[Construction of recombinant eukaryotic expression plasmid pcDNA3.1(+)-mtDNA of human colorectal carcinoma cells].
OBJECTIVE
To construct recombinant eukaryotic expression plasmid pcDNA3.1(+)-mtDNA for investigation of mutations in the D-loop region of mitochondrial DNA in human colorectal carcinoma.


METHODS
The D-loop region of 3 colorectal carcinoma cell lines (SW480, LoVo, and HT29) were amplified by PCR and sequenced. The mtDNA fragment was recombined in the eukaryotic expression plasmid pcDNA3. 1(+), and the resultant pcDNA3.1(+)-mtDNA recombinant was used to infect murine fibroblast cell line NIH3T3.


RESULTS
Among the 3 colorectal carcinoma cell lines (SW480, LoVo, HT29), 10, 9, 8 mutations, were identified, respectively. The 1119-bp fragment of mtDNA was successfully cloned. DNA sequencing analysis demonstrate total agreement of the sequence with that in GenBank. The mtDNA fragments were cloned into the multiple cloning sites of vector pcDNA3.1(+) properly and the recombinant was introduced into NIH3T3 cells.


CONCLUSIONS
The D-loop region of mitochondrial DNA is a highly polymorphoric and mutable region with high mutation rate in human colorectal carcinoma cells. The recombinant eukaryotic expression plasmid pcDNA3.1(+)-mtDNA is successfully constructed.
Wu
Qian-qian
QQ
Out-patient Department, Military College of Sports of PLA, Guangzhou 510515, China. swb535@126.com
Zhang
Qing-hua
QH
Ma
Li
L
D000818:Animals; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000473:pathology; D004272:DNA, Mitochondrial / Q000096:biosynthesis / Q000235:genetics*; D005057:Eukaryotic Cells / Q000378:metabolism*; D006801:Humans; D051379:Mice; D041681:NIH 3T3 Cells; D010957:Plasmids / Q000235:genetics; D017354:Point Mutation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
D004272:DNA, Mitochondrial; D011994:Recombinant Proteins