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: 6689539
• 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 2005-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6689539
• 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蛋白病（由tau蛋白组成的不溶性神经元内聚集体的形成）是多种神经退行性疾病的主要临床特征，包括额颞叶痴呆和与17号染色​​体相关的帕金森病（FTDP-17）。对 TDP-17 患者 tau 基因突变的鉴定将 tau 基因与这些疾病直接联系起来。其中一些突变改变了外显子 10 的剪接，导致 4R/3R tau 比率向有利于 4R tau 的方向转变。我们最近开发了一种基于细胞的系统来识别可以调节 SMN 基因外显子 7 剪接的化合物和小分子。基于细胞的系统已成功测试并用于化合物的鉴定。 SMN基因的外显子7剪接和tau基因的外显子10剪接之间的相似性使我们相信，也可以为tau基因的外显子10剪接建立基于细胞的系统，并且可以鉴定调节外显子10剪接的化合物。该提案的目标是开发基于细胞的检测方法来鉴定调节外显子 10 剪接的小分子。这些分子有可能用于纠正 FTDP-17 等神经退行性疾病中 4R/3R tau 异构体之间的比例。该项目的第一个目标是通过修改已发表的用于 tau 剪接研究的小基因，并附加报告基因和荧光素酶来生成小基因构建体，以便可以通过荧光素酶活性来测量外显子 10 剪接的效率。 RT-PCR 和荧光素酶活性将用于在瞬时转染至细胞系后验证这些构建体。与此同时，预计将外显子 10 纳入其 mRNA 的突变 tau 小基因也将被构建和测试。该项目的第二个目标是建立包含外显子 10（突变体）或外显子 10 排除（正常）小基因构建体的稳定细胞系。这些细胞系将通过 RT-PCR 和 SR 蛋白进行验证，这些蛋白已被证明会影响外显子 10 的剪接。优化后的细胞系将用于高（低）通量筛选（HTS、LTS）。