No-selectable Marker Gene Modified Cell Model Construction And Evaluation

In this study, we used PK15cells of porcine kidney as the cell models, setting up Functional MSTN targeting vector and TALEN effector protein expression vectors. Through electroporation transformation method, the targeting vector was transformed into PK15cells of porcine kidney, and we screened out a Monoclonal cells which was able to integrate the targeting vectors stably and express EGFP uniformly. Then, aimed at LoxP site anchored on targeting vector ΔMSTN,we designed Cre recombinase expression vectors. And the next, we translated the MSTN targeting vectors and TALEN expressing vectors into PK15cells of porcine kidney, screened monoclonal cells with MSTN gene knocked out, and knocked out selectable marker gene by Cre-LoxP recombination system to evaluate the efficiency of gene deletion and fixed-point replacement rate in order to provide new way to the study and use of gene recombination technology.By fluorescence-activated cell sorting(FACS), realtiefluores-cence quantitative PCR, TA cloning and sequencing, we verified the deleting efficiency of Cre-LoxP recombination system through which the endogenous selection marker gene was knocked out,and the results were listed as follows:1. We got monoclonal cell line by screening which also testified the effectiveness of selection marked neomycin phosphoric acid transferase and EGFP; Based on monoclonal cell line, we proved the feasibility and effectiveness of deleting endogenous selection marked expression box with the method, site-specific recombination mediated by Cre-LoxP.2. By flow cytometry, the total intensities of fluorescence expression for each group,GFP-A-Mean level, was gotten. The result shows, compared to negative control, Neg.Ctrl, fluorescence peak and shape of blank group, Mock, change little,while the Cre-loxP group change a lot with the shape narrowing down and fluorescence intensity declining. In addition, the analysis result of fluorescence expression variations for each group shows that expression level of PK-ΔMSTN fluorecyte in Cre-loxP group declines obviously, the rate of Fluorescent protein expression quantity dropped to46.1%.3. Extracting gDNA of the experimental cells for each group,and detecting CMV-NeoR-IRES-EGFP expression cassette gene in PK-ΔMSTN Monoclonal cell genome DNA by Taq digestion and Real-time quantitative PCR, a decline of gene dosage of CMV-NeoR-IRES-EGFP was found in PK-ΔMSTN Monoclonal cell genome DNA which had been transfected by Cre-loxP recombinant enzyme system. The quantitative PCR shows the rate of deleting green fluorescent protein gene intramolecularly by this system is97%.4. PCR products were recycled by gel recycling, then used TA clon and screening positive recon and sequencing.The result shows, Expression selection marked box were deleted through site-specific recombination, only residual a34bp LoxP site Between5ˊand3ˊhomologous arms, which proves furtherly that Cre-LoxP site-specific recombination system has good restructuring activity in pig cells, that means this system can delete the endogenous expression selection marked box efficiently.To sum up, this study set up a new system of deleting election markers for transgenic pig, opening up a feasible technical route to get no selection marked donor cells at the base of gene targeting.