It has now been almost a year since the cloning of the sheep Dolly from an adult ovine cell was announced (1). The year has brought much agonizing discussion, potential legislation, and some laurels, but no more Dollies. The principal scientist, Ian Wilmut, has announced (2) that he and his group have no intention of trying again (to clone using mammary DNA and a host denucleated ovine cell). Some "very soon" to be delivered (3) cows that were to be cloned from adult cells have yet to appear. Other rumored events seem also to have dissipated. It is a well-known tenet of science that a single observation is not to be codified until confirmed by someone in some way. The single observation gains some credence when well controlled or of a unique nature, or both. It is the lack of any confirmation that provokes our skepticism; here are some of the detailed reasons.
If we are to try to seriously analyze the mammalian cloning issue and its human implications, we should ask for details on points such as these, and for stronger statistics plus independent confirmation, before considering cloning of adult cells by means of nuclear transfer as a fait accompli.
- The cloning was done once out of some some 400 tries. Only one successful attempt out of some 400 is an anecdote, not a result. All kinds of imagined and unimagined experimental error can occur.
- The characterization of the mammary gland cells used as nucleus donors was poor; it could have been one of the donor's rare stem cells that was involved, as acknowledged in the paper (2).
- The reason why the donor ewe was pregnant was not explained (1). This is important, because the cell which led to Dolly could have been of fetal origin. Why was no analysis of the fetus and its father's genotype performed? Given these DNA fingerprints, or even the sex of the fetus, one could have excluded a fetal cell as donor.
- The demonstration that the four microsatellite marker DNAs seem the same in Dolly and in the donor mammary cells is good, but not sufficient; it would probably be rejected by a jury called to deliberate on Dolly's origin, not an unlikely event given Dolly's commercial potential. Sheep are highly inbred and there are, to our knowledge, no data on gene frequencies in sheep populations; differences in DNA fingerprints can provide exclusion, but similarities are only a statistic.
- An analysis of Dolly's mitochondrial DNA has not been given, although it could provide important clues to her origin; the genotype of the recipient oocyte and the mitochondrial genotype of the donor cell or that of any of the other players was also not given.
- Last summer (4), and again recently (A. E. Schnieke et al., Reports, 19 Dec., p. 2130), the same group announced the cloning of transgenic sheep, but from a fetal cell not an adult cell; Polly is not a Dolly. Remember, Dolly should be "aged" relative to her peer group. Barring new science, she must have retained any imprinted genes from the previous generation, she should have short telomeres, and her DNA should have an adult's worth of mutations; a special creature in more ways than one.
- No hint is given in the paper (1) that the donor ewe had apparently died a few years ago, thereby precluding pertinent immunological testing of genetic relationships.
Discussing such issues before they are immediately upon us is correct. However, indulging in endless debates is less so, when one considers both the scientific weaknesses of the experiment and the possible impact on the societal credibility of science itself of the "facts" on which they are purportedly based.
Vittorio Sgaramella
University of Calabria,
Cosenza 87030, ItalyNorton D. Zinder
Rockefeller University,
New York, NY 10021, USA
References
- I. Wilmut, A. E. Schnieke, J. McWhir, W. A. J. Kind, K. H. S. Campbell, Nature 385, 810 (1997).
- G. Kolata, New York Times, 29 July 1997, p. C3.
- ___, ibid., 8 August 1997, p. A10.
- ___, ibid., 25 July 1997, p. A18.
Response: With reference to the skepticism of Sgaramella and Zinder about the origins of Dolly the sheep, we would like to provide clarification about some of the points they raise. Dolly is the only live birth that resulted from the transfer of nuclei from the adult-derived mammary cell cultures. Admittedly, a single birth from 400 attempted fusions is not an efficient system. However, the suggestion that "experimental error does occur" can be answered in a number of ways. First, Dolly is a Finn Dorset ewe. At the time of these experiments there were no other Finn Dorset cells being cultured in the laboratory and no Finn Dorset embryos being used in any other experimental system. Thus, in terms of breed, Dolly can only have been derived from the cell culture established from the mammary gland. These cell cultures were not established for the purposes of nuclear transfer, but had been previously isolated for other studies. The reason that a pregnant donor ewe was prepared was to establish, in culture, a cell line that exhibited mammary epithelial-specific characteristics for long-term culture. This was part of a collaboration between PPL Therapeutics and the Hannah Research Institute. For this reason, the genotypes of the fetus and the ram used for insemination were not analyzed, and no fetal material was retained for analysis.Microsatellite analysis of Dolly mirrored exactly the pattern observed at both early (pre-nuclear donor) and late (post-nuclear donor) passages of the cell population. In addition, the cell population was predominately epithelial in nature. It is inconceivable that during the very short period of cell expansion, a rare fetal cell, if present, could have overgrown the mammary culture.
With regard to the mitochondrial DNA, samples from Dolly, all of the other lambs produced by nuclear transfer, the cell cultures, and representative samples from a number of randomly selected Blackface ewes (the breed used as oocyte donors) have been provided for analysis by independent third parties. When the results of these studies are available, they will be announced to the scientific community. Similarly, studies of the telomere length of the donor cells used for the production of Dolly, of Dolly herself, and of Finn Dorset sheep of representative ages are being analyzed at two centers. These studies are being coordinated with studies of all of the nuclear transfer offspring produced from embryo- and fetal-derived cell populations, of the cell populations themselves, and of the naturally produced offspring of those animals that have reached sexual maturity and have been bred.
We would like to point out that the methods described (1, 2) have been duplicated successfully by using cell populations derived from embryonic material (3). Other groups are attempting to repeat the technology using fetal and adult cell populations. It should be realized that only 11 months have elapsed since publication of our results (2); if one takes into account the time period required for gestation in sheep (5 months), one sees that it is unlikely that other authors would yet have had time to complete similar experiments and publish data.
Retrospectively, we and our co-authors realize that if the use of these cells for nuclear transfer had been anticipated, the skepticism of Sgaramella and Zinder could have been allayed by reference to an original donor tissue sample deposited with a respected neutral third party.
We were always aware that there would be some skepticism about our results and have been greatly encouraged by the positive reaction of the scientific community. We would like to think that this reflects the integrity with which we are accredited by our scientific peers. To us, as practicing scientists, this accolade is of paramount importance.
Keith H. S. Campbell
Alan Colman
PPL Therapeutics,
Roslin, Midlothian EH25 9PP,
Scotland, United KingdomIan Wilmut
Roslin Institute,
Roslin, Midlothian E-125 9PS,
Scotland, United Kingdom
References
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