RNA Interference Inhibition Of Sheep Inhibin Alpha Subunit Gene Expression

Inhibin is a kind of glycoprotein hormones secreted by the ovarian granulose cells and testis Sertolicells.which is composed of α-and β-subunits,they linked to form the heterodimer by a disulfidebond.α-subunit is the essential element for inhibin to carry on its function.Inhibin can inhibit FSHrelease,which regulate the formation of follicle.On male,it can inhibit spermatogenesis process.There-fore,we can use RNA interference technology to down-regulate the inhibin level in order to promotefollicular development and improve the production of sperm.RNA interference is a gene silencing mechanism which is triggered by endogeneous or exogeneousdouble-stranded RNA.It has high specificity. However,expession vector in previous reports could onlytranscript to generate one specific small interfering RNA(siRNA)and scilence single gene expressi-on,this has low interference efficiency.To improve the efficiency of stable knockdown with short hairpin RNA (shRNA), we insertedmultiple shRNA expression sequences into a single plasmid vector. In this study,We constructed shRNAexpression vector with dual-promoter aimed at INHA gene. The efficiency of knockdown was comparedamong single and two expression vectors.By detecting the change of biological phenotypes after thisvector was transfected into granulosa cells,then we observed and studied the gene silencing effe-ct.Moreover,the modeling tool carrier could lay the foundations for future studies on RNA interference.According to the cDNA sequences of INHA,the single shRNA recombinant plasmid pGenesil1.2-INHA,dual shRNA recombinant plasmid pGenesil1.2-INHA1+2and negative control pGenesil1.2-HKwere constructed respectively;Transfect pGenesil1.2-HK,pGenesil1.2-INHA, pGenesil1.2-INHA1+2into granulosa cells respectively. The relative amounts of INHA mRNA were detected by RT-PCR at48h after transfection respectively.Expression of the protein was estimated by Western lotting.The results are as follows:1.The results of sequencing and restrictive enzyme digestion confirmed that the single shRNArecombinant plasmid expression vector and the dual shRNA recombinant plasmid expression vectorwere constructed successfully．2.The RT-PCR showed that INHA mRNA expression were inhibited at48h post-transfection.Theinhibition rate of pGenesil1.2-INHA1+2mRNA in the INHA was83%,which was more effective thanthe single shRNA group．3.The single shRNA-expressing vector caused limited knockdown of the target protein in stabletransfectants.however, the dual expression vector apparently increased the effect of knockdowntransfectants.Conclution:Dual shRNA strategy was more effective than single shRNA in gene silencing.