Construction of Vector of Gene Targeting to Multiple Loci for Leghorn Chicken Based on BAC with Cre/lox P System

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 main material of gene targeting to multiple loci in Leghorn chicken 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 Not I/ Hind III and inserted into the upstream of the l ox 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 -Sce I 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 of gene-targeting vector to multiple loci is 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.