[Construction and identification of RNA interference vector for human tissue factor gene].
OBJECTIVE
To construct a RNA interference vector for human tissue factor (TF) gene.


METHODS
Human TF short hairpin RNA (shRNA) sequence was designed using online design software (Invitrogen) and synthesized into double-strand oligonucleotide (ds oligo), which was cloned into the pENTRTM/U6 plasmid, followed by transformation of the product into competent Top10 E. coli cells. After expansion of the transformed bacteria, the plasmid was extracted and sequenced, which was subsequently transfected into human umbilical vein endothelial cells (HUVECs). The interference effect of the vector on the target gene expression was detected by RT-PCR and immunofluorescence assay.


RESULTS
The sequencing result indicated that the plasmid pENTRTM/U6-RelB-shRNA was constructed correctly, which resulted in effective inhibition of TF expression in HUVECs after transfection.


CONCLUSIONS
The RNA interference vector against human TF gene has been constructed successfully, which may provide a stable transfection vector for potential treatment of blood coagulation abnormalities.
Department of Pediatrics, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, China. tangwenr@21cn.com
Wen
W
Tang
Shi-long
SL
Xiong
Hong-ling
HL
Yang
Jie
J
Bao
Jiang
J
DU
Shao-dong
SD
Hua
Xue-gang
XG
Sun
Zhi-chun
ZC
Feng
D001483:Base Sequence; D002460:Cell Line; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense / Q000235:genetics; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013925:Thromboplastin / Q000172:deficiency* / Q000235:genetics*; D014162:Transfection
D016376:Oligonucleotides, Antisense; D034741:RNA, Small Interfering; D013925:Thromboplastin