A Highly Efficient Method for Porcine Cloning by Nuclear Transfer Using In Vitro-Matured Oocytes

SC Walker, T Shin, GM Zaunbrecher… - Cloning & Stem …, 2002 - liebertpub.com
SC Walker, T Shin, GM Zaunbrecher, JE Romano, GA Johnson, FW Bazer, JA Piedrahita
Cloning & Stem Cells, 2002liebertpub.com
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 …
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.
Mary Ann Liebert