Development of In Vitro Porcine Oocytes Following Intracytoplasmic Injection of Sperm-Mediated GFP Gene

Kim, J.H.; Seong, H.H.; Park, J.K.; Im, S.K.; Kim, S.W.; Lee, Y.K.; Lee, P.Y.; Choi, Y.J.; Kim, Y.K.; Kim, J.H.; Chang, W.K.

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자료유형: 학술대회자료

저자정보: Kim, J.H. (Division of Animal Biotechnology, National Livestock Institute and Gyeongsang National University) Seong, H.H. (Division of Animal Biotechnology, National Livestock Institute and Gyeongsang National University) Park, J.K. (Division of Animal Biotechnology, National Livestock Institute and Gyeongsang National University) Im, S.K. (Division of Animal Biotechnology, National Livestock Institute and Gyeongsang National University) Kim, S.W. (Division of Animal Biotechnology, National Livestock Institute and Gyeongsang National University) Lee, Y.K. (Division of Animal Biotechnology, National Livestock Institute and Gyeongsang National University) Lee, P.Y. (Division of Animal Biotechnology, National Livestock Institute and Gyeongsang National University) Choi, Y.J. (Division of Animal Biotechnology, National Livestock Institute and Gyeongsang National University) Kim, Y.K. (Division of Animal Biotechnology, National Livestock Institute and Gyeongsang National University) Kim, J.H. (Division of Animal Biotechnology, National Livestock Institute and Gyeongsang National University) Chang, W.K.

저널정보: 한국동물번식학회 한국수정란이식학회 심포지움 한국수정란이식학회 2002년도 국제심포지엄

발행연도: 2002.1

수록면: 69 - 69 (1page)

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Transgenic animals production tools have been valuable for research and purpose. The current methods of gene transfer, microinjection and nuclear transfer, which are widely used in transgenic animal production, but all most methods has only had limited success in production of larger species. Here, we report the possibility of a sperm-mediated gene transfer method in porcine embryos. Oocytes were collected from ovaries harvested at a local slaughterhouse were matured in 500${mu}ell$ drops of TCM-199 under mineral oil at 38.5$^{\circ}C$ in a humidified atmosphere of 5%CO2 in air. After 42-43h of in vitro maturation oocytes were denuded. for sperm injection into the cytoplasm of the porcine oocytes, sperm suspension in NIM medium are subjected extraction with TritonX-100 before mixing with a green fluorescent gene (GFP). Sperm with Tritonx-100 were prepared by adding TritonX-100 to a final volume of 0.05% in the sperm suspension and mixing by trituration for 60s before two wishes in NIM medium at 2$^{\circ}C$. A(ter wishing, sperm were mixed with TritonX-100 at $25^{\circ}C$ followed by washes at 2$^{\circ}C$. Sperm were resuspended in ice cold NIM to a final volume of 400${mu}ell$ and 2-20ng/${mu}ell$ DNA were triturated on ice for 60s. All microinjection was performed in HEPES-buffered CZB medium at room temperature within 2h. After culture in NCSU-23 for 72h, percent of porcine embryos transfected GFP gene are 20.7%(6/29) in 20ng/${mu}ell$ sperm-DNA mixed group and other groups were 3.7 %(2/54)and 4.7%(3/67). These data suggests that sperm-mediated gene transfer method should be used to the production tool of transgenic pig efficiently.

#sperm #GFP #microinjection #transgenic #porcine

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