[Construction of RNA interference expression vectors of human neuropathy target esterase and its inhibition for expression of NTE in mammalian cells].
OBJECTIVE
To construct the RNA interference expression vector for expression of human neuropathy target esterase (NTE) gene in mammalian cells.


METHODS
Spe 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.


RESULTS
pSUPER/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.


CONCLUSIONS
Stable eukaryotic expression vector of double-stranded RNA of NTE, pSUPER/neo-NTE, has been constructed successfully with promoter substitution strategy.
Chang
Ping-an
PA
Laboratory of Molecular Toxicology, State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, China.
Chen
Rui
R
Li
Wei
W
Wu
Yi-jun
YJ
D000818:Animals; D019556:COS Cells; D002265:Carboxylic Ester Hydrolases / Q000235:genetics / Q000502:physiology*; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D005822:Genetic Vectors; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000096:biosynthesis* / Q000235:genetics; D014162:Transfection
D034741:RNA, Small Interfering; D002265:Carboxylic Ester Hydrolases; C021859:neurotoxic esterase