The Study Of Seeking An Effective SiRNA Targeting Mouse CatSper1 By Using A PEGFP-N1-Catsper1 Eukaryotic Expression Vector

PartⅠThe construction and expression of the mouse CatSper1 recombinant plasmidObjective: To construct a recombinant plasmid by which the mouse CatSper1 mRNA can be transcribed for siRNA screening.Methods: Two pairs of primers were designed to amplify the different part of open reading frame of mouse CatSper1. With the two PCR products as templates, then the whole open reading frame was amplfied by recombination PCR, and cloned into the eukaryotic expression vector pEGFP-N1. DNA sequencing was applied to confirm the correct clone.Results: Full lenth (2061bp) of the opening reading frame of CatSper1 was successfully amplified by recombination PCR. The DNA sequencing confirmed the correction of the cloning of pEGFP-N1-CatSper1.Conclusion: The opening reading frame of mouse CatSper1 was successfully inserted into vector pEGFP CatSper1 multiple cloning sites, and obtained the recombinant plasmid pEGFP-N1-CatSper1.PARTⅡScreening for the effective siRNA sequences in vitroObjective: To design three chemically synthesized small interfering RNAs (siRNAs) and evaluate their inhibitory effect on the expression of exogenous gene CatSper1 in mouse neuroblastoma N2a cells.Methods: Three siRNA sequences, which aimed at gene CatSper1 in male mice, were designed with the assistant of Bioinformatics analysis software and cotransfected into mouse neuroblastoma N2a cell line, with the recombinant plasmid pEGFP-N1-CatSper1. In order to screen for the RNAi sequence which can significantly reduce the N2a cell expression of exogenous CatSper1, the inhibitory effect was evaluated by observing EGFP fluorescence intensity, Real-time quantitative PCR, Western Blotting Analysis, etc.Results: Compared with the blank control group, mRNA and protein expression decreased in three experimental groups with the interference of siRNAs we designed. According to our observation, the most active siRNA was the one with 3'end (1870-CCTTGAAGATGCAGCTTAT-1889). Compared with the control group ,siRNA group was the best interference sequence and decreased by 79%.While, in negative control siRNA group, mRNA and protein expression did not decrease.Conclusion: Effective siRNAs were selected by using eukaryotic expression of exogenous CatSper1. Research CatSper1 functions with the application of RNAi may provide a reference for contraception.