AI Article Synopsis
- - The study aimed to create an RNA interference vector to express the human neuropathy target esterase (NTE) gene in mammalian cells.
- - Researchers cloned an insert with a promoter and multiple cloning sites into a vector (pSUPER/neo), followed by ligating oligos that target NTE, leading to the creation of the pSUPER/neo-NTE vector, which was then introduced into specific cell types.
- - The resulting vector successfully inhibited NTE activity in mammalian cells, demonstrating that a stable expression vector for double-stranded RNA of NTE was effectively constructed.
Article Abstract
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.
Conclusion: Stable eukaryotic expression vector of double-stranded RNA of NTE, pSUPER/neo-NTE, has been constructed successfully with promoter substitution strategy.
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