Construction of eukaryotic cell expression recombinant pcDNA3ª±1(+) ª²mtDNA in colon tumors and transfection of mtDNA into murine fibroblast cells NIH3T3

    SONG Weiª²Bing1,XIAO Bing1,YAN Li1 ,LI Liangª²Ren1,PENG Danª²Dan1,WU Qianª²Qian2£¬YE Fangª²Peng1,MA Li3

    1Institute of Digestive Diseases,Nanfang Hospital,Nanfang Medical University£¬Guangzhou 510515£¬China,2Department of Outª²patient,Military Physical Education College of PLA£¬Guangzhou 510515£¬China,3Cadre¬ðs Sanatorium,Second Artillery of PLA,Beijing 100086,China

    ¡¾Abstract¡¿ AIM£º To investigate mutations in the Dª²loop region of mitochondrial DNA in colon tumors,to construct eukaryotic cell expression recombinant pcDNA3ª±1(+) ª²mtDNA and to transfect pcDNA3ª±1(+) ª²mtDNA in murine fibroblast cells NIH3T3.METHODS: The Dª²loop region of mtDNA of the three colonic tumors cells (SW480,Lovo and HT29) was amplified by PCR and sequenced.The fragments of mtDNA were inserted into the eukaryotic cell expression plasmid pcDNA3ª±1(+) and the pcDNA3ª±1(+) ª²mtDNA recombinant was used to infect the murine fibroblast cell NIH3T3.RESULTS: Among the three colonic tumors cells (SW480,Lovo,HT29),10,9 and 8 mutations were identified respectively.The 1119 kb fragment of mtDNA was successfully cloned.DNA sequencing analysis proved that the sequence of the gene cloned was totally consistent with the data of GenBank.The mtDNA fragments were cloned into the multiple cloning sites of vector pcDNA3ª±1(+) properly.The recombinant was then introduced into NIH3T3 cells.CONCLUSION: The Dª²loop region of mitochondrial DNA is a highly polymorphoric and mutable region and the mutation rate is relatively high in cells with colon tumors.The pcDNA3ª±1(+) ª²mtDNA eukaryotic cell expression recombinant is successfully constructed and mtDNA is successfully transfected into NIH3T3 cells. ......