Construction and identification of siRNA expression vectors for silencing Ca~(2+) transport protein gene

Objective:To construct specific small interfering RNA(siRNA) expressing vectors of intracellular Ca2+ transport protein(CaT1)gene and detect its silencing effects.Methods:The hairpin sequences of siRNAs targeting CaT1 gene were designed,synthesized and cloned into pSlincer 3.1-H1 plasmids after annealing.The vectors were then enriched in E.coli.The recombinant pSlincer 3.1-H1 plasmids were identified by restriction endonuclease cutting and DNA sequencing and then transfected into Human Gastric Carcinoma Cell Line BGC-823.The expression of CaT1 mRNA was examined by RT-PCR.Results:The siRNA oligonucleotides of CaT1 were correctly cloned into the pSlincer 3.1-H1 plasmids and confirmed by restriction endonuclease cutting and DNA sequencing.RT-PCR analysis revealed that the expression of CaT1 mRNA in BGC-823 cells transfected with the pSlincer 3.1-H1 constructs of siRNA was significantly decreased compared with that of the negative control and untransfected group.Conclusion:siRNA expression plasmids for silencing Ca2+ transport protein gene are successfully constructed,and they effectively inhibit the CaT1 gene expression.