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学科主题: Biochemical Research Methods; Biochemistry & Molecular Biology; Biotechnology & Applied Microbiology
题名: Retention of the developmental pluripotency in medaka embryonic stem cells after gene transfer and long-term drug selection for gene targeting in fish
作者: Yunhan Hong ; Songlin Chen ; Jianfang Gui & Manfred Schartl
通讯作者: Hong, YH, Natl Univ Singapore, Dept Biol Sci, 10 Kent Ridge Crescent, Singapore 119260, Singapore
关键词: cell transfection ; drug selection ; embryonic stem cells ; gene transfer ; Oryzias latipes
刊名: TRANSGENIC RESEARCH
发表日期: 2004
卷: 13, 期:1, 页:41-50
收录类别: SCI
部门归属: Natl Univ Singapore, Dept Biol Sci, Singapore 119260, Singapore; Univ Wurzburg, Bioctr, D-97074 Wurzburg, Germany; Chinese Acad Sci, Inst Hydrobiol, State Key Lab Ecol & Biotechnol, Wuhan 430072, Peoples R China; Chinese Acad Fisheries Sci, Yellow Sea Fisheries Res Inst, Qingdao 266071, Peoples R China
摘要: Embryonic stem (ES) cells provide a unique tool for introducing random or targeted genetic alterations, because it is possible that the desired, but extremely rare recombinant genotypes can be screened by drug selection. ES cell-mediated transgenesis has so far been limited to the mouse. In the fish medaka (Oryzias latipes) several ES cell lines have been made available. Here we report the optimized conditions for gene transfer and drug selection in the medaka ES cell line MES1 as a prelude for gene targeting in fish. MES1 cells gave rise to a moderate to high transfection efficiency by the calcium phosphate co-precipitation (5%), commercial reagents Fugene (11%), GeneJuice (21%) and electroporation (>30%). Transient gene transfer and CAT reporter assay revealed that several enhancers/promoters and their combinations including CMV, RSV and ST (the SV40 virus early gene enhancer linked to the thymidine kinase promoter) were suitable regulatory sequences to drive transgene expression in the MES1 cells. We show that neo, hyg or pac conferred resistance to G418, hygromycin or puromycin for positive selection, while the HSV-tk generated sensitivity to ganciclovir for negative selection. The positive-negative selection procedure that is widely used for gene targeting in mouse ES cells was found to be effective also in MES1 cells. Importantly, we demonstrate that MES1 cells after gene transfer and long-term drug selection retained the developmental pluripotency, as they were able to undergo induced differentiation in vitro and to contribute to various tissues and organs during chimeric embryogenesis.
WOS记录号: WOS:000189176400006
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内容类型: 期刊论文
URI标识: http://ir.ihb.ac.cn/handle/152342/9548
Appears in Collections:中科院水生所知识产出(2009年前)_期刊论文

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Yunhan Hong; Songlin Chen; Jianfang Gui & Manfred Schartl.Retention of the developmental pluripotency in medaka embryonic stem cells after gene transfer and long-term drug selection for gene targeting in fish,TRANSGENIC RESEARCH,2004,13(1):41-50
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