中文摘要：

以奶牛为研究对象,以其重复的rRNA基因间的间隔序列为靶位点,基于BAC重组酶系统构建多位点基因打靶载体,为建立体内多位点基因打靶技术获得关键材料.首先构建BAC-TDN筛选载体,然后构建pYLVS-GD表达载体.将BAC-TDN筛选载体和pYLVS-GD表达载体共转化至大肠杆菌NS3529中,通过Cre重组酶的作用形成BAC-TDN-VS-GD质粒,采用归位内切酶I-SceI切除pYLVS载体骨架,构建奶牛多位点基因打靶载体BAC-TDN-GD,利用接头LS使之环化.BAC-TDN-GD打靶载体与pYLSV质粒组成了通用型奶牛多位点打靶载体系统.以重复序列为靶位点的多位点基因打靶技术,将部分解决目前存在的打靶效率低、安全性差等问题.

英文摘要：

The Bos taurus was the study object in this research.The vectors of multiple locus gene targeting on the repetitive internal transcribed spacers between rRNA genes in Bos taurus were constructed based on the BAC with Cre/loxP system.The key material of multiple locus gene targeting in vivo would be got.The plasmid of pYLSV-TDN with TK,HRDS2 and NEO genes has been constructed firstly.The TK-HRDS2-NEO DNA fragment digested from the plasmid of pYLSV-TDN by Not I/Hind III was inserted into the upstream of the l...