Attempt on targeted integration of DNA in transgenic mice system

建立了携带CAG启动子+ lox71+bsr基因单拷贝的转基因小鼠系，通过杂交获得纯合子雄性小鼠。收集过卵母鼠与纯合子雄鼠杂交的受精卵，注入Cre表达载体pCAGGS-Cre和靶向载体lox66+NLSlacZ+MC1neo+pBluescript。当靶向载体插入小鼠基因组的lox71位点时，由于NLSLacZ基因被置于CAG启动子下，胚胎被X-gal染成蓝色。将注射的卵子移植到养母的输卵管中，12.5 dpc收集胚胎，用X-gal染色。同时提取胎盘基因组dna，用PCR和Southern blotting检测靶向载体的整合模式。在不同条件下进行了约3500个卵的微注射。在携带转基因的486个胚胎中，有5个胚胎显示X-gal阳性，表明靶向效率为1%。这些X-gal阳性胚胎的DNA分析证实了靶向整合。然而，1%的效率不足以有效生产转基因动物。我们将完善制度，提高目标整合的效率。我们还利用基因诱捕方法获得了携带lox71位点作为启动子资源的转基因小鼠系。到目前为止，已经建立了17个小鼠系并分析了转基因的表达模式。

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.