An In Vitro Study Of Cre-on Conditional EGFP-RNAi

RNA interference (RNAi) is a naturally occurring post-transcriptional gene silencing mechanism mediated by double-stranded RNA (dsRNA),has been adapted as a genetic tool for gene function study.It was reported in some researches that the silence of certain genes will affect embryogenesis[1,2,3,4].To overcome these limitations,a conditional RNAi model using a combination of RNAi and Cre/LoxP system is to be constructed.The aim of this subject is to construct and identify plasmids,pCMV-Cre-EGFP which expressing both Cre and EGFP respectively,universal conditional RNAi vector pU6-floxedRFP for target gene,and conditional silencing vector pU6-floxedRFP-shEGFP for EGFP gene.Either pU6-floxedRFP or pU6-floxedRFP-shEGFP was cotransfected with pCMV-Cre-EGFP into 293T cells to detect its function.The LoxP-TS-CMV-RFP-LoxP fragment was constructed and inserted into the ApaⅠrestriction site of pSilencer1.0-U6 to obtain pU6-floxedRFP.The cre insert was cloned into the Multiple Cloning Site (MCS) of pIRES2-EGFP to construct pCMV-Cre-EGFP.After transfecting 293T cells mediated by FuGENE 6,the expression and recombination effect of Cre were analyzed.pCMV-Cre-EGFP and pU6-floxedRFP were co-transfected into 293T cells in Cre(+) group,pIRES2-EGFP and pU6-floxedRFP were co-transfected in Cre(-) group. 48h later,cells of both groups were visualized for EGFP or RFP by fluorescence microscopy and detected with flow cytometry.It was shown that the cre gene was cloned into the MCS of the vector successfully.The expression of RFP in Cre(+) group was less than in Cre(-) group(7.54 %±2.85% vs 3.53 %±1.62% , p<0.01) . This indicates that pCMV-Cre-EGFP can express active Cre and recombine pU6-floxedRFP. The short hairpin RNA (shRNA) targeting to EGFP and its control were designed , chemically synthesized and inserted into pU6-floxedRFP to obtain pU6-floxedRFP-shEGFP and pU6-floxedRFP-shEGFP-C.pCMV-Cre-EGFP was co-transfected into 293T cells,mediated by FuGENE 6,with different re-constructed vectors in different groups respectively , as follows : pCMV-Cre-EGFP and pU6-floxedRFP-shEGFP were co-transfected in siRNA group,pCMV-Cre-EGFP and pU6-floxedRFP were co-transfected in siRNA(-) group,pCMV-Cre-EGFP and pU6-floxedRFP-shEGFP-C were co-transfected in siRNA(control) group.In 72h after transfection,the expression of GFP in siRNA group was less than siRNA(-) group and siRNA(control) group.In the first section the expression of GFP in siRNA group and siRNA(-) group were 20.43%±1.37% and 29.57%±4.54% (p<0.05).In the second section the expression of GFP in siRNA group,siRNA(-) group and siRNA(control) group were 15.37%±7.45%,27.88%±9.92%,22.7%±7.53%(p<0.01).GFP in siRNA group was less than siRNA(control) group(p<0.05) . It is indicated that pU6-floxedRFP-shEGFP placed a Cre-onconditional inhibition for the expression of EGFP.In conclusion, the plasmid pCMV-Cre-EGFP which expressing both Cre and EGFP respectively was constructed and identified.The universal conditional RNAi vector for target gene , pU6-floxedRFP , and conditional silencing vector pU6-floxedRFP-shEGFP for EGFP gene were constructed.Either pU6-floxedRFP or pU6-floxedRFP-shEGFP was co-transfected with pCMV-Cre-EGFP into 293T cells to detect its function.pCMV-Cre-EGFP eukaryotic expression plasmid can express active recombinase Cre in vitro . The experiment results showed that pU6-floxedRFP-shEGFP can inhibit 58.3% of the expression of EGFP when recombinase Cre existed indicating the strategy of Cre-On conditional RNAi transgenic animal is feasible and reliable.