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Frontiers of Medicine

ISSN 2095-0217

ISSN 2095-0225(Online)

CN 11-5983/R

Postal Subscription Code 80-967

2018 Impact Factor: 1.847

Front Med Chin    2009, Vol. 3 Issue (1) : 57-60     DOI: 10.1007/s11684-009-0003-z
RESEARCH ARTICLE |
Construction and identification of lentiviral RNA interference vector of rat leptin receptor gene
Zhengjuan LIU(), Jie BIAN, Yuchuan WANG, Yongli ZHAO, Dong YAN, Xiaoxia WANG
Department of Pediatrics, the Second Affiliated Hospital, Dalian Medical University, Dalian 116027, China
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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     
Corresponding Authors: LIU Zhengjuan,Email:liuzj_1963@yahoo.com.cn   
Issue Date: 05 March 2009
URL:  
http://academic.hep.com.cn/fmd/EN/10.1007/s11684-009-0003-z     OR     http://academic.hep.com.cn/fmd/EN/Y2009/V3/I1/57
Fig.1  PCR expression of OBRb in pRNA-lentivector-VGFP. PCR amplification products of OBRb in pRNA-lentivector-VGFP were electrophoresed on a 2% agarose gel and stained with ethidium bromide and the product length was 302 bp. M: 100 bp DNA ladder; 1, 2: OBRb-siRNA; 3, 4: Control-siRNA
Fig.2  DNA sequencing result of pRNA-Lenti-OBRb (part)
Fig.3  Rat glioma cells transfected with pRNA-Lenti-OBRb-VGFP (×250). Green fluorescence was observed 48 hours after transfection with pRNA-Lenti-OBRb-VGFP.
Fig.4  Relative OBRb mRNA levels in rat glioma cells. The rat glioma cells transfected with pRNA-Lenti-OBRb-VGFP were collected and total RNA was isolated 48 h after infection. The amount of OBRb gene expression was normalized to β-actin gene expression. The data are shown as . *: <0.01 control.
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