Camus A, Kress C, Babinet C, Barra J
Département d'Immunologie, URA CNRS 1960, Institut Pasteur, Paris, France.
Mol Reprod Dev. 1996 Nov;45(3):255-63. doi: 10.1002/(SICI)1098-2795(199611)45:3<255::AID-MRD1>3.0.CO;2-R.
A new gene trap vector has been designed, comprised of a fusion between the Sh ble gene, which confers resistance to the antibiotic phleomycin, and the lacZ gene (phleal fusion gene). A synthetic splice acceptor, made of the yeast branchpoint followed by a pyrimidin-rich sequence of 27 nucleotides, is included at the 5' extremity. The linearized gene trap vector was introduced into mouse embryonic stem cells (ES cells), and 40 phleomycin resistant (phleo') cell lines possessing a single copy of the insert were selected. They were stable in expressing the lacZ gene. Reporter gene expression was studied at days 8.5 and 10.5 of embryonic development in chimeric embryos obtained after injection of phleo' ES clones into 8-cell stage embryos. Out of 20 phleal lines examined, 14 exhibited beta-galactosidase expression at day 10.5. Use of the phleal fusion gene trap vector to select genes expressed in ES cells, therefore, is compatible with the isolation of genes expressed at midgestation. However, and most intriguingly, 10 out of these 14 cell lines (71%) displayed reporter gene expression mostly in heart and liver. Two of them exhibited, in addition, expression in central nervous system (CNS) or in CNS and limb buds, respectively. Germline chimeras were subsequently obtained and 15 mouse lines have been established. Intercrosses of animals heterozygous for the insertion revealed a mutant phenotype in several lines.
一种新的基因捕获载体已被设计出来，它由赋予对博来霉素抗生素抗性的Sh ble基因与lacZ基因（phleal融合基因）融合而成。在5'末端包含一个由酵母分支点后接27个核苷酸的富含嘧啶序列组成的合成剪接受体。将线性化的基因捕获载体导入小鼠胚胎干细胞（ES细胞），并筛选出40个具有单个插入拷贝的博来霉素抗性（phleo'）细胞系。它们稳定表达lacZ基因。在将phleo' ES克隆注射到8细胞期胚胎后获得的嵌合胚胎的胚胎发育第8.5天和10.5天研究报告基因的表达。在所检测的20个phleal系中，有14个在第10.5天表现出β-半乳糖苷酶表达。因此，使用phleal融合基因捕获载体筛选ES细胞中表达的基因与分离妊娠中期表达的基因是相容的。然而，最有趣的是，这14个细胞系中有10个（71%）的报告基因表达主要在心脏和肝脏。其中两个分别还在中枢神经系统（CNS）或CNS和肢芽中表达。随后获得了种系嵌合体并建立了15个小鼠品系。对插入杂合的动物进行杂交，在几个品系中发现了突变表型。
