Recently, the transgenic animal production technique is very important for the production of bio-parmaceutical as animal bio-reactor system. However, the absence of survival evaluation in in vitro produced transgenic embryos has been a problem of the ...
Recently, the transgenic animal production technique is very important for the production of bio-parmaceutical as animal bio-reactor system. However, the absence of survival evaluation in in vitro produced transgenic embryos has been a problem of the low productivity of transgenic animal because of absent of pre-estimate of pregnancy after transgenic embryos transferred into recipient. Therefore, this study is conducted to improve efficiency of transgenic cattle production by transgenic technique. Firstly, this study is to develop transgenic cell line expressing targeted human granulocyte colony stimulating factor (hGCSF) and green fluorescence protein (GFP) genes as well as production of Somatic Cell Nuclear Transfer (SCNT) embryos derived from co-expressed transgenic donor cells. Constructed pPiggy-mWAP-hGCSF-EF1-GFP vector was chemically transfected into bovine fetus cells and then, only GFP expressed cells were selected as donor cells for SCNT. Cleavage and blastocyst rates of parthenogenetic, SCNT embryos using non-TG cell and hGCSF-GFP dual expressed SCNT embryos were examined (cleavage rate: 78.0±2.8 vs. 73.1±3.2 vs. 70.4±4.3%, developmental rate: 27.2±3.2 vs. 21.9±3.1 vs. 17.0±2.9%). Result indicated that cleavage and blastocyst rates of TG embryos were significantly lower (P<0.05) than those of parthenogenetic and non-TG embryos, respectively. Also, transgenic bovine embryos were produced by injection of FIV-EGFP lentiviral vector into perivitelline space of in vitro matured MІІ stage oocytes, and then in vitro fertilization (IVF) was occured. Normal IVF and EGFP expressing blastocysts were transferred into recipients. Results indicated that 2 expanded blastocysts (34.7%) transferred group showed significantly (p < 0.05) higher pregnancy rate than 1 expanded blastocyst (26.8%) transferred group. In case of parity of recipient, ET to heifer (34.9%) showed significantly (p < 0.05) higher pregnancy rate than ET to multiparous recipient (21.2%). However, there are no significant differences of pregnancy rate between natural induced estrus and artificial induced estrus groups. Significantly (p < 0.05) higher pregnancy rate was obtained from recipient group which have normal corpus luteum with crown group (34.8%) than normal corpus luteum without crown (13.6%). Additionally, treatment of 100 ㎍ Gn-RH injection to recipient group (38.6%) 1 day before ET significantly (p < 0.05) increase pregnancy rate than non- Gn-RH injection to recipient group (38.6%). We also transferred 2 EGFP expressing expanded blastocysts to each 19 recipients, 7 recipients were pregnant and finally 5 EGFP transgenic cattle were produced under described ET condition. Therefore, our result suggested that transfer of 2 good-quality expanded blastocysts to 100㎍ of Gn-RH injected recipient which have normal corpus luteum with crown is feasible to produce transgenic cattle.