Retention of the developmental pluripotency in medaka embryonic stem cells after gene transfer and long-term drug selection for gene targeting in fish
文献类型：期刊论文
| 作者 | Hong, YH; Chen, SL; Gui, JF; Schartl, M |
| 刊名 | TRANSGENIC RESEARCH
 |
| 出版日期 | 2004-02-01 |
| 卷号 | 13期号:1页码:41-50 |
| ISSN号 | 0962-8819 |
| 关键词 | cell transfection drug selection embryonic stem cells gene transfer Oryzias latipes |
| 通讯作者 | Hong, YH, Natl Univ Singapore, Dept Biol Sci, 10 Kent Ridge Crescent, Singapore 119260, Singapore |
| 中文摘要 | 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. |
| 英文摘要 | 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. |
| 学科主题 | Biochemical Research Methods; Biochemistry & Molecular Biology; Biotechnology & Applied Microbiology |
| WOS标题词 | Science & Technology ; Life Sciences & Biomedicine |
| 类目[WOS] | Biochemical Research Methods ; Biochemistry & Molecular Biology ; Biotechnology & Applied Microbiology |
| 研究领域[WOS] | Biochemistry & Molecular Biology ; Biotechnology & Applied Microbiology |
| 关键词[WOS] | GERM-LINE CHIMERAS ; ORYZIAS-LATIPES ; TRANSIENT EXPRESSION ; FUNCTIONAL-ANALYSIS ; MOUSE EMBRYOS ; ZEBRAFISH ; ESTABLISHMENT ; TRANSPLANTS ; DERIVATION ; ELEMENTS |
| 收录类别 | SCI |
| 语种 | 英语 |
| WOS记录号 | WOS:000189176400006 |
| 公开日期 | 2010-10-13 |
| 源URL | [http://ir.ihb.ac.cn/handle/152342/9548]  |
| 专题 | 水生生物研究所_中科院水生所知识产出（2009年前）_期刊论文 |
| 作者单位 | 1.Natl Univ Singapore, Dept Biol Sci, Singapore 119260, Singapore 2.Univ Wurzburg, Bioctr, D-97074 Wurzburg, Germany 3.Chinese Acad Sci, Inst Hydrobiol, State Key Lab Ecol & Biotechnol, Wuhan 430072, Peoples R China 4.Chinese Acad Fisheries Sci, Yellow Sea Fisheries Res Inst, Qingdao 266071, Peoples R China |
| 推荐引用方式 GB/T 7714 | Hong, YH,Chen, SL,Gui, JF,et al. Retention of the developmental pluripotency in medaka embryonic stem cells after gene transfer and long-term drug selection for gene targeting in fish[J]. TRANSGENIC RESEARCH,2004,13(1):41-50. |
| APA | Hong, YH,Chen, SL,Gui, JF,&Schartl, M.(2004).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,13(1),41-50. |
| MLA | Hong, YH,et al."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 13.1(2004):41-50. |
入库方式： OAI收割
来源：水生生物研究所
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