军事医学2011，Vol.35Issue(4)：272-277,6.
内含子序列对CART基因转录调控作用的初步研究
Regulation role of intron sequence in CART gene transcription
摘要
Abstract
Objective To explore the regulation role of intron sequence in CART gene transcription. Methods CART Intron and CART NRSEnon Intron fragments were respoctively amplified from human genomic DNA isolated from HeLa cells and mesenchymal stem ceils before being cloned into pGL3-Basic-CART32 vector to generate pGL3-Basic-CART32-Intron and pGL3-Basic-CART32-NRSEnon Intron reporter gene constructs. Restriction enzyme digestion and plasmid sequencing methods were used to verify the construction. Subsequently, pGL3-Basic-CART32-Intron, pGL3-Basic-CART32-NRSEnon Intron reporter gene constructs and the vector pGL3-Basic-CART32 were transfected into NTera2/CloneD1 cells and pRLCMV plasmid was co-transfected to normalize for luciferase activity. 1 × 107 NT2/CloneD1 ceils were used for chromatin immunoprecipitation assay with anti-NRSF or anti-IgG antibodies to validate the interaction of neuron-restrictive silencer element (NRSF) protein to the CART intron in vivo. Results Restriction enzyme digestion and plasmid sequencing confirmed the successful construction of pGL3-Basic-CART32-Intron and pGL3-Basic-CART32-NRSEnon Intron luciferase reporters. The activity of the pGL3-Basic-CART32-Intron reporter was decreased by 57.9％ compared to that of pGL3-Basic-CART32 vector (P ＜ 0.05 ). The activity of pGL3-Basic-CART32-NRSEnon Intron reporter was increased by 53.8％relative to pGL3-Basic-CART32-Intran vector. ChIP PCR products were detected with the anti-NRSF antibody in NTera2/CloneD1 cells rather than by control anti-IgG antibody. Conclusion These results indicate CART intron negatively regulates transcription of CART gene through the internal NRSE motif by specific binding to NRSF.关键词
可卡因-苯丙胺调节转录肽/内含子/转录调控/神经元限制性沉默因子/神经元限制性沉默元件Key words
cocaine-and amphetamine-regulated transcript/ intron/ transcriptional regulation/ neuron-restrictive silencer factor/ neuron-restrictive silencer element分类
生物科学引用本文复制引用
张静,裴雪涛,王思涵,杨印祥,姚海雷,陈琳,吕洋,南雪,岳文,李艳华..内含子序列对CART基因转录调控作用的初步研究[J].军事医学,2011,35(4):272-277,6.基金项目
国家高技术研究发展计划(863计划)领域重大项目(2006AA02A107) （863计划）
国家重大科学研究计划项目(2010CB945500) （2010CB945500）
国家自然科学基金项目(81070618) （81070618）
北京市自然科学基金项目(5102036) （5102036）
