A highly efficient method for porcine cloning by nuclear transfer using in vitro-matured oocytes.
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
Shawn C
SC
Center for Animal Biotechnology and Genomics, Texas A&M University, College Station, Texas 77843-4458, USA.
Shin
Taeyoung
T
Zaunbrecher
Gretchen M
GM
Romano
Juan E
JE
Johnson
Greg A
GA
Bazer
Fuller W
FW
Piedrahita
Jorge A
JA
D000818:Animals; D019976:Cloning, Organism / Q000379:methods*; D046508:Culture Techniques; D004624:Embryo Transfer; D005260:Female; D018895:Microsatellite Repeats / Q000235:genetics; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000166:cytology*; D011247:Pregnancy; D012744:Sex Ratio; D013552:Swine / Q000235:genetics*