基于BAC重组酶系统构建莱航鸡多位点基因打靶载体的研究

Construction of Vector of Multiple Loci Gene Targeting in Leghorn chicken Based on BAC with Cre/lox P System

摘要:

以莱航鸡重复的rDNA 基因间的间隔序列为靶位点，利用BAC重组酶系统构建含人干扰素基因的多位点基因打靶载体，为建立莱航鸡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-GID表达载体。将BAC-TDN筛选载体和pYLVS-GID表达载体共转化至大肠杆菌NS3529中，通过其Cre重组酶的作用形成BAC-TDN-VS-GID质粒，采用归位内切酶I-SceⅠ切除pYLVS质粒骨架，利用接头LS使之环化，构建成莱航鸡大容量多位点基因打靶载体BAC-TDN-GID。每次克隆均经酶切或PCR、测序等鉴定DNA片段的插入及插入方向。以该载体为材料的多位点基因打靶技术将提高基因定点整合效率，解决外源基因不能稳定表达、安全性等部分问题，突破了DNA重复序列不能作为外源基因整合靶位点的禁区。

Abstract:

Based on the sequence of BAC (Bacterial Artificial Chromosome) along with the Cre/lox P system, the gene-targeting vectors to multiple loci of the repetitive internal transcribed spacers between rDNA genes in Leghorn chicken were constructed. The key material of multiple loci gene targeting in vivo would be obtained. First, the plasmid of pYLSV-TDN with TK, HRDS2, and Neo genes was constructed. The TK-HRDS2-Neo DNA fragment obtained from the plasmid of pYLSV-TDN was digested by NotⅠ/HindⅢ and inserted into the upstream of the lox P site of BAC plasmid for obtaining the selective vector of BAC-TDN. The expression vector of pYLVS-GID with EGFP, hIFN genes, and HRDS1 was then obtained. The plasmid of BAC-TDN-VS-GID was obtained by cotransformation of the selective vector of BAC-TDN and the expression vector of pYLVS-GID to E. coli NS3529 through the action of Cre/lox P system. The gene_targeting vector of BAC-TDN-GID to multiple loci of the ITS region in Leghorn chicken was obtained by cleaving the sequence of pYLVS with the homing endonuclease of Ⅰ-SceⅠ and ligating with the linker of LS. The insertion and the insert direction of DNA fragments were identified by restriction digestion or PCR and sequencing in each clone. The significance of the technique ofgene-targeting vector to multiple loci are shown as follows. First, the targeting loci were increased to 100～300. Second, the problems of unstable expression of inserted genes were partially solved. Third, the need for safety against toxicity integration was resolved. Fourth, the forbidden zone of gene integrating on the repetitive DNA sequences was broken through.