[Construction and identification of matrix metalloproteinase inhibitor-1 siRNA eukaryotic expression vectors].
OBJECTIVE
To construct TIMP-1 siRNA eukaryotic expression vectors and evaluate their effect on TIMP-1 mRNA expression in hepatic stellate cells.


METHODS
The combinant lone DNA with cutting sites of BamH I and Xho I enzyme according to the sequences of 447-465, 552-540 TIMP-1 of rats and nonspecific sequence were selected and cloned to pGEM-T vector and sub-cloned to pRNAT-U6.2. They were then identified by double enzyme digestion analysis and DNA sequencing. Three plasmids were transfected into T6 separately through an oligofectamine package. TIMP-1 mRNA expression was evaluated by RT-PCR.


RESULTS
Targeting sequences of TIMP-1 siRNA eukaryotic expression vectors were correct. TIMP-1 mRNA expression was significantly reduced by transfecting them into the T6.


CONCLUSIONS
We successfully constructed two TIMP-1 siRNA eukaryotic expression vectors and the transfected cells can significantly suppress the TIMP-1 expression.
Tian
Li
L
Department of Gastroenterology, First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China. tianyu615999@sina.com
Li
Ji-chang
JC
Zhao
Guo-qiang
GQ
Chen
Kui-sheng
KS
Fan
Wen-hui
WH
Lou
Xin
X
Sun
Miao-miao
MM
Cao
Xue-quan
XQ
D000818:Animals; D002460:Cell Line; D020868:Gene Silencing; D005822:Genetic Vectors; D055166:Hepatic Stellate Cells; D010957:Plasmids; D034741:RNA, Small Interfering; D051381:Rats; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000235:genetics*; D014162:Transfection
D034741:RNA, Small Interfering; D019715:Tissue Inhibitor of Metalloproteinase-1