[Construction of eukaryotic expression vector of hMTH1 gene antisense RNA].
OBJECTIVE
To construct pEGFP-C1-T vector, an eukaryotic expression plasmid of hMTH1 gene antisense RNA.


METHODS
The conservative region of hMTH1 gene was amplified by RT-PCR after total RNA being extracted from human embryo lung fibroblast (HLF) and then cloned into pGEM-T vector. After the recombinant plasmid was certified by DNA sequencing, the conservative region of hMTH1 gene was inserted into pEGFP-C1 vector reversedly and pEGFP-C1-T vector was constructed. The efficiency of antisense inhibition was verified by Western blotting after cell transfection.


RESULTS
423 bp fragment including conservative region of hMTH1 gene was obtained by RT-PCR. After cloned by pGEM-T vector and certified by DNA sequencing, pEGFP-C1-T vector was successfully constructed by means of recombinant DNA technology. Additionally pEGFP-C1-T vector could efficiently decrease hMTH1 protein level by 46%.


CONCLUSIONS
The efficient expression vector of hMTH1 gene antisense RNA, pEGFP-C1-T has been constructed successfully.
Jiang
Gao-feng
GF
Department of Health Toxicology, School of Public Health, Sun Yat-sen University, Guangzhou 510080, China.
Zhuang
Zhi-xiong
ZX
Liu
Qi-zhan
QZ
He
Yun
Y
Du
Liu-tao
LT
D045643:DNA Repair Enzymes; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics*; D010957:Plasmids; D016372:RNA, Antisense / Q000096:biosynthesis*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D016372:RNA, Antisense; D010744:Phosphoric Monoester Hydrolases; C078207:8-oxodGTPase; D045643:DNA Repair Enzymes