Construction Of Gab1-targeting Lentiviral RNA Interference Vectors And Investigation Of Its Influence On Proliferation Of Human Vascular Endothelial Cell

ObjectiveTo construct and identify a lentiviral vector harboring RNAi sequence targetinghuman Gab1gene and investigate its proliferation effect on human vascularendothelial cell.MethodsIn the first part，We designed and synthesized five pairs of complementary doublestrand DNA oligos，which could express short hairpin RNA targeting the Gab1mRNA，and linked them into the lentiviral plasmid vector GV118；The positiveclone was chosen and confirmed by PCR and DNA sequencing; next we constructedanother Gab1over-expression eukaryotic plasmid，which was cotransfected togetherwith the recombined lentiviral plasmid into293T cell in order to screen and verify theknockdown effect of the RNA interference by Western-blot； and then we packagedthe most efficient lentiviral plasmid vector assisted with the other twoplasmids(pHelper1.0and pHelper2.0) in293T cell line，and detected the viral titerby dilution method．In the second part，we infected the vascular endothelialcells with RNAi lentivirus，Expression of Gab1in endotheliaI cells was detectedusing quantitative real-time PCR and Western blot. Finally, The proliferation ratefor transfected cells was validated by MTT assay performed at days1,2,3,4,and5after cell seeding.Results1、 PCR analysis and DNA sequencing demonstrated that the RNAi sequencetargeting the human Gab1gene was successfully inserted into the lentiviralvector．2、 The recombinant lentivirus was harvested from293T cells with a viral titer of 3×10~9TU/ml．3、 We detected a highly efficient inhibition to the expression of Gab1gene throughRT-PCR and Western blot after infecting the specific RNAi lentivirus toendotheliaI cell．4、Down regulating the expression of Gab1gene by Gab1-targeting RNAi lentiviruscould obviously suppress the proliferation of endothelial cell．ConclusionThe Gab1-targeting RNAi lentivirus is efficient to inhibit the expression ofGab1gene in endothelial cell，which Can be used as an important technical tool tostudy the change of biological behavior before and after RNA interference andfurther study of the role of Gab1gene in the development of angiogenesis. TheGab1gene plays an important role in the proliferation of endothelial cell．...