Autologous somatic cell nuclear transfer in pigs using recipient oocytes and donor cells from the same animal.
The objective of the present study was to examine the feasibility of the production of autologous porcine somatic cell nuclear transfer (SCNT) blastocysts using oocytes and donor cells from slaughtered ovaries. Therefore, we attempted to optimize autologous SCNT by examining the effects of electrical fusion conditions and donor cell type on cell fusion and the development of SCNT embryos. Four types of donor cells were used: 1) denuded cumulus cells (DCCs) collected from in vitro-matured (IVM) oocytes; 2) cumulus cells collected from oocytes after 22 h of IVM and cultured for 18 h (CCCs); 3) follicular cells obtained from follicular contents and cultured for 40 h (CFCs); and 4) adult skin fibroblasts. The DCCs showed a significantly (p < 0.01) lower rate of fusion than the CCCs when two pulses of 170 V/mm DC were applied for 50 microsec (19 +/- 2% vs. 77 +/-3%). The rate of DCC fusion with oocytes was increased by the application of two DC pulses of 190 V/mm for 30 microsec, although this was still lower than the rate of fusion in the CCCs (33 +/- 1% vs. 80 +/- 2%). The rates of cleavage (57 +/- 5%) and blastocyst formation (1 +/- 1%) in the DCC-derived embryos did not differ from those (55 +/- 6% and 3 +/- 1%, respectively) in the CCC- derived SCNT embryos. Autologous SCNT embryos derived from CFCs (5 +/- 2%) showed higher levels of blastocyst formation (p < 0.01) than CCC-derived autologous SCNT embryos (1 +/- 0%). In conclusion, the results of the present study show that culturing cumulus and follicular cells before SCNT enhances cell fusion with oocytes and that CFCs are superior to CCCs in the production of higher numbers of autologous SCNT blastocysts.
Lee
Eunsong
E
School of Veterinary Medicine and Institute of Veterinary Science, Kangwon National University, Chunchon 200-701, Korea. eslee@kangwon.ac.kr
Song
Kilyoung
K
D000818:Animals; D030801:Animals, Genetically Modified; D019976:Cloning, Organism; D054885:Cumulus Cells / Q000378:metabolism; D004558:Electric Stimulation; D046149:Embryo Culture Techniques / Q000662:veterinary; D047108:Embryonic Development; D005260:Female; D005347:Fibroblasts / Q000378:metabolism; D053652:Nuclear Transfer Techniques / Q000662:veterinary*; D009865:Oocytes / Q000378:metabolism*; D006080:Ovarian Follicle / Q000378:metabolism; D013552:Swine / Q000196:embryology / Q000502:physiology*