Efficient sequential gene regulation via FLP- and Cre-recombinase using adenovirus vector in mammalian cells including mouse ES cells
Efficient sequential gene regulation via FLP- and Cre-recombinase using adenovirus vector in mammalian cells including mouse ES cells
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DOI：
10.1111/j.1348-0421.2006.tb03850.x
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发表时间：
2006-01-01
影响因子：
2.6
通讯作者：
Saito, Izumu
中科院分区：
文献类型：
--
作者：
Kondo, Saki;Takahashi, Yuzuka;Saito, Izumu
位点特异性重组酶因其调控作用严格、高效而被广泛应用于基因表达调控。然而，每个系统一次只能调节一个基因。在这里，我们展示了利用表达Cre和flp的重组腺病毒（rAd）在两种不同的监测细胞系中有效的“顺序”基因调控，分别调控一个基因（OFF-ON- off）和两个基因（ON-OFF和OFF-ON，独立）。一般来说，连续使用表达Cre和flp的rAd往往会引起显著的细胞毒性，但我们在这里描述了连续调节rAds的最佳剂量。我们还建立了一种去除饲养细胞后的rAd感染小鼠胚胎干细胞系的有效方法，表明该系统可用于在将胚胎干细胞导入胚细胞用于嵌合小鼠生产之前，从重组胚胎干细胞中去除frt -侧翼耐药基因盒。由于我们的序列基因调控系统提供了高效的目的基因调控和严格的off调控，因此它不仅对利用cDNA微阵列分析阐明新的基因功能，而且对发育和再生研究中的“基因开关”具有潜在的价值。
Site-specific recombinase is widely applied for the regulation of gene expression because its regulatory action is strict and efficient. However, each system can mediate regulation of only one gene at a time. Here, we demonstrate efficient "sequential" gene regulation using Cre- and FLP-expressing recombinant adenovirus (rAd) in two different monitor cell lines, for regulation of one gene (OFF-ON-OFF) and for two genes (ON-OFF and OFF-ON, independently). Generally, serial use of Cre- and FLP-expressing rAd tends to cause significant cytotoxicity, but we here described optimum dose of the rAds for serial regulation. We also established an efficient method of rAd infection to mouse ES cell lines after removing feeder cells, showing that this system is useful for removal of FRT-flanked drug-resistance gene cassette from recombinant ES cells prior to introduction of ES cells into blastocytes for chimeric mice production. Because our sequential gene-regulation system offers efficient purpose-gene regulation and strict OFF-regulation, it is potentially valuable for elucidating not only novel gene functions using cDNA microarray analysis but also for "gene switching" in development and regeneration research.