“Successful Serial Recloning in the Mouse over Multiple Generations”, 2013-03-07 ():
Successful recloning of viable mice over 25 generations
Cloning efficiency remains consistent over 25 iterations
No evidence for the accumulation of reprogramming or genomic errors
Serially recloned mice have the same characteristics as standard clones
Previous studies of serial cloning in animals showed a decrease in efficiency over repeated iterations and a failure in all species after a few generations. This limitation led to the suggestion that repeated recloning might be inherently impossible because of the accumulation of lethal genetic or epigenetic abnormalities.
However, we have now succeeded in carrying out repeated recloning in the mouse through a somatic cell nuclear transfer method that includes a histone deacetylase inhibitor. The cloning efficiency did not decrease over 25 generations, and, to date, we have obtained more than 500 viable offspring from a single original donor mouse. The reprogramming efficiency also did not increase over repeated rounds of nuclear transfer, and we did not see the accumulation of reprogramming errors or clone-specific abnormalities.
Therefore, our results show that repeated iterative recloning is possible and suggest that, with adequately efficient techniques, it may be possible to reclone animals indefinitely.
…The success rates of serial recloning varied between generations; for example, the average success rates of recloning attempts in G3, G7, and G11 were very low (4%–5%), whereas the success rates for the next generations of each of these (G4, G8, and G12) were 1.5–2× higher. G16 showed the highest success rate, but in the next generation, the success rate decreased by 1⁄3rd (Figures 1A & 1B). This variation was observed not only between generations but also within experiments. In G10, G18, and G25, the maximum success rate was over 20%, but the minimum rate was only 3%–4%. (Table S1 available online). Therefore, although we saw substantially higher cloning success rates in recent generations (G16, G24, and G25) than in G1, the high variation even within generations makes it difficult to draw any clear conclusions about changes in success rate. Nevertheless, we have been able to conduct repeated recloning over 25 generations, and, to date, 581 recloned mice have been generated from one original donor mouse (Table S1 & Figure 2B).
…Fertility can also be used as an indicator of normal development in mice. To examine the fertility of our cloned mice, we selected 4 G20 clones randomly at the time of weaning and mated them with normal BDF1 male mice produced via natural mating. All the clones gave birth naturally to normal litter sizes, and pups lacked any abnormalities; the mean age at first birth was about 2 months, similar to that of naturally generated mice (Table S2).
…In this study, we examined telomere lengths in the recloned mice at 3 months of age and compared them with those of age-matched control mice. We also collected samples from earlier generations of recloned mice still living at the same time, which were older at the time of collection. As shown in Figure 2A, these experiments revealed that there was no evident shortening of telomeres in the recloned mice of any generation or at any age.
…In this study, we also found that successive recloning over multiple generations produced phenotypically normal fertile mice with normal lifespans. Thus, there seems to be no inherent reason why recloning in mice should fail, and it seems most likely that the previous failures in serial recloning ( et al 2000) can be attributed to the low success rate of the cloning techniques being used at that time, leading to an accidental end to the serial recloning experiment. Even with our improved procedure, the cloning success rate varied 2%–25% through the 25 generations that we examined. Thus, with further improvement to nuclear-transfer cloning techniques, unlimited animal recloning in many animal species might in fact be possible.