[Inhibition of tissue factor expression in endothelial cells by lentivirus mediated shRNA].
OBJECTIVE
To construct the recombinant lentivirus RNAi vector, and to determine whether the lentivirus mediated short hairpin RNA (shRNA) can inhibit the tissue factor (TF) expression in endothelial cells.


METHODS
Two short hairpin RNAs targeting to human TF were cloned into pENTRTM/U6 plasmid to obtain an entry clone, and the positive clones were verified by sequencing. A recombination reaction was performed between the pENTR/U6 entry construction and pLenti6/BLOCKiTTM-DEST vector, and then the positive clones were confirmed by sequencing. The 293FT cell line was transfected by the above recombined plasmid and lentivirus packing materials, the culture supernatant was harvested, and the virus titer was determined. RT-PCR and ELISA were used to observe the inhibition of TF gene expression after the lentivirus transduction in human umbilical vein endothelial cells.


RESULTS
The shRNA sequences targeting to human TF were cloned into the vectors, and an entry clone and an expression clone were constructed successfully, which were proved by sequence determination. Viral particles were packaged in the 293FT cell line, all virus stocks were collected, and the transfection titer was 5*10(5)/transduced unit. RT-PCR and enzyme linked immunosorbent assay demonstrated that the lentivirus stocks could suppress the TF expression in endothelial cells remarkably.


CONCLUSIONS
Lentivirus RNAi vectors containing human TF gene are successfully constructed, and lentivirus mediated shRNA can inhibit the TF expression in endothelial cells, which may provide a highly effective method for the prevention and treatment of thrombo-embolic diseases.
Xiong
Shi-long
SL
Laboratory Medicine Center, Nangfang Hospital, Southern Medical University, Guangzhou 510515, China.
Wang
Qian
Q
Zheng
Lei
L
Bao
Jie
J
Huang
Xian-zhang
XZ
Liu
Jing-zheng
JZ
Zeng
Fang-yin
FY
Qiu
Yu-rong
YR
D001483:Base Sequence; D015536:Down-Regulation; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D008969:Molecular Sequence Data; D034622:RNA Interference; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D013925:Thromboplastin / Q000096:biosynthesis* / Q000235:genetics; D014471:Umbilical Veins / Q000166:cytology
D012333:RNA, Messenger; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D013925:Thromboplastin