A highly efficient method for porcine cloning by nuclear transfer using in vitro-matured oocytes
Cloning and stem cells
To date, the efficiency of pig cloning by nuclear transfer of somatic cell nuclei has been extremely low, with less than 1% of transferred embryos surviving to term. Even the utilization of complex procedures such as two rounds of nuclear transfer has not resulted in greater overall efficiencies. As a result, the applicability of the technology for the generation of transgenic and cloned animals has not moved forward rapidly. We report here a simple nuclear transfer protocol, utilizing commercially available in vitro-matured oocytes, that results in greater than 5% overall cloning efficiency. Of five recipients receiving nuclear transfer embryos produced with a fetal fibroblast cell line as nuclear donor, all five established pregnancies by day 28 (100%), and 4/5 (80%) went to term. Efficiencies for each transfer were 7% (9 piglets/128 doublets transferred), 5% (5/100), 12% (7/59), and 6.6% (7/106). The overall efficiency in all recipients was 5.5% and in pregnant recipients 7.7%, with a total of 28 cloned piglets produced. With the average fusion rate being 58%, the percentage of fused doublets producing a live piglet approached 12%. The method described here can be undertaken by a single micromanipulator at a reasonable cost, and should facilitate the broad utilization of porcine cloning technology in transgenic and nontransgenic applications.
Walker, S. C., Shin, T., Zaunbrecher, G. M., Romano, J. E., Johnson, G. A., Bazer, F. W., & Piedrahita, J. A. (2002). A highly efficient method for porcine cloning by nuclear transfer using in vitro-matured oocytes. Cloning and stem cells, 4 (2), 105-12. https://doi.org/10.1089/153623002320253283