Objective:To construct the RNAi vectors targeting mouse NOMO1 gene and compare the silence effects of these small interference RNAs,and to study the effects of NOMO1 gene on the differentiation of P19 cells to cardiac myocytes. Methods:Short hairpin RNAs of mouse NOMO1 gene were designed by Designer3.0 software,synthesized and cloned into the pGPU6/Hygro plasmid. Connection products were transfected into competent cells and identified by PstⅠ+BamHⅠ double digestion and DNA sequencing. Then the vectors were transfected into P19 cell. The level of NOMO1 mRNA was evaluated by RT-PCR. The expression of α-MHC mRNA in P19 cell,which was induced by DMSO,was evaluated by RT-PCR. Results:shRNA was inserted into the pGPU6/Hygro plasmid correctly,confirmed by double digestion and DNA sequencing. The strains of P19 cell stably expressing shRNA were selected. RT-PCR and Western blot showed that 4 shRNAs silenced the expression of NOMO1 gene markedly. α-MHC was down-regulated in transfected P19 cell. Conclusion:pGPU6/Hygro vectors carried mouse NOMO1 shRNA are constructed successfully. It provides us a useful tool for the study of NOMO1 gene function in the differentiation of P19 cell into cardiac myocyte. NOMO1 may be involved in α-MHC gene expression,and the differentiation of P19 stem cell into mesodermal cell.