介绍一种鉴定胚胎干细胞基因打靶快速、经济的筛选新方法(英文)

A RAPID AND COST-EFFECTIVE SCREENING METHOD FOR IDENTIFICATION OF TARGETED EMBRYONIC STEM CELLS*

小鼠基因剔除动物模型越来越广泛地应用于哺乳动物基因功能与疾病的研究。然而每当胚胎于细胞同源重组的效率过低时,鉴定与分离带有定位变异的阳性克隆就会既费力又昂贵。本工作以类固醇受体共激活子基因为例,研究出一种快速鉴定阳性克隆的新方法。在构造重组载体时,将一段编码半乳糖苷酶的DNA序列整合到共激活子基因的蛋白起始码后面。于是,在干细胞内同源重组发生以后,半乳糖苷酶的表达就会受控于内源性共激活子基因的启动子。在载体与半乳糖苷酶DNA随机整合的大多数非特异克隆中,因为缺少启动子或由于不正确的氨基酸编码连接,导致合成牛乳糖苷酶的可能性较小。因此,在半乳糖苷酶染色阳性的克隆中,具有特异突变的阳性克隆可以富集30倍以上。从牛乳糖苷酶的阳性克隆中,再用Southern Blot方法进一步确认带有基因剔除的阳性克隆就大大减少了工作量。因为半乳糖苷酶的细胞化学染色法简便而可靠,所以在重组效率低时,可以用这种方法在短期内筛选大量克隆。但是应该注意,应用该方法的前提条件是所研究的基因必须在胚胎干细胞内表达。这种方法更为重要的意义在于当带有基因剔除的胚胎干细胞发育成小鼠后,牛乳糖苷酶的组化染色法可以轻而易举地用来揭示所研究基因在动物不同组织与细胞中的表达水平。

Generation and characterization of knockout mice from targeted emhryonic stem (ES) cells have become one of the most powerful approaches to study gene function in vivo. However, experiments to identify targeted ES clones can be both labor-intensive and expensive when the targeting efficiency is low. Using the steroid receptor coactivator-3 (SRC-3) gene as a model, we have developed a rapid and cost-effective method for identification of targeted ES clones. Specifically, a promoterless LacZ sequence was fused to the 5'-coding sequence of SRC-3 in the targeting vector. After homologous recombination in ES cells, LacZ expression was regulated by the endogenous gene promoter. In cases of random insertions the β-galactosidase would more likely not be produced due to lacking promoter or missing amino acid-reading frame. Accordingly, targeted ES clones were enriched more than 30 times in the population of hlue clones positive to X-gal staining. .Therefore, targeted ES clones can be selected quickly and economically by X-gal staining in large scale followed by .Southern blot analysis on small number of blue clones if the gene of interest is expressed in ES cells. This strategey is particularly useful when the targeting efficiency is low and a reporter such as LacZ or GFP for mouse gene expression is desired.