[Construction of shRNA expression vector pWH1 and its function in silencing the HIF1 gene].
OBJECTIVE
To construct the expression vector of small hairpin RNA(shRNA) and to test its efficacy in silencing the Hypoxia-inducible Factor-1 (HIF1) gene.


METHODS
The H1 gene promoter was amplified from the genome of the human blood cells by PCR. Then the promoter was cloned into the pEGFP-C1 vector digested with the restriction enzyme. The constructed vector was named pWH1. The primer was designed to target the human HIF1 cDNA gene. The annealed primer fragment was cloned into pWH1. The new constructed plasmid was transfected into the SGC7901 cell line. Then the expression level of HIF1 gene was assayed by RT-PCR and Western blot.


RESULTS
The newly constructed plasmid expressed shRNA to target the HIF1 gene.The results of RT-PCR and Western blot showed the expression of HIF1 gene was reduced dramatically in mRNA and at protein level.


CONCLUSIONS
The successful construction of shRNA expression vector (pWH1) provides a tool for further research into the function of a novel gene.
Wu
Yuan-ming
YM
Department of Pathology and Pathophysiology, Fourth Military Medical University, Xi'an 710032, China. wuym@fmmu.edu.cn
Zhang
Xiao-nan
XN
Han
Zhe-yi
ZY
Li
Chun-ying
CY
Chen
Nan-chun
NC
D015153:Blotting, Western; D020868:Gene Silencing / Q000502:physiology; D005822:Genetic Vectors; D006801:Humans; D051793:Hypoxia-Inducible Factor 1 / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D051793:Hypoxia-Inducible Factor 1; D034741:RNA, Small Interfering