Construction And Knock Down Efficiency Evaluation Of Synaptotagmin1siRNA Expressing Vector

Synaptotagmins (Syt) are a family of type I membrane proteins with evolutionarilyconserved cytoplasmic C2domains. They are located on secretory vesicles and granules inneuron, neuroendocrine and other cells. As the primary Ca2+sensor for neurotransmitterrelease and regulated exocytosis, synaptotagmins bind calcium with its C2domian to triggerand regulate the fusion process between vesicle membrane and target membrane. There areover17isoforms of synaptotagmin in mammalian, they may have different roles in secretoryregulation because of different localizaiton and Ca2+binding affinity. It is eargely needed tofind very useful and efficient methods to identify roles of each Syt isoform, and thus clarifyrelationships between their structure and function. RNA interfering (RNAi) is a potenttechnique for silencing the expression of target gene at the step of post-transcription, whichmay be useful for us to identify the regulatory function of Syts in vesicle fusion, endocytosisand vesicle recycling.pRNAT-H1.1-Shuttle-RFP was constructed by replacing EGFP in pRNAT-H1.1-Shuttlewith RFP, and the construction was verified by digestion of restriction enzyme, gelelectrophoresis and sequencing analysis. And then, we designed and synthesized the shRNAfor Syt1and that for scrambled sequence which has no target genes in human, rat and mouse.The DNA fragments were inserted into BamHⅠand HindⅢ of pRNAT-H1.1-Shuttle-RFP,and the construction was confirmed right.pIRES2-EGFP was employed to optimize the transfection of human embryonic kidney293a cells by cationic lipids VigoFect. The results demonstrated that transfection efficiencyincreased in a dose-dependent manner with the amount of DNA plasmid used, and optimaltransfection time and cell density should be identified to reach a compromise of highertransfection efficiency and lower toxicity. Co-transfection experiments of pIRES2-EGFP andpRNAT-H1.1-Shuttle-RFP-Scram indicated that the two co-transfected plasmids wereequivalently delivered into the same cells, and the co-transfection efficiency was rarelyaffected by cell density and proportion of the two plasmids. However, plasmid receipted cellsseemed to be indisposed to accept plasmid again during the second transfection and very lowco-transfection efficiency was observed in tandem-transfection. Syt1and its siRNA expressing plasmids were co-transfected or tandem-transfected intoHEK293a cells to evaluate knock down efficiency of the two siRNAs. Results of real timePCR and western blotting indicated the higher knock down efficiency of these two siRNAs,while the expression level of Syt1mRNA decreased to42%and34%of that in Syt1expressing cells, and the protein levlel of Syt1decreased to36.1%and10.3%of that in Syt1expressing cells, respectively. However, the knock down efficiency of the two siRNAexpressing plasmids decreased dramaticly, which may be due to the lower co-transfectionefficiency of the two tandem transfected plasmids.