[Effects of different donor cells and passages on the development of nuclear transfer porcine embryos].
This study was undertaken to systematically examine effects of different donor cells and passages on the development of nuclear transfer porcine embryos, so as to establish a preliminary procedure for porcine cloning. Porcine oocytes obtained from ovaries at slaughter were matured in vitro for 40-44h and then enucleated in manipulation medium containing 5 microg/mL cytochalasin B. Fibroblast cells (FC), oviduct epithelial cells (OEC), granulosa cells (GC) and cumulus cells(CC) after 3-9 passages in TCM + 10% FBS were treated by serum starvation (0.5% FBS for 2-9 days), 0.1 microg/mL aphidiconlin (APD) for 1 day and 0.5% FBS for 2-9 days or continue culturing in 10% FBS for 2-9 days, then, were transferred into enucleated oocytes by microinjection or electronic fusion (100 V/mm, 30 micros and 1 pulses). Reconstituted embryos were activated with a combination of calcium ionophore A23187 or electric pulse and 6-DMAP, and cultured for 6 days, to evaluate their cleavage and embryonic development. The cleavage rate of embryos reconstructed with FC and GC pretreated with 0.1 microg/mL APD + 0.5% FBS were significantly higher than that of serum starvation group and control group (P<0.01). There was significantly difference in the cleavage rate and embryonic development among embryos derived from GC, CC and FC, OEC pretreated with 0.1 microg/mL APD + 0.5% FBS. The cleavage rate of embryos reconstructed with GC by electrofusion was significantly higher than that by microinjection (P<0.05), but no difference was found in their proportion developing to blastocysts. 75% to 85% of GC at 3 and 6 passages, and FC at 6 and 10 passages had normal karyotype, which did not show significant difference among them (P>0.05). When GC at G3, G4, G5 and G6 of passages were used as donor cells, the cleavage rate and blastocyst rate was similar, moreover, FC at G6, G7, G8 and G9 of passages also resulted in a similar cleavage rate and blastocyst development. These results indicate that: (1) FC and GC can be cultured up to 9 passages and keep relatively stable karyotepe; (2) Using 0.1 microg/mL APD to treat donor cells prior to nuclear transfer can improve the efficiency of somatic cell nuclear transfer in buffalo but serum starvation is inefficient in our system; (3) Both of FC and GC cells can be used as the donor karyoplasts for nuclear transfer, and their efficiency are not influenced by the culture passages; (4) The development of reconstructed embryos by electrofusion is higher than that by microinjection, but there is no difference in the total efficiency between the two methods.
Zhang
De Fu
DF
Animal Husbandry and Veterinary Research Institute, Shanghai Academy of Agricultural Sciences.
Liu
Dong
D
Tang
Lin Lin
LL
Wang
Ying
Y
Chen
Yin
Y
Wang
Kai
K
Schellander
Karl
K
Lin
Cai Lu
CL
D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001755:Blastocyst / Q000166:cytology / Q000502:physiology; D000001:Calcimycin / Q000494:pharmacology; D017690:Cell Transplantation / Q000379:methods*; D002478:Cells, Cultured; D054885:Cumulus Cells / Q000166:cytology; D046149:Embryo Culture Techniques; D004622:Embryo, Mammalian / Q000166:cytology* / Q000502:physiology; D047108:Embryonic Development / Q000187:drug effects; D005260:Female; D005347:Fibroblasts / Q000166:cytology; D006107:Granulosa Cells / Q000166:cytology; D007476:Ionophores / Q000494:pharmacology; D008845:Microinjections; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000166:cytology* / Q000502:physiology; D010053:Ovary / Q000166:cytology; D010057:Oviducts / Q000166:cytology; D013552:Swine
D007476:Ionophores; D000001:Calcimycin; C001020:N(6),N(6)-dimethyladenine; D000225:Adenine