Screening For Highly Transductive Adenoassociated Viruses In Mammalian Endometrial Cells And The Effect Of Dcaf1 Gene Knockdown On Decidualization

The Adeno-associated virus(AAV)is a non-enveloped virus measuring 26 nm with a genome of 4.7 kb.AAV exhibits various serotypes,and their transduction efficiency varies greatly among different cell types.At present,lentivirus and adenovirus have been commonly used to study gene expression and regulation in mammalian uterine epithelial and stromal cells.However,there is limited study on the use of adeno-associated viruses in these specific cell.The DCAFs family comprises a class of proteins that can bind with DDB1 to form E3 ligase complex,which are involved in regulating a variety of cell activities,including proliferation,cell cycle progression and DNA damage response.As a member of the DCAFs protein family,DCAF1(also known as Vpr BP)plays an important role in the reproductive fields such as oocyte development,early embryo development and female fertility.However,the specific function and role of Dcaf1 in embryo implantation and decidualization during early pregnancy remain unclear.In this study,we initially isolated mammalian uterine epithelial and stromal cells using enzyme digestion method.We then utilized Real-Time PCR,fluorescence microscopy and Western-Blot technology to measure viral titers and explore the transduction of 10 serotypes of AAV in mammalian uterine primary cells.serotypes with high transduction efficiency were identified through screening.The expression of Dcaf1 during early pregnancy was investigated by Real-Time PCR and immunohistochemistry.Furthermore,we constructed AAV virus containing si RNA targeted mouse Dcaf1 and human DCAF1 and transduced uterine stromal cells,and the si RNA with High knockdown efficiency were selected.Additionally,in vitro models of artificial induced mouse and human uterine stromal cell decidualization were successfully established,and the effect of AAV-mediated Dacf1 knockdown on decidualization was assessed by Real-Time PCR.The results showed that AAV-DJ was capable of transducing mouse uterine epithelial cells.in mouse uterine stromal cells,the transduction efficiency was AAV-DJ >AAV1,AAV2,AAV5>AAV9.The transduction efficiency in rabbit uterine epithelial cells was AAV5,AAVDJ>AAV2>AAV9.The transduction efficiency in rabbit uterine stromal cells was AAVDJ >AAV1,AAV2,AAV5>AAV9.The transduction efficiency in porcine uterine epithelial and stromal cells was AAV-DJ>AAV2,AAV5>AAV1.The transduction efficiency in human uterine stromal cell lines was AAV-DJ>AAV2,AAV9>AAV1,AAV3>AAV8.The overall expression of Dcaf1 m RNA showed an upward trend during the early gestation in mice,with low during the1 st to 4th day of early gestation,and gradually increased during the 5th to 8th day,Dacf1 protein is highly expressed in uterine stromal cells and demulsification area.AAV-DJ transduction effectively knocked the expression of Dcaf1 in mouse and human deciduating cells,resuting in reduced degree of decidualization.In conclusion,AAV-DJ,AAV5 and AAV2 exhibited superior transduction efficiency in uterine epithelial cells.AAV-DJ,AAV2,AAV9,AAV5 and AAV1 showed better transduction effects in uterine stromal cells.Dcaf1 was found to be highly expressed in the decidualization process of early pregnancy in mice,suggesting its potential involvement in the regulation of decidualization process.AAV transduction can effectively knock down the expression level of Dcaf1 and reduce the degree of decidualization.