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題 名 | 顯微注射反轉錄病毒轉染早期小鼠胚以產生基因轉殖小鼠=Generation of Transgenic Mice by Subzonal Infection Methods after Microinjection of the Retroviral Particles Into Perivitelline Space of Mouse Early Embryos |
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作 者 | 杜清富; 黃麗華; 鄭登貴; | 書刊名 | 中國畜牧學會會誌 |
卷 期 | 28:3 1999.09[民88.09] |
頁 次 | 頁381-394 |
分類號 | 437.21 |
關鍵詞 | 小鼠胚; 反轉錄病毒; 透明帶下轉染法; 基因轉殖小鼠; Mouse embryos; Retrovirus; Subzonal infection; Transgenic mice; |
語 文 | 中文(Chinese) |
中文摘要 | 本研究應用顯微注射法,將反轉錄病毒顆粒,直接注入透明帶下卵黃膜間隙,進 行1-或2-細胞期小鼠胚之轉染,冀能產生基因轉殖小鼠,並提高病毒轉染分溝細胞之機率, 且增加外源基因在生殖細胞系被傳承之效果。 試驗一,比較助細胞條件化培養液(helper cell conditioned medium)中添加0、4、8、 16、與20μg/ml之polybrene(PB),對小鼠胚存活率之影響。注射之小鼠胚,經24h之轉染 後,結果分別有0.0%(n=86)、2.5%(n=129)、54.7%(n=100)、100.0%(n=43)死亡。將試驗中存 活之小鼠胚移置後,仍未獲得仔小鼠之出生。 試驗二,第一部份將助細胞條件化培養液,舉行離心濃縮病毒顆粒及去除其中可能具毒 性之代謝物質。共516個2-細胞期小鼠胚,被注入病毒顆粒懸浮液及轉染16-24h後,在移 置後產下2隻基因轉殖小鼠。第二部份,縮短濃縮病毒顆粒轉染時間至2~3h,不論對2-或1- 細胞期小鼠胚,均可提高其受胚母小鼠懷孕率。且在1-細胞期轉染之小鼠胚中,共擭得3隻 基因轉殖小鼠,其中兩隻活的公基因轉殖小鼠之F1後裔為基因轉殖者,僅7.4%與2.7%。綜 合上述:以濃縮之病毒顆粒轉染2-或1-細胞期小鼠胚,均可獲得基因轉殖小鼠;小鼠胚對PB 之毒性敏感,添加濃度應低於8μg/ml;惟此法並未提高轉殖效率與外源基因進入生殖細胞之 機率。 |
英文摘要 | The purposes of this study were to establish an easy way for the production of transgenic mice by using retrovirus-contained porcine growth hormone cDNA (PSN) to transfected 1- and/or 2-cell mouse embryos via the subzonal infection (SUZIN), and to increase the rate of transgene germ line transmitted. In the initial experiment, the toxicity of Polybrene (PB), which is supposed to have the advantage of improving the transfection efficiency of retroviral particles (RVP) to mouse embryos was studied. Various concentrations (0, 4, 8, 16 and 20 g/ml) of PB-contained medium, conditioned by PSN helper cells, was injected directly into the perivitelline space of mouse embryos and the appearance of embryos were evaluated after they had been further cultivated at a condition of 37℃ and 5% CO2 in air for 24 h in vitro. Experimental result revealed that 0, 2.5, 54.7, 91.2 and 100% of embryos were lysed after they had been injected with 0, 4, 8, 16 and 20 g/ml of PB, respectively. Therefore, two concentration of PB (0 and 4 g/ml) were used in subsequent studies. In the later experiments, concentrated RVP with or without PB were used for generating transgenic mice by SUZIN. To achieve the subzonal infection, an approximate amount of 200 p1 RVP solution was injected into the perivitelline space of each embryo. All embryos after RVP injection were implanted into fallopian tubes of foster dams and integration of PSN transgene within the new born mice were analyzed by both PCR and Southern blot analysis. Experimental results appeared that the transfer of 703 embryos, SUZIN at 2-cell stage, into 38 foster dams resulted in 10 of them becoming pregnant and two out of the 71 new borns confirmed to be transgenic. Similarly, transfer of 329 embryos, SUZIN at 1-cell stage, into 14 foster dams also resulted in 11 of them pregnant and 3 of the 42 new born mice were found to be transgenic. Of the 5 transgenic mice, 3 died soon after birth. Two were now sexually matured and confirmed to be fertile. The transgene was found to be germline-transmitted to between 2.7 and 7.4%. The conclusion reached in the present studies was that subzonal infection of retrovirus particles being carried with the transgene did provide an alternative approach for the production of the transgenic animals. |
本系統中英文摘要資訊取自各篇刊載內容。