• Reproductive and Developmental Biology
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• 제31권3호
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• pp.153-159
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• 2007

본 연구에서: hG-CSF의 발현을 유도적으로 조절하기 위한 FIV-Tet-On lentivirus vector system을 구축하고자 하였다. hG-CSF는 호중성구 계열 세포의 증식과 분화, 생존에 영향을 미치는 물질로서, 이 유전자의 발현을 증가시키기 위하여 FIV-Tet-On vector 상의 hG-CSF나 $rtTA2^SM2$ 서열의 3' 위치에 WPRE 서열을 도입하였다. 구축된 각각의 vector는 293FT 세포에 일시적으로 transfection하여 virus를 생산하였으며, 이 virus를 일차 배양 세포인 CEF와 PFF에 감염시켰다. 각 세포에 전이되 hG-CSF의 발현 양상을 관찰하기 위하여 doxycycline을 첨가하거나 첨가하지 않은 배지에서 배양한 후 quantitative real-time PCR, Western blot과 ELISA를 이용하여 hG-CSF 유전자의 발현 정도를 비교 측정한 결과, CEF에서는 WPRE가 hG-CSF의 3' 위치에 도입된 경우에 발현량과 유도율이 가장 높은 것으로 나타났고, PFF에서는 rtTA 서열의 3'위치에 도입된 경우에 발현량과 유도율이 가장 큰 것으로 확인되었다. 이 FIV-Tet-On vector system은 형질 전환 동물의 생산이나 유전자 치료에서 문제시되는 외래 유전자의 지속적인 과다 발현에 의한 개체의 생리적인 부작용을 최소화하기 위한 해결 방법으로 제시될 수 있을 것이다.

• Reproductive and Developmental Biology
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• 제32권1호
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• pp.15-20
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• 2008

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%.

• 한국수정란이식학회지
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• 제28권3호
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• pp.169-175
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• 2013

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 MII 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 $100{\mu}g$ 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 $100{\mu}g$ of Gn-RH injected recipient which have normal corpus luteum with crown is feasible to produce transgenic cattle.

• 한국수정란이식학회지
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• 제28권3호
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• pp.185-191
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• 2013

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 MII 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 serum-containing and-free medium into blastocyst were not significantly different (22.3 vs 21.5%) and those of GFP-expressing 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.