Construct A Recombinase Delete Vector And Application In The Animal Cells

With the development and application,of transgenic technology,despite its great potential benefit, its negative effects gradually attract more and more attention.. As a important technology for prolonging human lifespan, transgenic animals for organ transplantation attracted a wide spread attention,however, its safety should never be neglected.Pig is an important animal model for organ transplantation research for the great similarity between human and pig in physiology .The present researches focus on the donor organ and donor animals of Swine and porcine kidney by its particularity.After obtain the cloning products, the problem that the exogenous gene especially marker and reporter gene should be saved or dismissed attracts many researcher's research interest. It's necessary to delete the exogenous gene, in order to minimize the risk of potential from the marker and reporter genes. This experiment try to construct a plasmid which contains the recombinase recognition sites by using the site-specific recombination system and to verify the deletion effects in animal cells.The main results includes:(1) Based on the test required, we designed a sequence that contains the multiple cloning sites in the middle and the double recombinase (CreFLP)recognition site on both end, and obtained the target fragment with 211bp full-length by overlap extension PCR(SOE PCR), then the target fragment was cloned into pMD18-T Simple backbone vector;(2) Taking pCDNA3.1, pEGFP-N3, pIRES plasmid as a template, marker and reporter genes were amplified:including neomycin resistance (neomycin) gene, Enhanced green fluorescent protein (EGFP) gene, strong promoter (CMV) gene and the sequence of internal ribosome entry site (IRES);(3) The two marker and reporter genes (Neomycin and EGFP) were connected to the backbone vector for expression different protein respectively or co-expression of the two protein by IRES system.(4) As the two vector backbones from the step(3), we delete the Cre recombinase recognition site and obtain another two different vectors that have only one FLP recombinase recognition site. Finally we constructed four different vectors that have different recombinase recognition sites and different protein expression pattern;(5) The above-mentioned four kinds of plasmid were used to transfect the epithelial cells of pig kidneys(PK15), a stable monoclonal cell line was selected by G418 selection and results indicated that the four constructed vectors could express the marker and reporter gene successfully; (6) The FLP recombinase plasmid was used to transfect the selected monoclonal cell line, 3 passages later, RNAs from transfected cells and the control group were extracted, cDNA was obtained by RT-PCR.(7) The expression difference of the marker and reporter gene between deletion group and non-deletion group was verified by relative quantitative PCR , significant difference was observed between two groups, which confirm the success of our experiment(P<0.05).(8) The delete efficiency of the plasmid of pCF,pCF-pA,pF and pF-pA are 21%,25%,18%,16%.