are capable of evoking the entire developmental process from the nucleus of a differentiated adult cell. Since the initial report of cloned sheep, other investigators have reported bovine cloning. More recently, calves have been cloned that have been genetically modified to produce pharmaceuticals: a step toward "pharming".
In a recent development, Wakayama et al. (1998) have reported Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei. This result is particularly significant because the mouse is a superior experimental animal, although the practical applications are not as readily apparent as they are with agriculturally significant animals such as sheep and cows.
Wilmut and his colleagues have shown that developmental potential of transplanted nuclei is enhanced when donor nuclei are in either the G0 or G1 phase of the cell cycle (Campbell et al., 1996, 1996a). Hence, Wakayama et al. (1998) selected nuclei of cells known to be at G0/G1 for nuclear transfer. Sertoli cells (accessory cells in testes) and neuronal cells from adult mice are in the G0 phase, whereas more than 90% of cumulus cells (which surround the developing oocyte in the ovary) are in the G0/G1 phase. Wakayama e t al. (1998) transferred nuclei from these cells into enucleated oocytes by a simple microinjection procedure.
Enucleated oocytes were injected with a single nucleus from one of these three cell types, and the oocytes were activated either simultaneously with injection or after a delay of from 1-6 hours. Oocytes were activated in culture medium containing Sr^++ and cytochalasin B. The Sr^++ caused activation, whereas cytochalasin B prevented the formation of polar bodies, thus retaining chromosomes in the oocyte. Delayed activation was significantly more effective in permitting normal development to the morula/blastocyst stages than was simultaneous activation. This delay facilitated the formation of structures resembling pronuclei (pseudo-pronuclei).
Cumulus cell nuclei resulted in development of live born mice, whereas attempts to use nuclei from Sertoli cells (accessory cells in testes) and neuronal cells were unsuccessful. (Some nuclear transplant recipients developed, but none developed past 8.5 days of development.) It is unclear why the latter two cell types were not successful in promoting development, but it has been proposed that their G0 status is not sufficient to allow them to be reprogrammed.
Applications of Cloning Technology
The latent totipotency of adult mammalian nuclei suggests that it may be feasible to reprogram adult human cells for use in the treatment of disease. Thus, investigators may be able to develop strategies to facilitate the repair and regeneration of human tissues. Nucleo-cytoplasmic interactions that restore potency to differentiated cells are an important research focus with great potential in treating diseases such as cancer, diabetes and neurodegeneration.
Another potential application of mammalian cloning is the production of clones of genetically-engineered domestic animals, such as sheep, pigs and cattle. For example, bovine nuclei could be engineered so that medically-significant proteins would be selectively secreted into the milk of cattle produced by nuclear transplantation.
Campbell, K.H.S., Loi, P., Otaegui, P.J. and Wilmut, I. 1996. Cell cycle co-ordination in embryo-cloning by nuclear transfer. Rev. Reprod. 1: 40-45.
Campbell, K.H.S., McWhir, J. Ritchie, W.A. and Wilmut, I. 1996 a. Sheep cloned by nuclear transfer from a cultured cell line. Nature 38-: 64-66.
Wakayama, T., Perry, A.C.F, Zuccotti, M., Johnson, K.R. and Yanagimacchi, R. 1998. Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei. Nature 394: 369-374.
Wilmut, I., Schnieke, A.E., McWhir, J., Kind, A.J. and Campbell, K.H.S. 1997. Viable offspring derived from fetal and adult mammalian cells. Nature 385: 810-813.
Web links to Cloning Literature and Media Reports
Web Links to Applications of Cloning Technology
In another twist, Tanja Dominko and collaborators in Wisconsin have reported that cow eggs can serve as surrogates for nuclei of other species, such as rats, pigs, sheep and monkeys. This surprising result indicates that maternal factors in the egg can function across species to activate the zygotic genome. For more on this topic, see the following:
Follow the links below to explore these fascinating topics and their ethical implications in more detail. The links are arranged roughly in descending chronological order.
Two books have recently been released discussing cloning and its implications:
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Leon Browder & Laurie Iten (Ed.) Dynamic Development
Last revised Wednesday, October 7, 1998