Construction and identification of RNA interference vector for human tissue factor gene.
- Author:
Wen TANG
1
;
Shi-long XIONG
;
Hong-ling YANG
;
Jie BAO
;
Jiang DU
;
Shao-dong HUA
;
Xue-gang SUN
;
Zhi-chun FENG
Author Information
- Publication Type:Journal Article
- MeSH: Base Sequence; Cell Line; Genetic Engineering; methods; Genetic Vectors; genetics; Humans; Molecular Sequence Data; Oligonucleotides, Antisense; genetics; RNA Interference; RNA, Small Interfering; genetics; Reverse Transcriptase Polymerase Chain Reaction; Thromboplastin; deficiency; genetics; Transfection
- From: Journal of Southern Medical University 2007;27(7):1065-1067
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo construct a RNA interference vector for human tissue factor (TF) gene.
METHODSHuman 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.
RESULTSThe 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.
CONCLUSIONThe 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.
