Screen Friendly Gene Locus By Cre/lox Recombination System

In order to make the whole experimental operation process that foreign genes are introduced into animal bodies and express in high-level be more efficiently and more easily, the Cre/lox system was used to select in the chromosome of animal cells.LoxP and loxSll sites were synthesized, and cloned into the vector pEGFP-1, a new vector plox. Then some necessary elements such as GFP gene, neo gene were inserted between the loxP site and the loxS 11 site and obtained a new gene targeting vector pIoxGFP. On the other hand, the chicken v -IFN cDNA was cloned into the vector plox between the loxP site and the lox511 site and got another gene targeting vector ploxIFN. The plasmid ploxGFP DNA was transfected into bovine fetal fibroblast cells, then on the selected G418 medium, the green fluorescent cell clones were gained. After multiple, plasmid ploxIFN and pBS185 DNA were co-transfected into the green fluorescent cell clones, then filtrated the non-fluorescent cell clones.Two non-fluorescent cell clones, the green fluorescent cell clones and the cells which were co-transfected by the plasmid ploxIFN and pBSISS DNA were checked up by PCR. The primers were in the sequence of vector pEGFP-1 outside the lox sites, and the whole sequence of the vector were transfected into the fibroblast cells. After the examination of PCR, one non-fluorescent cell clone had the same result as the plasmid ploxIFN-a 1000bp product; another non-fluorescent cell clone showed a 500bp product, a deletion reaction was thought to happen between the two lox sites in the genome under the reaction of Cre recombinase so that the GFP gene and neo gene etc were cut. Using the green fluorescent cell clones as the template, a product of 4Kb which was the same as the result of PCR of the plasmid ploxGFP was got. On the other hand, not only a 4Kb product but also a 1000bp product and a 500bp product were showed when the co-transfected cells were used to be the template. Because there is no plasmid sequence in bovine genome, nothing was gained by the PCR reaction of bovine genome.From the result, it is feasible to use the Cre/lox system to screen friendly gene locus.Furthermore, more and more researchers tend to use animal mammary gland bioreactor to highly express foreign genes in the latex. The aim of this part is that using bovine aS1-casein 5'regulation region and 3'flanking region to construct mammary gland expressing vectors.The bovine aS1-casein 5'regulation region about 2.08Kb and 3'flanking region about were got by PCR. Firstly, the bovine aS1-casein 5'regulation region was cloned into the vector pEGFP-1 and constructed a new vector P-5; then the bovine aSi-casein 3'flanking region was inserted into the position of GFP gene in the vector of P-S and a mammary gland universal expressing vector pbCAS was created.Secondly, the chicken r-IFN cDNA was introduced into the vector pbCAS between the bovine aSi-casein 5'regulation region and 3'flanking region to create a vector pbCASIFN to examine if this bovine aSi-casein 5'regulation region and 3'flanking region could regulate chicken Y -IFN to express in the latex by producting transgenic mice.