The purpose of 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. In this study, we successfully produced hGCSF-GFP dual expressed SCNT embryos and cryopreserved to produce transgenic cattle for bioreactor system purpose. Further process of our research will transfer of transgenic embryos to recipients and production of hGCSF secreting cattle.

The purpose of this study is to improve production efficiency of vitrified-thawed transgenic bovine embryos. Transgenic bovine embryos were produced by injection of FIV-GFP lentiviral vector into perivitelline space of in vitro matured MІІ stage oocytes, and then in vitro fertilization. EGFP-expressing transgenic bovine blastocysts were cultured in serum-containing and serum–free medium. These blsatocysts were vitrified by pull and cut (PNC) container made with 0.25 cm plastic straw. Results indicate that total developmental rates of normal IVF embryo cultured in serumcontaining and–free medium into blastocyst were not significantly different (22.3 vs 21.5%) and those of GFPexpressing transgenic bovine embryo into blastocyst showed no significant difference between serum-containing (13.9%) and–free medium (13.1%). However, developmental rate of GFP transgenic embryo was significantly (P<0.05) lower than its of normal IVF embryos. In additional study, we vitrified GFP transgenic normal bovine blastocysts using PNC vitrification method. Survival rate of vitrified-thawed GFP transgenic blastocyst (23.1%) was significantly (P<0.05) lower than its of normal blastocysts (68.9%). Although, survival rate of vitrified-thawed GFP transgenic blastocyst was lower than its of normal blastocyst, our result may suggested that PNC vitrification method is feasible to cryopreserve transgenic embryos. Our next plan will be the production of GFP express transgenic bovine derived from vitrified-thawed embryos using PNC method.

This study was conducted to investigate optimal time of artificial insemination (AI) to Hanwoo female after natural estrus. AI was occurred 12 and 24 hours after natural estrus in both heifer and multiparous recipient then pregnancy and parturition rates were estimated. Results indicated that AI performed at 24 hours after natural estrus showed significant (p<0.05) higher pregnancy rate in both heifer and multiparous recipient groups with significantly (p<0.05) higher abortion rate. However, there are no significant differences of parturition rate, twin birth and sex ratio in both heifer and multiparous recipient groups. Therefore, our results may suggest that performance of AI at 24 hours after natural estrus promise higher pregnancy rate than AI at 12 hours after natural estrus in both heifer and multiparous recipient.

Pronuclear DNA microinjection has been the most universal method in transgenic animal production but its success rate of transgenesis in mammals are extremely low. To address this long-standing problem, we used retrovirus- and lentivirus-based vectors carrying the enhanced green fluorescent protein (EGFP) gene under the control of ubiquitously active cytomegalovirus (CMV) promoter to deliver transgenes to bovine embryos. The rate of transgenesis was evaluated by counting EGFP positive blastocysts after injection of concentrated virus stock into the perivitelline space of the bovine oocytes in metaphase II. Among two different types of lentivirus vectors derived from FIV (feline immunodeficiency virus) and HIV (human immunodeficiency virus), the former scored the higher gene transfer efficiency; almost 100% of the blastocysts developed from the oocytes infected with FIV-based vector were EGFP positive. As for the vectors derived Com HIV lentivirus, the transgenesis rate of the blastocysts was reduced to 39%.