Construction And Verification Of AAV Specific Expression Vector In Mouse Spermatogenic Cells

Objective: 1.To construct AAV-specific expression vectors containing mouse spermatogenic cellspecific promoters Dazl,Stra8,Hspa2,Pgk2 and Prm1 respectively.And to verify and screen valid specific promoters.2.To construct the mouse spermatogenic cell-specific expression vector AAV-Dazl-RFPFlag,and to verify its expression specificity in vitro and vivo.Methods: 1.Screening of mouse spermatogenic cell-specific promoter.We constructed AAV specific expression vectors containing mouse spermatogenic cellspecific promoters Dazl,Stra8,Hspa2,Pgk2 and Prm1 respectively through homologous recombination,and transfected them into GC1 cells.Effective mouse spermatogenic cellspecific promoters were screened out by comparing the RFP expression of five vectors.2.Construction of mouse spermatogenic cell-specific expression vector AAV-Dazl-RFPFlag.The homologous recombination was used to insert the mouse spermatogenic cellspecific promoter Dazl fragment into AAV-CMV-RFP-Flag vector of which the CMV promoter was removed by enzymatic digestion.We used double digestion and gene sequencing to verify the construction of vector AAV-Dazl-RFP-Flag.3.Verification of AAV-Dazl-RFP-Flag vector expression in vitro.Adeno-associated virus vector AAV-CMV-RFP-Flag and mouse spermatogenic cellspecific expression vector AAV-Dazl-RFP-Flag were transfected into GC1 cells and 293 T cells respectively.We collected cell slides and detected the expression of RFP through immunofluorescence.Cell pellets were also collected and the expression of RFP was detected by RT-qPCR and western blot.4.Verification of AAV-Dazl-RFP-Flag vector expression in vivo.Adeno-associated virus vector AAV-CMV-RFP-Flag and mouse spermatogenic cellspecific expression vector AAV-Dazl-RFP-Flag were packaged into viruses.These viruses were injected into seminiferous tubules of three weeks ICR mouse testis by microinjection.After one week,testicular tissue was took out to detect the RFP expression through stereomicroscope and immunofluorescence.RNA from testis tissue was extracted to detect expression of RFP using RT-qPCR.Results: 1.The mouse spermatogenic cell-specific promoters Dazl,Stra8,Hspa2,and Pgk2 could initiate the expression of RFP in GC1 cells,and Dazl promoter was the strongest among them.2.The mouse spermatogenic cell-specific expression vector AAV-Dazl-RFP-Flag was constructed successfully.3.The AAV-Dazl-RFP-Flag vector can express RFP both in GC1 cells and 293 T cells,but the expression level in 293 T cells was lower than that in GC1 cells.4.The expression of RFP of AAV-Dazl-RFP-Flag vector can be detected at RNA level in mouse testis tissue,but in the phenotypic level.Conclusion: 1.The Dazl promoter is screened from five spermatogenic cell-specific promoters.2.Spermatogenic cells specifically express mouse spermatogenic cell-specific expression vector AAV-Dazl-RFP-Flag at RNA level.