Improvement of a porcine somatic cell nuclear transfer technique by optimizing donor cell and recipient oocyte preparations.
This study was conducted to improve a porcine somatic cell nuclear transfer (SCNT) technique by optimizing donor cell and recipient oocyte preparations. Adult and fetal fibroblasts, and cumulus and oviduct cells were used as donor cells, and in vivo- and in vitro-matured oocytes were employed as recipient oocytes. The percentages of fusion and development to the blastocyst stage, the ratio of blastocysts to 2-cell embryos, and cell number of blastocysts were monitored as experimental parameters. In Experiment 1, donor cells of four different types were transferred to enucleated oocytes matured in vitro, and more (P < 0.05) blastocysts were derived from SCNT of fetal fibroblasts than from that of other cells (15.9% versus 3.1-7.9%). For SCNT using fetal fibroblasts, increasing the number of subcultures up to 15 times did not improve developmental competence to the blastocyst stage (12.2-16.7%). In Experiment 2, fetal fibroblasts were transferred to enucleated oocytes that matured in vivo or in vitro. When parthenogenetic activation of both types of oocytes was conducted as a preliminary control treatment, a significant increase in blastocyst formation was found for in vivo-matured compared with in vitro-matured oocytes (36.4% versus 29.5%). However, no improvement was achieved in SCNT using in vivo-matured oocytes. In conclusion, the type of donor somatic cell is important for improving development after porcine SCNT, and fetal fibroblasts were the most effective among examined cells. A system with good reproducibility has been established using fetal fibroblasts as the donor karyoplast after subculturing 1-10 times, and using both in vivo and in vitro-matured oocytes as the recipient cytoplast.
Lee
Gab-sang
GS
Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University, Seoul 151-742, South Korea.
Hyun
Sang-hwan
SH
Kim
Hye-soo
HS
Kim
Dae-young
DY
Lee
So-hyun
SH
Lim
Jeong-mook
JM
Lee
Eun-song
ES
Kang
Sung-keun
SK
Lee
Byeong-chun
BC
Hwang
Woo-suk
WS
D000818:Animals; D001755:Blastocyst / Q000502:physiology; D002478:Cells, Cultured; D019976:Cloning, Organism / Q000379:methods*; D004622:Embryo, Mammalian; D005187:Fallopian Tubes / Q000648:ultrastructure; D005260:Female; D005347:Fibroblasts / Q000648:ultrastructure; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000648:ultrastructure*; D006080:Ovarian Follicle / Q000648:ultrastructure; D013552:Swine