Liao Lan, Kuang Shao Qing, Yuan Yu Hui, Xu Jian Ming
Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030, USA.
Shi Yan Sheng Wu Xue Bao. 2002 Sep;35(3):163-8.
Generation and characterization of knockout mice from targeted embryonic 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 beta-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 blue 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 strategy is particularly useful when the targeting efficiency is low and a reporter such as LacZ or GFP for mouse gene expression is desired.
从靶向胚胎干细胞（ES细胞）生成基因敲除小鼠并对其进行表征，已成为研究体内基因功能最强大的方法之一。然而，当靶向效率较低时，鉴定靶向ES细胞克隆的实验既费力又昂贵。以类固醇受体辅激活因子3（SRC-3）基因为模型，我们开发了一种快速且经济高效的方法来鉴定靶向ES细胞克隆。具体而言，在靶向载体中，将无启动子的LacZ序列与SRC-3的5'编码序列融合。在ES细胞中进行同源重组后，LacZ的表达由内源基因启动子调控。在随机插入的情况下，由于缺乏启动子或缺失氨基酸阅读框，β-半乳糖苷酶很可能不会产生。因此，在对X-gal染色呈阳性的蓝色克隆群体中，靶向ES细胞克隆的富集倍数超过30倍。所以，如果感兴趣的基因在ES细胞中表达，通过大规模的X-gal染色，然后对少数蓝色克隆进行Southern印迹分析，可以快速且经济地筛选出靶向ES细胞克隆。当靶向效率较低且需要如LacZ或GFP这样的小鼠基因表达报告基因时，这种策略特别有用。
