LONDON - Dolly the sheep, the first animal to be cloned by transferring a nucleus from an adult cell, has confirmed predictions that the protective "caps" on the ends of her chromosomes - her telomeres - are shorter than would be expected for an animal her age.
For cells in culture, shorter than usual telomeres are associated with aging. The reduction seen in Dolly, who is nearly three, is attributed to both the age of the animal from whom the adult cells were taken (six years), and the culturing of the cells before the cloning process began.
Dolly appears to be perfectly healthy; this year she gave birth to triplets. Nevertheless, said the team which created her, the finding suggests that it might be wise to keep culture times of cells being grown for nuclear
transfer techniques to a minimum.
Furthermore, said Alan Colman, research director of PPL Therapeutics, of Roslin, Scotland, the discovery could have implications for researchers examining the proposed technique of "therapeutic cloning." This futuristic idea would involve taking a healthy cell from someone with leukemia, for example, transferring its nucleus into an enucleated human oocyte, creating an embryo, and then deriving and culturing blood stem cells from that embryo. These could then be returned to the cancer patient after he or she had received a lethal dose of radiation, in order to repopulate the bone marrow.
Colman told BioWorld International: "The implication is that anything which involves culture will result in telomere sizes going down, and that could prejudice the future uses of that material. If it was going to age prematurely, then that would be an issue, although we don't know for certain at present that this is the case. In terms of cloning livestock, the message is that cells which are being cultured should be dealt with as fast as possible."
Telomeres are repeated sequences on the ends of the chromosomes, which are thought to protect the chromosomes from inadvertently fusing during cell division. In cells in culture, some are lost each time the cell divides, until eventually the cells become senescent. Such observations have led to the hypothesis that the telomeres act as a kind of molecular clock: When most have been lost, this is a signal to the cell that it has reached old age and should stop dividing.
Germ cells have genes encoding a protein complex called telomerase, which can manufacture telomeres and add them back onto the chromosomes. Somatic cells do not have an active telomerase gene, but in some tumor cells the gene is reactivated. This suggests that some cancers might occur because their reactivation of the telomerase gene allows the cells to replicate indefinitely. One possible strategy to fight cancer might therefore be to knock out this gene.
The length of Dolly's telomeres has therefore been of some interest. Data obtained by Colman and his colleagues have now been published in a letter to Nature, dated May 27, by Paul Shiels, also of PPL, and colleagues from the same company and from the Roslin Institute, also in Roslin.
They used a restriction enzyme to cut off the mean terminal restriction fragment (TRF). Studies of control animals showed that the TRF length decreased with increasing age, at a mean rate of 0.59 kilobases (kb) per year. The team compared the TRF length of Dolly and two other animals produced by the nuclear transfer technique, with those of age-matched controls.
Dolly's TRF size was 20 percent less than that of sheep of similar age: 19.14 kb compared with an expected length of 23.9, plus or minus 0.18 kb. This difference was statistically significant (p<0.005).
Shiels and his co-authors write: "The smaller TRF in [Dolly] is consistent with the age of her progenitor mammary tissue (six years old) and with the time that ovine mammary epithelial cells derived from that tissue spent in culture before nuclear transfer."
Another animal, 6LL6, where the nucleus was derived from sheep embryonic cells, also showed a significant decrease in TRF size, they said. However, in the third animal, where the nucleus was obtained from fetal fibroblasts, there was no significant difference in the TRF size - possibly, said the researchers, because fetal tissue and minimal culturing had been used in its production.
The team concluded: "As the number of animals analyzed was small, it is possible that the difference was due to natural variation of the mean TRF size in these individuals, but the statistical significance of the data argues against this."
The next question is whether the reduction in TRF size has implications for the longevity of these animals. Colman told BioWorld International the answer is, "in Dolly's case, we don't know. We know that she is perfectly healthy, as are all the other animals, and she is not showing any signs of aging, but of course we don't know what the future holds."
In one experiment carried out by other researchers, the gene for telomerase was knocked out in mice. These animals were able to reproduce without apparent ill effect for five generations; the sixth was infertile. Thus, said Colman, the implication is "that the reduction in telomere length may not be enough to have an effect on the generation we are dealing with. In fact, reduction in telomere length during a lifetime may be a consequence of the aging process rather than its cause."
Much interesting scientific work on this phenomenon remains to be done, he added. "We can't afford to put a lot of resources into this area, but we would like to think that other people might contact us, who could do the work if we gave them the material. We feel we have to focus on the business side and the methods of commercializing the technology which are appropriate for us."
One experiment Colman expects will be easy to carry out is to mate Dolly with a male animal produced by nuclear transfer, which also has shorter telomeres. "The offspring that Dolly has had so far has been produced in the normal way by mating with a local ram," he said. "What we don't know is whether, in her daughters, the telomere sizes have been restored to normal. It would be interesting to see whether the germ cells have an ability to not just maintain - as they do - telomere sizes, but to restore them to what they should be. If we mated two animals produced by nuclear transfer, it should be easy to find this out."
A statement released by PPL said the results do not appear to have any practical consequences for cloning of livestock, since by using fetal cells and minimal culture time, any potential effects could be circumvented. It added, "Once a limited number of founder animals have been produced by nuclear transfer, flocks would normally be produced by natural breeding, and it is entirely possible that telomere shortening would be corrected in the germ line, so that the offspring of clones would have normal telomere length."