Dietrich P, Dragatsis I, Xuan S, Zeitlin S, Efstratiadis A
Department of Genetics and Development, Columbia University, Russ Berrie Medical Sciences Pavilion, New York, New York 10032, USA.
Mamm Genome. 2000 Mar;11(3):196-205. doi: 10.1007/s003350010037.
To explore the potential of a simple and rapid approach for ubiquitous conditional gene disruption, we have generated Cre-producer mouse transgenic lines (Hs-cre1, 6 and 7) expressing a recombinase transgene (cre) from a heat shock gene promoter and tested their performance in Cre-mediated excision of target DNA in crosses with Cre-responder strains carrying loxP-modified alleles of the genes encoding the Huntington's disease gene homolog (Hdh), the epidermal growth factor receptor (Egfr), and the type 1 insulin-like growth factor receptor (Igf1r). Analyses of progeny possessing various transgene/reporter combinations showed that cre expression can occur without heat shock in early embryos, but this constitutive transcription is stochastic and transgene dependent. Thus, Hs-cre1 behaves predominantly as a "deleter" strain, since the majority of progeny (approximately 70-85%) exhibit complete recombination, regardless of reporter locus. Lines Hs-cre6 and Hs-cre7, however, function successfully as "mosaicking" strains because, in addition to two extreme classes of progeny with 0% or 100% recombination, they generate an intermediate class of mosaics exhibiting various degrees of partial DNA excision. Notably, the frequency of offspring in each class varies between reporters, but mosaic embryos are consistently obtained in adequate numbers (approximately 30-60%). The Hs-cre6 transgene is also inducible and can be used to introduce mosaicism into adult tissues at preselected developmental times by heat shock treatment of mice with 0% recombination in tail DNA. By bypassing the lethality resulting from some gene knockouts, mosaic embryos and mice make particular mutational analyses possible and are also very useful for the identification of cell lineage-specific gene functions.
为了探索一种简单快速的方法实现普遍条件性基因敲除的潜力，我们构建了表达来自热休克基因启动子的重组酶转基因（cre）的Cre产生小鼠转基因品系（Hs-cre1、6和7），并在与携带亨廷顿舞蹈病基因同源物（Hdh）、表皮生长因子受体（Egfr）和1型胰岛素样生长因子受体（Igf1r）基因的loxP修饰等位基因的Cre应答品系杂交中，测试了它们在Cre介导的靶DNA切除中的性能。对具有各种转基因/报告基因组合的后代的分析表明，cre表达可以在早期胚胎中无热休克的情况下发生，但这种组成型转录是随机的且依赖于转基因。因此，Hs-cre1主要表现为“敲除”品系，因为大多数后代（约70-85%）表现出完全重组，而与报告基因位点无关。然而，Hs-cre6和Hs-cre7品系成功地作为“镶嵌”品系发挥作用，因为除了两类极端的后代，一类重组率为0%，另一类为100%，它们还产生了一类中间的镶嵌体，表现出不同程度的部分DNA切除。值得注意的是，每个类别的后代频率在不同报告基因之间有所不同，但始终能获得足够数量（约30-60%）的镶嵌胚胎。Hs-cre6转基因也是可诱导的，通过对尾部DNA重组率为0%的小鼠进行热休克处理，可用于在预选的发育时间将镶嵌性引入成年组织。通过绕过一些基因敲除导致的致死性，镶嵌胚胎和小鼠使得特定的突变分析成为可能，并且对于鉴定细胞谱系特异性基因功能也非常有用。
