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.

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 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 improving the non-surgical embryo transfer (ET) method. Transgenic bovine embryos were produced by injection of feline immunodeficiency virus enhanced green fluorescent protein (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 μg 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 μg of Gn-RH injected recipient which have normal corpus luteum with crown is feasible to produce transgenic cattle.

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.

“Hwanggeumchal”, a new covered waxy cultivar derived from the cross between “Chalssalbori” and “Milyang 65” with waxy endosperm and early maturing cultivars, respectively was developed at Honam Agricultural Research Institute (HARI), NICS, RDA, 2006. The origin of “Hwanggeumchal” is “Suwon 403” (SB942090-B-B-B-42-1). The initial cross was made in 1994 and the selected line showed a high yield and good quality characteristics under yield trial test in 2003. “Suwon 403” consistently performed well for three years (2004-2006) from the four locations of regional yield trial (RYT) in Korea and released as “Hwanggeumchal”. The characteristics of “Hwanggeumchal” were the following: rate I growth habit, green leaf and stem, compact spike and with long rough awns. The heading date was April 30 in upland and April 28 in paddy field, which was 1 and 4 days later than that with check cultivar, “Seodunchalbori”. The culm length was 81 cm which was 3 cm shorter than those with check cultivar. It showed spike length of 4.5 cm and 633 spikes per m2, 64 grains per spike, 27.6 g of 1,000-grain weight and 632 g of test weight. It showed stronger winter hardiness and higher resistance to barley yellow mosaic virus (BaYMV) than those with check cultivar. It had yellow aleurone and showed higher water absorption, expansion rate and total phenol content than those of check cultivar, but lower protein, β-glucan content and pearling rate. The average yield of the pearled grain in the RYT was 4.04 ton ha-1 in upland and 4.12 ton ha-1 in paddy field, which was 10% higher and 4% lower than that of the check cultivar, respectively. This cultivar would be suitable for the area above the daily minimum mean temperature of -8℃ in January in Korean peninsula.

“Hyemi”, a new covered cultivar derived from the crosses between “Suwon 298” and “Milyang 34/Albori//Dongbori” developed at the Honam Agricultural Research Institute (HARI), NICS, RDA in 2006. The origin of “Hyemi” is “Suwon 400” (SB951032-B-B-B-28). The initial cross was made in 1995 and the selected line showed a high yield and good quality characteristics under yield trial test in 2003. “Suwon 400” consistently performed well for three years (2004-2006) from the four locations of regional yield trial (RYT) in Korea and released as “Hyemi”. The characteristics of “Hyemi” were the following: rate Ⅳ growth habit, green leaf and stem, compact spike and with long rough awns. The heading date was April 29 in upland and April 23 in paddy field, which was similar and 2 days earlier than that with check cultivar, “Olbori”. The culm length was 81 cm which was 4 cm shorter than those with check cultivar. It showed spike length of 4.1 cm and 646 spikes per m2, 52 grains per spike, 33.4 g of 1,000-grain weight and 672 g of test weight. It showed similar winter hardiness and higher resistance to barley yellow mosaic virus (BaYMV) than those with check cultivar. It showed higher protein content, whiteness and total phenol content than those of the check cultivar, but lower β-glucan content and pearling rate. The average yield of the pearled grain in the RYT was 3.95 ton ha-1 in upland and 4.38 ton ha-1 in paddy field, which was 13% and 16% higher than that of the check cultivar, respectively. This cultivar would be suitable for the area above the daily minimum mean temperature of -8℃ in January in Korean peninsula.