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| Construction of Cannabinoid Receptor 1 Gene(CNR1) siRNA Expression Vector and Screening of CNR1-interference Positive L6 Cell Clones |
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Abstract The cannabinoid receptor type 1 (CNR1) is a key component of the endocannabinoid system, which has been reported to play a pivotal role in modulating feeding behavior and energy balance. In order to further study on gene function of CNR1, this study was conducted to construct and identify CNR1 gene small interfering(siRNA) expression vectors and screened the stable CNR1- interference positive L6 cell clones. Three pairs of CNR1-specific double-strand siRNAs were designed and inserted into the pYr-1.1vector. The CNR1 gene siRNA expression vectors were identified by restriction enzyme digestion and sequencing. After that siRNAs were transfected with L6 cells by LipofectamineTM(Lip)2000. Then, the transfection efficiency was detected by EGFP and FCM. CNR1 gene expression was determined by Real-time PCR and the stable transgenic L6 cell clones were screened by G418. The results revealed that the CNR1 gene siRNA expression vectors have been constructed successfully. The transient transfection efficiencies of L6 cells were 10.45%(P<0.01), 8.57%(P<0.01)and 8.71%(P<0.01) respectively, and the silencing efficacies of the transient transfected L6 cells were 39%(P<0.05), 64%(P<0.01) and 68%(P<0.01), respectively. The optimal selection concentration of G418 for stable transfected L6 cell clones was 800 μg/mL. The silencing efficacies of CNR-1-positive transgenic cell clones were 43%(P<0.05), 78%(P<0.01) and 91%(P<0.01), respectively. The results showed that CNR1-3 expression vector was optimal silencing vector and CNR1-3 stable transgenic cell clones were best silencing cell line. This study successfully provides CNR1 gene silencing method by siRNA and the screening of CNR1-interference positive L6 cell clones renders basic tools for further studying the functions of CNR1 gene.
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Received: 28 February 2012
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