Attempt on targeted integration of DNA in transgenic mice system

建立了携带CAG启动子+lox 71 +bsr基因的转基因小鼠品系，并通过杂交获得纯合雄性小鼠。从与纯合子雄性小鼠杂交的超排卵雌性小鼠收集受精卵，并注射Cre表达载体pCAGGS-Cre和靶向载体lox 66 +NLSlacZ+ MC 1 neo +pBluescript。当靶向载体插入小鼠基因组中的lox 71位点时，胚胎用X-gal染成蓝色，因为NLSLacZ基因位于CAG启动子下。将注射的卵移植到寄养母亲的输卵管中，并在12.5dpc收集胚胎，用X-gal染色。同时，从胎盘组织中提取基因组DNA，用PCR和Southern杂交检测目的载体的整合模式。在各种条件下显微注射约3500个卵。在486个携带转基因的胚胎中，5个胚胎显示出阳性X-gal染色，证明1%的靶向效率。这些X-gal阳性胚胎的DNA分析证实了靶向整合。然而，1%的效率对于有效生产转基因动物是不够的。我们将对该系统进行改进，提高靶向整合的效率，并通过基因捕获法获得了以lox 71位点为启动子资源的转基因小鼠品系。目前已建立了17个转基因小鼠品系，并对转基因小鼠的表达模式进行了分析。

A transgenic mice line carrying a single copy of the transgene that is CAG promoter + lox71+bsr gene was established, and the homozygous male mice were produced through intercrossing. Fertilized eggs were collected from superovalated females crossed with the homozygous male mice, and injected with the Cre expression vector, pCAGGS-Cre, and the targeting vector that is lox66+NLSlacZ+MC1neo+pBluescript. When the targeting vector was inserted into the lox71 site in the mouse genome, the embryos were stained into blue with X-gal because the NLSLacZ gene was placed under the CAG promoter. The injected eggs were transferred into the oviducts of foster mothers, and the embryos were collected at 12.5 dpc, stained with X-gal. At the same time, the genomic DNAs were extracted from the placentas, and the integration pattern of the targeting vector was examined with PCR and Southern blotting. About 3500 eggs were microinjected under various conditions. Out of 486 embryos carrying the transgene, 5 embryos showed positive X-gal staining, demonstrating 1% of targeting efficiency. The DNA analyses of these X-gal positive embryos confirmed the targeted integration. However, the efficiency of 1% is not enough for effective production of transgenic animal. We are going to improve the system and raise the efficiency of targeting integration.We have also produced transgenic mice lines carrying a lox71 site as promoter resources by gene trapping method. Until now, 17 mice lines have been established and analyzed the expression pattern of transgene.