J4 ›› 2011, Vol. 37 ›› Issue (4): 670-674.
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ZHAO Dong-Hai1, ZHU An-Feng2, YANG Shu-Yan1, ZHONG Xiu-Hong1, ZHANG Yi-Zhong1, DIAO Li-Wei1
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Abstract:Objective To construct the rat NKX2.5-pEGFP fusion eukaryotic expression plasmid and screen bone marrow mesenchymal stem cells(MSCs) xpressing 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 arrow mononuclear cells were obtained by gradient centrifugation and adherent culture.The recombinant plasmids NKX2.5-pEGFP were ransfected into rat MSCs by Lipo2000 and screened with G418 for 2 eeks.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.
Key words: mesenchymal stem cells;NKX2.5;enhancement type green fluorescent protein
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ZHAO Dong-Hai, ZHU An-Feng, YANG Shu-Yan, ZHONG Xiu-Hong, ZHANG Yi-Zhong, DIAO Li-Wei. Establishment of |marrow mesenchymal stem cells expressing rat NKX2.5-pEGFP fusion protein[J].J4, 2011, 37(4): 670-674.
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http://xuebao.jlu.edu.cn/yxb/EN/Y2011/V37/I4/670
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