Selecting functional siRNA target sites of hUBE2W based on H1-U6 dual promoter RNAi plasmid.
- Author:
Yingying ZHANG
1
;
Chao LI
;
Zhixin YANG
;
Long XU
;
Hengqi ZHU
;
Xiaowei ZHOU
;
Peitang HUANG
Author Information
1. Beijing Institute of Biotechnology, The Academy of Military Sciences, Beijing 100071, China.
- Publication Type:Journal Article
- MeSH:
Base Sequence;
Humans;
Molecular Sequence Data;
Plasmids;
genetics;
Promoter Regions, Genetic;
genetics;
RNA Interference;
RNA, Messenger;
biosynthesis;
genetics;
RNA, Small Interfering;
genetics;
physiology;
RNA, Small Nuclear;
genetics;
Ubiquitin-Conjugating Enzymes;
genetics;
metabolism
- From:
Chinese Journal of Biotechnology
2008;24(11):1975-1980
- CountryChina
- Language:Chinese
-
Abstract:
hUBEW, a newly identified class I ubiquitin conjugating enzyme, probably plays an important role in tumorigenesis and DNA repair processes. RNA interference (RNAi) is a process in cells to degrade specific homologous mRNA by forming duplex RNA and has been developed into a powerful tool to study gene functions. In this study, the H1-U6 dual promoter RNAi plasmid was constructed and the target sequence for hUbe2w could be transcribed from both strands and form a double stranded RNA with two 5'Uridine overhangs, which closely resembles endogenous functional siRNA. The hUbe2w cDNA was amplified from reverse transcription of the 293FT total RNA by RT-PCR, and then cloned into the pGL3-Control, pCMV-myc and pDsRed-express-C1 plasmids respectively, which were selected as report vectors to detect the RNAi effects. The plasmids were co-transfected into HEK293FT cells, and then the luciferase activity and hUBE2W protein expression were measured respectively. The Resulted reduction of mRNA and protein level demonstrate that the targets of 125 and 259 could significantly inhibit the hUbe2w expression.
