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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    https://doi.org/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 Author(s): LIU Zhengjuan,Email:liuzj_1963@yahoo.com.cn   
Issue Date: 05 March 2009
 Cite this article:   
Jie BIAN,Yuchuan WANG,Yongli ZHAO, et al. Construction and identification of lentiviral RNA interference vector of rat leptin receptor gene[J]. Front Med Chin, 2009, 3(1): 57-60.
 URL:  
https://academic.hep.com.cn/fmd/EN/10.1007/s11684-009-0003-z
https://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.
1 Robbins J M, Khan K S, Lisi L M, Robbins S W, Michel S H, Torcato B R. Overweight among young children in the Philadelphia health care centers: incidence and prevalence. Arch Pediatr Adolesc Med , 2007, 161(1): 17–20
2 Bueno G, Bueno O, Moreno L A, Garcia R, Tresaco B, Garagorri J M, Bueno M. Diversity of metabolic syndrome risk factors in obese children and adolescents. J Physiol Biochem , 2006, 62(2): 125–133
3 Nakanishi T, Li R, Liu Z, Yi M, Nakagawa Y, Ohzeki T. Sexual dimorphism in relationship of serum leptin and relative weight for the standard in normal-weight, but not in overweight children as well as adolescents. Eur J Clin Nutr , 2001, 55(11): 899–993
4 Fleisch A F, Agarwal N, Roberts M D, Han J C, Theim K R, Vexler A, Troendle J, Yanovski S Z, Yanovski J A. Influence of serum leptin on weight and body fat growth in children at high risk for adult obesity. J Clin Endocrinol Metab , 2007, 92(3): 948–954
5 Augustine R A, Grattan D R. Induction of central leptin resistance in hyperphagic pseudopregnant rats by chronic prolactin infusion. Endocrinology , 2008, 149(3): 1049–1055
6 Liu Z J, Bian J, Liu J, Endoh A. Obesity reduced the gene expression of leptin receptors in hypothalamus and liver. Horm Metab Res , 2007, 39(7): 489–494
7 Liu Z J, Endoh A, Li R S, Ohzeki T. Effects of leptin and dexamethasone on long and short leptin receptor mRNA. Pediatr Int , 2004, 6(5): 561–564
8 Leung R K, Whittaker P A. RNA interference: from gene silencing to gene-specific therapeutics. Pharmacol Ther , 2005, 107(2): 222–239
9 M?kinen P I, Koponen J K, K?rkk?inen A M, Malm T M, Pulkkinen K H, Koistinaho J, Turunen M P, Yl?-Herttuala S. Stable RNA interference: comparison of U6 and H1 promoters in endothelial cells and in mouse brain. J Gene Med , 2006, 8(4): 433–441
10 Nishitsuji H, Ikeda T, Miyoshi H, Ohashi T, Kannagi M, Masuda Y. Expression of small hairpin RNA by lentivirus-based vector confers efficient and stable gene-suppression of HIV-1 on human cells including primary non-dividing cells. Microbes Infect , 2004, 6(1): 76–85
11 Yamamoto T, Miyoshi H, Yamaoto N, Yamamoto N, Inoue J, Tsunetsugu-Yokota Y. Lentivirus vectors expressing short hairpin RNAs against the U3-overlapping region of HIV nef inhibit HIV replication and infectivity in primary macrophages. Blood , 2006, 108(10): 3305–3312
12 Baile C A, Bella-Fera M, Martin R J. Regulation of metabolism and body fat mass by leptin. Annu Rev Nutr , 2000, 20(1): 105–127
13 Bjorbaek C, Kahn B B. Leptin signaling in the central nervous system and the periphery. Recent Prog Horm Res , 2004, 57(2): 305–331
14 Funahashi H, Yada T, Suzuki R, Shioda S. Distribution, function, and properties of leptin receptors in the brain. Int Rev Cytol , 2003, 224(1): 1–27
15 El-Haschimi K, Lehnert H. Leptin resistance—or why leptin fails to work in obesity. Exp Clin Endocrinol Diabetes , 2003, 111(1): 2–7
16 Burguera B, Couce M E, Curran G L, Jensen M D, Lloyd R V, Cleary M P, Poduslo J F. Obesity is associated with a decrease leptin transport across the blood-brain barrier in rats. Diabetes , 2000, 49(7): 1219–1223
17 Widdowson P S, Upton R, Buckingham R, Arch J, Williams G. Inhibition of food response to intracerebroventricular injection of leptin is attenuated in rats with diet-induced obesity. Diabetes , 1997, 46(11): 1782–1785
18 Martin R L, Perez E, He Y J, Dawson R Jr, Millard W J. Leptin resistance is associated with hypothalamic leptin receptor mRNA and protein down-regulation. Metabolism , 2000, 49(11): 1479–1484
19 Schuber S, Grunert H P, Zeichhardt H, Werk D, Erdmann V A, Kurreck J. Maintaining inhibition: siRNA double expression vectors against coxsackieviral RNAs. J Mol Biol , 2005, 346(2): 457–465
20 Haraguchi S, Saga Y, Natio K, Inoue H, Seto A. Specific gene silencing in the pre-implantation stage mouse embryo by an siRNA expression vector system. Mol Reprod Dev , 2004, 68(1): 17–24
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