Retention of the developmental pluripotency in medaka embryonic stem cells after gene transfer and long-term drug selection for gene targeting in fish
Retention of the developmental pluripotency in medaka embryonic stem cells after gene transfer and long-term drug selection for gene targeting in fish
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DOI：
10.1023/b:trag.0000017172.71391.fa
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发表时间：
2004-02-01
影响因子：
3
通讯作者：
Schartl, M
中科院分区：
文献类型：
--
作者：
Hong, YH;Chen, SL;Schartl, M
胚胎干（ES）细胞提供了一种引入随机或靶向遗传改变的独特工具，因为可以通过药物选择来筛选所需的但极其罕见的重组基因型。 ES细胞介导的转基因迄今为止仅限于小鼠。在青鳉鱼 (Oryzias latipes) 中，已经获得了几种 ES 细胞系。在这里，我们报告了青鳉 ES 细胞系 MES1 中基因转移和药物选择的优化条件，作为鱼类基因靶向的前奏。 MES1 细胞通过磷酸钙共沉淀 (5%)、商业试剂 Fugene (11%)、GeneJuice (21%) 和电穿孔 (>30%) 产生中等到高的转染效率。瞬时基因转移和 CAT 报告基因测定表明，几种增强子/启动子及其组合，包括 CMV、RSV 和 ST（与胸苷激酶启动子连接的 SV40 病毒早期基因增强子）是驱动 MES1 细胞中转基因表达的合适调节序列。我们表明，neo、hyg 或 pac 赋予对 G418、潮霉素或嘌呤霉素的阳性选择抗性，而 HSV-tk 产生对更昔洛韦的敏感性以进行阴性选择。广泛用于小鼠 ES 细胞基因靶向的正负选择程序被发现在 MES1 细胞中也有效。重要的是，我们证明MES1细胞在基因转移和长期药物选择后保留了发育多能性，因为它们能够在体外进行诱导分化，并在嵌合胚胎发生过程中形成各种组织和器官。
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.