Construction and identification of lentiviral RNA interference vector of rat leptin receptor gene

Abstract

Leptin resistance is a main mechanism of acquired childhood obesity, and the suppression of long form of leptin receptor (OBRb) gene expression in diet-induced obese rats indicates that the down-regulation of OBRb gene expression plays a pivotal role in the mechanism of leptin resistance. The aim of the present study was to construct the lentiviral RNA interference (RNAi) vector of rat OBRb gene and evaluate the effects of siRNA on silencing OBRb gene expression. The target sequence of siRNA-OBRb was designed, and the complementary DNA containing both sense and antisense oligonucleotides was synthesized. After phosphorylation and annealing, these double-stranded DNA was cloned to pRNA-lentivector-VGFP to construct pRNA-Lenti-OBRb-VGFP recombinants with U6-containing promoter, target sequence and Poly III terminator. Then, the products were confirmed by electrophoresis and sequencing analysis, and the effects of RNAi on reducing gene expression were further confirmed by real-time polymerase chain reaction in transfected rat glioma cells expressing OBRb. The target sequence of siRNA-OBRb was successfully cloned to pRNA-lentivector-VGFP, and the RNAi protocol specifically reduced the expression of OBRb mRNA by approximately 80% compared with controls in transfected rat glioma cells. The successful construction of rat lentivirus vectors expressing OBRb-specific shRNA may be useful for further investigation in vivo.

Keywords

receptors, leptin / RNA interference / lentivirus vector

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Acknowledgements

The study was supported by the National Natural Science Foundation of China (Grant Nos. 30571577 and 30771798).

RIGHTS & PERMISSIONS

2014 Higher Education Press and Springer-Verlag Berlin Heidelberg