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Attempt on targeted integration of DNA in transgenic mice system

转基因小鼠系统中DNA定向整合的尝试

基本信息

批准号：
09558107
负责人：
ARAKI Kimi
金额：
$ 6.91万
依托单位：
Kumamoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1999
项目状态：
已结题

项目摘要

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.

建立了携带单拷贝转基因CAG启动子+lox71+BSR基因的转基因小鼠品系，通过杂交产生纯合子雄性小鼠。用Cre表达载体pCAGGS-Cre和靶向载体lox66+NLSlacZ+MC1neo+pBluescrip分别与纯合子雄性小鼠杂交，采集受精卵。将NLSLacZ基因插入小鼠基因组的lox71位点后，由于NLSLacZ基因位于CAG启动子下方，X-Gal将胚胎染成蓝色。将注射的卵子移植到寄养母亲的输卵管中，在12.5DPC时收集胚胎，X-Gal染色。同时，从胎盘组织中提取基因组DNA，并通过聚合酶链式反应和Southern印迹杂交检测靶向载体的整合模式。在不同条件下显微注射约3500个卵子。在携带转基因的486个胚胎中，有5个胚胎X-Gal染色呈阳性，显示了1%的靶向效率。对这些X-半乳糖阳性胚胎的DNA分析证实了靶向整合。然而，1%的效率不足以有效地生产转基因动物。我们将改进系统，提高靶向整合的效率。我们还通过基因捕获的方法获得了以lox71位点为启动子资源的转基因小鼠株系。到目前为止，已经建立了17个小鼠系，并对转基因的表达模式进行了分析。