Hernandez Diana, Chandan Pritpal, Janmohamed Azara, Phillips Ian R, Shephard Elizabeth A
Department of Biochemistry and Molecular Biology, University College London, London, UK.
School of Biological Sciences, Queen Mary, University of London, London, UK.
Methods Mol Biol. 2006;320:307-319. doi: 10.1385/1-59259-998-2:307.
The steps required to delete genes from the mouse genome are illustrated by showing how a cluster of three flavin-containing monooxygenase (Fmo) genes (Fmo1, Fmo2, and Fmo4) were deleted from mouse chromosome 1. Such large deletions are accomplished using loxP/Cre recombinase technology. Genomic clones corresponding to the genes to be deleted are first isolated, and then appropriate genomic fragments are cloned into vectors containing a loxP site. This produces targeting vectors, which are electroporated into mouse embryonic stem (ES) cells to allow a homologous recombination event to take place between the mouse genomic fragment, present within the vector, and the homologous sequences in the ES cell genome. Screening of ES cells for recombinants in which loxP sites have been inserted on either side of the gene cluster to be deleted is described. Recombination by Cre recombinase to produce ES cell lines carrying the deletion on chromosome 1 is also described.
通过展示如何从小鼠1号染色体上删除一组三个含黄素单加氧酶（Fmo）基因（Fmo1、Fmo2和Fmo4），来说明从小鼠基因组中删除基因所需的步骤。这种大的缺失是使用loxP/Cre重组酶技术完成的。首先分离与要删除的基因相对应的基因组克隆，然后将适当的基因组片段克隆到含有loxP位点的载体中。这产生了靶向载体，将其电穿孔导入小鼠胚胎干细胞（ES细胞），以使载体中存在的小鼠基因组片段与ES细胞基因组中的同源序列之间发生同源重组事件。描述了对ES细胞进行筛选，以寻找loxP位点已插入到要删除的基因簇两侧的重组体。还描述了通过Cre重组酶进行重组以产生在1号染色体上携带缺失的ES细胞系。
