A cell base system for compounds regulating tau splicing

调节 tau 剪接的化合物的细胞基础系统

• 批准号: 6581033
• 负责人: JIANHUA ZHOU
• 金额: $ 18.6万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-12-15 至 2004-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6581033
• 关键词: RNA splicing; cell line; drug screening /evaluation; gene expression; gene mutation; genetic regulation; immunofluorescence technique; immunoprecipitation; microarray technology; neural degeneration; neuropathology; polymerase chain reaction; tau proteins; transfection; western blottings

DESCRIPTION (provided by applicant): Tauopathies, formation of insoluble intraneuronal aggregates made of tau protein are major clinical features of several neurodegenerative disorders including frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17). Identification of mutations in the tau gene from TDP-17 patients directly linked the tau gene to these diseases. Several of these mutations alter splicing of exon 10, resulting in shifting of 4R/3R tau ratios in favor of 4R tau. We have recently developed a cell-based system to identify compounds and small molecules that can regulate exon 7 splicing of the SMN genes. The cell- based system has been successfully tested and used for identification of compounds. Similarities between exon 7 splicing of the SMN gene and exon 10 splicing of the tau gene lead us to believe that cell-based systems can also be established for exon 10 splicing of the tau gene and compounds can be identified to regulate exon 10 splicing. The goal of this proposal is to develop the cell-based assays for identification of small molecules that modulate exon 10 splicing. These molecules are potentially used to correct the ratios between 4R/3R tau isoforms in neurodegenerative diseases such as FTDP-17. The first aim of this project is to generate mini-gene constructs by modifying published mini-genes used for tau splicing studies with a reporter gene, luciferase attached so that efficiency of exon 10 splicing can be measured by luciferase activities. RT-PCR and activities of luciferase will be used to validate these constructs after transient transfection into cell lines. At the same time, mutant tau mini-genes that are expected to include exon 10 into their mRNAs will be also constructed and tested. The second aim of this project will be to establish stable cell lines with exon 10 included (mutant) or exon 10 excluded (normal) mini-gene constructs. The cell lines will be validated by RT-PCR and by SR proteins, which have been shown to affect splicing of the exon 10. The optimized cell lines will be then used in high (low) throughput screens (HTS, LTS).

描述(由申请人提供)： Tau病，即由tau蛋白组成的不可溶的神经元内聚集体的形成是几种神经退行性疾病的主要临床特征，包括额颞叶痴呆和17号染色体连锁帕金森病(FTDP-17)。发现TDP-17患者的tau基因突变直接将tau基因与这些疾病联系在一起。其中几个突变改变了外显子10的剪接，导致4R/3R tau比率向4R tau转变。我们最近开发了一种基于细胞的系统来识别能够调节SMN基因外显子7剪接的化合物和小分子。基于细胞的系统已经成功地进行了测试，并用于化合物的鉴定。SMN基因外显子7的剪接和tau基因的外显子10剪接之间的相似性使我们相信，也可以建立基于细胞的系统来剪接tau基因的外显子10，并且可以鉴定出调节外显子10剪接的化合物。这项建议的目标是开发基于细胞的分析方法，以识别调节外显子10剪接的小分子。这些分子有可能用于纠正神经退行性疾病(如FTDP-17)中4R/3R tau亚型之间的比率。该项目的第一个目标是通过修改已发表的用于tau剪接研究的微型基因来产生微型基因结构，该微型基因带有附加的荧光素酶报告基因，从而可以通过荧光素酶活性来衡量外显子10的剪接效率。RT-PCR和荧光素酶活性将在瞬时转染细胞系后用于验证这些构建体。与此同时，突变的tau微型基因也将被构建和测试，这些基因预计将包括外显子10到其mRNAs中。该项目的第二个目标是建立包含外显子10(突变)或排除外显子10(正常)微基因结构的稳定细胞系。这些细胞系将通过RT-PCR和SR蛋白质进行验证，这些蛋白质已被证明影响外显子10的剪接。然后，优化的细胞系将用于高(低)通量筛选(HTS，LTS)。