Construction of RNA interference expression vectors of human neuropathy target esterase and its inhibition for expression of NTE in mammalian cells.
- Author:
Ping-an CHANG
1
;
Rui CHEN
;
Wei LI
;
Yi-jun WU
Author Information
- Publication Type:Journal Article
- MeSH: Animals; COS Cells; Carboxylic Ester Hydrolases; genetics; physiology; Cell Line, Tumor; Cercopithecus aethiops; Genetic Vectors; RNA Interference; RNA, Small Interfering; biosynthesis; genetics; Transfection
- From: Chinese Journal of Industrial Hygiene and Occupational Diseases 2006;24(1):27-30
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo construct the RNA interference expression vector for expression of human neuropathy target esterase (NTE) gene in mammalian cells.
METHODSSpe I and Xho I-digested insert from pSUPER, which comprised H1 RNA polymerase III promoter and the multiple cloning sites, were cloned into the compatible in the pcDNA3.1 (+) to generate pSUPER/neo that could express small interfering RNA in mammalian cells. The annealed oligos targeting the expression of NTE were ligated into pSUPER/neo vector digested with Bgl II and Hind III to generate pSUPER/neo-NTE, which was transfected into COS7 and SH-SY5Y cells. The inhibitory effect of the expression of NTE was detected by western blot analysis and the enzyme activity assay.
RESULTSpSUPER/neo-NTE could stably express double-stranded RNA of NTE. The expression of pSUPER/neo-NTE in COS7 and SH-SY5Y cells could efficiently inhibit the activity of NTE in the mammalian cells.
CONCLUSIONStable eukaryotic expression vector of double-stranded RNA of NTE, pSUPER/neo-NTE, has been constructed successfully with promoter substitution strategy.
