Establishment of marrow mesenchymal stem cells expressing rat NKX2.5-pEGFP fusion protein

Objective To construct the rat NKX2.5-pEGFP fusion eukaryotic expression plasmid and screen bone marrow mesenchymal stem cells(MSCs) expressing rat fusion protein NKX2.5-pEGFP and provide theoretical foundation for the research of cell and gene therapy of myocardial infarction.Methods The total RNAs were extracted from myocardial cells of embryo rats,RT-PCR amplification was performed to produce rat gene of NKX2.5.The recombinant plasmid NKX2.5-pEGFP was obtained by inserting the aim gene into pEGFP-N3.The MSCs derived from bone marrow mononuclear cells were obtained by gradient centrifugation and adherent culture.The recombinant plasmids NKX2.5-pEGFP were transfected into rat MSCs by Lipo2000 and screened with G418 for 2 weeks.The expression of recombinant plasmid in MSCs was detected by Western blotting and fluoroscope analysis.Results The length of NKX2.5 gene obtained by RT-PCR was 981 bp which was consistent with theoretical numerus.The recombinant plasmid NKX2.5-pEGFP was identified by endonuclease digestion and PCR,which could be digested into two fragments with BamHⅠ and SalⅠ,about 981 and 4 700 bp,the results were coincident with the anticipated result.The protein with molecular weight of 65 000 was found in MSCs transfected with the recombinant plasmid NKX2.5-pEGFP by Western blotting which was coincident with theoretical numerus.Green fluorescence was positive expression in MSCs transfected with the recombinant plasmid NKX2.5-pEGFP by fluorescence detection.Conclusion The rat MSCs expressing NKX2.5-pEGFP fusion protein is successfully constructed.