Determination of the Optimal Concentration of Several Selective Drugs Useful for Generating Multi-Transgenic Porcine Embryonic Fibroblasts
Determination of the Optimal Concentration of Several Selective Drugs Useful for Generating Multi-Transgenic Porcine Embryonic Fibroblasts
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DOI：
10.1111/j.1439-0531.2011.01964.x
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发表时间：
2012-10-01
影响因子：
1.7
通讯作者：
Watanabe, S.
中科院分区：
文献类型：
--
作者：
Sato, M.;Ohtsuka, M.;Watanabe, S.
内容猪胚胎成纤维细胞（Porcine embryonic fibroblasts，PEFs）是猪体细胞核移植（somatic cell nuclear transfer，SCNT）的供体细胞。用外源DNA转染PEFs对于通过SCNT生产遗传修饰（GM;转基因或敲除）猪是必不可少的。在这种情况下，严格需要选择标记来选择和富集稳定转染的细胞。用于此目的的最常用的选择性药物是新霉素类似物（G418/遗传霉素）; neo已被广泛用作猪基因组操作中的选择性标记基因。然而，对其他选择药物的最佳浓度知之甚少。这通常会阻碍猪基因组的功能分析和个体转基因猪的发育。本研究探讨了选择性药物的最佳浓度，而不是新霉素，可用于选择转染的PEFs。将猪胚胎成纤维细胞在含有不同浓度药物的培养基中孵育长达10天，以确定PEFs致死的最佳药物浓度。发现下列浓度是用于PEF的最佳选择性浓度：G418/遗传霉素，400 μ g/ml;杀稻瘟菌素S，8 μ g/ml;潮霉素B，40 μ g/ml;嘌呤霉素，2 μ g/ml;和博莱霉素，800 μ g/ml。用携带选择标记的质粒重复转染导致产生多重耐药猪转染子。此外，发现这些标记是独立的。目前的信息将是有用的生产SCNT介导的转基因仔猪表达多种转基因。
Contents Porcine embryonic fibroblasts (PEFs) are widely used as donor cells for somatic cell nuclear transfer (SCNT) in pigs. Transfection of PEFs with exogenous DNA is essential for producing genetically modified (GM; transgenic or knockout) pigs via SCNT. In this case, selectable markers are strictly required selecting and enriching stably transfected cells. The most frequently used selective drug for this purpose is a neomycin analogue (G418/geneticin); neo has been widely used as a selectable marker gene in the genomic manipulation of pigs. However, little is known about optimal concentrations of other selection drugs. This often hampers functional analysis of the porcine genome and development of individual GM pigs. This study explores the optimal concentrations of selective drugs, other than neomycin, that can be used for the selection of transfected PEFs. Porcine embryonic fibroblasts were incubated in media containing different concentrations of drugs for up to 10 days, to determine the optimal drug concentrations fatal for PEFs. The following concentrations were found to be optimal selective concentrations for use with PEFs: G418/geneticin, 400 mu g/ml; blasticidin S, 8 mu g/ml; hygromycin B, 40 mu g/ml; puromycin, 2 mu g/ml; and zeocin, 800 mu g/ml. Repeated transfections with plasmids carrying selectable markers resulted in the generation of multidrug-resistant swine transfectants. Furthermore, these markers were found to be independent. The present information will be useful for the production of SCNT-mediated GM piglets that express multiple transgenes.