Construction Of FLP Recombinase Expression Vector And Its Application In FLP/FRT System

The use of site-specific recombinases (SSRs) both in vitro and in vivo,haveproven to be useful tools in the analysis of gene function.Upon binding to their targetrecognition sequences, SSRs can induce the deletion, insertion, or inversion of DNAsequences leading to conditional gene inactivation or expression.The first widelyused SSR in mammalian cultured cells and animals was the P1bacteriophage-derivedCre, a member of the λ integrase family that recognizes homotypic34bp loxPrecognition sites. To date, Cre recombinase remains one of the most efficient SSR toefficiently mediate DNA recombination both in vitro and in vivo.A second SSR fromSaccharomyces cerevisiae. A third SSR, ФC31from Streptomyces lividans, alsodisplays activity in mammalian cells.In this study,we constructed three vectors in order to validate the genes deletionefficiency of FLP/FRT system in PK-15cells.FLP expression vector pCEP4-FLP.To improve FLP translational efficiency in PK-15cells, FLP0which can pressefficient in37℃recombinases were reengineered according to the native amino acidsequence but with pig codon usage.This vector can express FLP whitout into thecell′s chromosomes.We constructed a vector,in this vector,the EGFP gene isregulated by a EF1α promoter,but its expression is inhibited by a FRT-flankedintervening cassette containing the stop gene,followed by transcriptional terminationsequences.The transcriptional termination sequences will be excised under theexistence of FLP,and the expression of EGFP was promotered.The final vector wasnamed pEF1α-FRT-stop-FRT-GFP.We contruct a deletion vector called pcDNA3.1-FRT-EF-GFP-FRT,in this vector two FRT sequences with the same orientation wereintroduced on either side of the EF1α promoter and EGFP genes. The EF1α promoterand EGFP genes will be excised under the existence of FLP.The express of EGFP was detected after the cycled plasmid of pEF1α-FRT-stop-FRT-GFP and pCEP4-FLP transfected in the epithelial cells of Pig kidneys and PEF.The plasmid of pcDNA3.1-FRT-EF-GFP-FRT was transfected into the epithelialcells of Pig kidneys (PK-15),a stable monoclonal cell line was seleeted by G418andEGFP.Then,the EF1α promoter and EGFP was deleted after the FLP pressing vectorpCEP4-FLP transfected into the monoclonal cell line. The cells of not express EGFPwas indicated by fluorescence activated cell sorting(FACS).We expect that PK-15cells vcould facilitate the research of transgenic pigs without marker genes.