Cellular composition and viability of cloned bovine embryos using exogene-transfected somatic cells.
The present study compared the efficiency of transgenic (TG) cloned embryo production by somatic cell nuclear transfer (SCNT) with fetal-derived fibroblast cells (FFCs) which were transfected with pEGFP-N1 to in vitro-fertilized (IVF), parthenogenetic and SCNT counterparts by evaluating the rates of cleavage and blastocyst formation, apoptosis rate at different developmental stages, cell number, ploidy and gene expression in blastocysts. In SCNT and TG embryos, the rates of cleavage and blastocyst formation were significantly lower (p < 0.05) than those of IVF controls, but it did not differ between SCNT and TG embryos. In IVF control, 86.7% embryos displayed diploid chromosomal complements and the rates were significantly (p < 0.05) higher than those of SCNT and TG embryos. Most TG embryos (79%) with FFCs expressed the gene by both PCR and under fluorescence microscopy. The expression of apoptosis by TUNEL was first detected at six to eight cell stages in all embryos of IVF, SCNT and TG groups, but the expression rate at each developmental stages was significantly higher (p < 0.05) in SCNT and TG embryos than in IVF counterparts. The expression rate in inner cell mass (ICM) of TG embryos was significantly higher (p < 0.05) than in SCNT and IVF embryos. These results indicate that the high occurrence of apoptosis observed in SCNT and TG embryos compared with IVF counterparts might influence the developmental competence. Moreover, the SCNT embryos derived using non-transfected donor cells exhibited a lower apoptosis expression in ICM cells than in TG embryos derived using pEGP-N1-transfected donor cells suggesting a possible role of negative gene effect in TG embryos.
Lee
S-L
SL
Institute of Animal Medicine, College of Veterinary Medicine, Gyeongsang National University, Chinju, Korea.
Kumar
B M
BM
Kim
J-G
JG
Ock
S-A
SA
Jeon
B-G
BG
Balasubramanian
S
S
Choe
S-Y
SY
Rho
G-J
GJ
D000818:Animals; D030801:Animals, Genetically Modified; D017209:Apoptosis; D001755:Blastocyst / Q000166:cytology*; D002417:Cattle / Q000196:embryology*; D002470:Cell Survival; D019976:Cloning, Organism / Q000662:veterinary; D004622:Embryo, Mammalian / Q000166:cytology*; D047108:Embryonic Development / Q000502:physiology; D005307:Fertilization in Vitro / Q000662:veterinary; D020287:In Situ Nick-End Labeling / Q000662:veterinary; D053652:Nuclear Transfer Techniques / Q000662:veterinary; D010312:Parthenogenesis; D014162:Transfection / Q000379:methods / Q000662:veterinary*