Friedrich G, Soriano P
Howard Hughes Medical Institute, Baylor College of Medicine, Houston, Texas 77030.
Genes Dev. 1991 Sep;5(9):1513-23. doi: 10.1101/gad.5.9.1513.
A general strategy for selecting insertion mutations in mice has been devised. Constructs lacking a promoter and including a beta-galactosidase gene, or a reporter gene encoding a protein with both beta-galactosidase and neomycin phosphotransferase activity, were designed so that activation of the reporter gene depends on its insertion within an active transcription unit. Such insertion events create a mutation in the tagged gene and allow its expression to be followed by beta-galactosidase activity. Introduction of promoter trap constructs into embryonic stem (ES) cells by electroporation or retroviral infection has led to the derivation of transgenic lines that show a variety of beta-galactosidase expression patterns. Intercrossing of heterozygotes from 24 strains that express beta-galactosidase identified 9 strains in which homozygosity leads to an embryonic lethality. Because no overt phenotype was detected in the remaining strains, these results suggest that a substantial proportion of mammalian genes identified by this approach are not essential for development.
已经设计出一种在小鼠中选择插入突变的通用策略。构建了缺乏启动子并包含β-半乳糖苷酶基因或编码具有β-半乳糖苷酶和新霉素磷酸转移酶活性的蛋白质的报告基因的载体，设计使得报告基因的激活取决于其插入到一个活跃的转录单元内。这种插入事件在标记基因中产生突变，并允许通过β-半乳糖苷酶活性追踪其表达。通过电穿孔或逆转录病毒感染将启动子捕获构建体导入胚胎干细胞（ES细胞），已导致产生显示出各种β-半乳糖苷酶表达模式的转基因品系。对来自24个表达β-半乳糖苷酶的品系的杂合子进行杂交，鉴定出9个品系，其中纯合性导致胚胎致死。由于在其余品系中未检测到明显的表型，这些结果表明通过这种方法鉴定的相当一部分哺乳动物基因对于发育并非必不可少。
