Construction Of TDRG1shRNA Expression Vector And Study On The Interfering Effect Of TDRG1shRNA Expression Vector In NTERA-2Cells

Objective:To construct short hairpin RNA recombinant plasmid expression vector targeting of TDRG1, and detect the specific interfering effect of TDRG1-shRNA expression vector in NTERA-2cells. Screen the recombinant plasmid pGPU6/GFP/Neo-TDRG1-shRNA which can block the expression of TDRG1gene high effectively and specificly.Then this study laid the foundation for further research about the biological behavior role of that TDRG1in testicular tumor.Methods:Oligos for short hairpin RNA targeting for TDRG1were designed and connected to the expression vector pGPU6/GFP/Neo to construct the TDRG1shRNA expression vectors. The recombinant plasmid TDRG1-shRNA486, TDRG1-shRNA738,TDRG1-shRNA921and shNC. The four recombinant expression vectors were transformed into E.coli JM109and then, extracted, digested and identified. lipofectamineTM2000were used to generated and transfected shRNA into NTERA-2cells respectively. Expression of TDRG1mRNA was assayed by real-time reverse transcription-polymerase chain reaction.(Real time-PCR).Results:Restriction enzyme cutting verify the three recombinant plamids were positive.TDRGl-shRNA expression vector was successfully constructed. TDRG1-shRNA486was more effective in the suppression of TDRG1with significant reduction of TDRG1mRNA(p<0.01), and inhibitory rates to TDRG1gene mRNA was84.89%.Conclusion:the three recombinant plamids TDRG1-shRNA could interfere the expression of TDRG1in NTERA-2cells, the pGPU6/GFP/Neo-TDRG1-shRNA486Was the one that had the strongest inhibitory effect.14figures,12tables,41references.