Lepper Christoph, Fan Chen-Ming
Department of Embryology, Carnegie Institution of Washington, Baltimore, MD, USA.
Methods Mol Biol. 2012;798:297-308. doi: 10.1007/978-1-61779-343-1_17.
Gene inactivation has become the gold standard for determining gene function in the mouse. Many genes inactivated in the germ line cause early lethality that precludes phenotypic assessment at a later time point. Conditional gene inactivation using Cre recombinase expressed via a tissue specific promoter/enhancer allows phenotypic analyses of selected tissues, but lacks temporal control. Recent development of the tamoxifen-inducible Cre-ER (T2) offers both cell type-specific and temporal control of conditional gene inactivation. As an example, we describe the design and step-wise construction of a Cre-ER (T2) knock-in allele at the Pax7 locus using the recombineering method - Pax7 is selectively expressed in embryonic muscle progenitors and adult muscle stem cells. The resulting Pax7-Cre- ER (T2) (Pax7 (CE)) allele has been successfully applied to embryos and adults for tamoxifen-regulated myogenic lineage tracing and gene inactivation (Nature 460:627-631, 2009; Genesis 48:424-436, 2010).
基因失活已成为确定小鼠基因功能的金标准。许多在生殖系中失活的基因会导致早期致死，从而排除了在稍后时间点进行表型评估的可能性。使用通过组织特异性启动子/增强子表达的Cre重组酶进行条件性基因失活，可以对选定组织进行表型分析，但缺乏时间控制。最近开发的他莫昔芬诱导型Cre-ER（T2）实现了条件性基因失活的细胞类型特异性和时间控制。例如，我们描述了使用重组工程方法在Pax7基因座设计和逐步构建Cre-ER（T2）敲入等位基因的过程——Pax7在胚胎肌肉祖细胞和成年肌肉干细胞中选择性表达。所得的Pax7-Cre-ER（T2）（Pax7（CE））等位基因已成功应用于胚胎和成年小鼠，用于他莫昔芬调节性肌源性谱系追踪和基因失活（《自然》460:627 - 631，2009；《基因发生》48:424 - 436，2010）。
