Molecular Approaches to Understanding Global Regul...

了解全球监管的分子方法...

• 批准号: 7017030
• 负责人: SUSAN Celia ROBERTS
• 金额: $ 23.03万
• 依托单位: UNIVERSITY OF MASSACHUSETTS AMHERST
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-01-15 至 2008-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7017030
• 关键词: Conifera; affinity chromatography; antiAIDS agent; antineoplastics; bioinformatics; biotechnology; chemical registry /resource; clone cells; complementary DNA; drug design /synthesis /production; functional /structural genomics; gene expression; gene expression profiling; genetic library; genetic regulation; genetic screening; genetically modified plants; messenger RNA; method development; paclitaxel; plants; subtraction hybridization; tissue /cell culture

DESCRIPTION (provided by applicant): Plant cell cultures can be used to alleviate the demands placed on harvest for the supply of important plant-derived medicinals. Due to economic constraints, there are currently few plant cell culture processes used commercially for the manufacture of such products. Limitations in low volumetric productivity and variability in productivity can be overcome through a better understanding of global metabolic control. Plant cell culture has received significant attention as a supply method for the anti-cancer agent paclitaxel, which has been approved by the FDA for the treatment of breast, ovarian and lung cancers as well as the AIDS-related Kaposi's sarcoma. The proposed research project centers on the use of molecular approaches to delineate global metabolic control of paclitaxel accumulation in Taxus cell cultures. We propose a significantly different approach than other research groups, one that uses transcription profiling to identify genes involved in global metabolic control. Such genes will be involved not just in paclitaxel biosynthesis (where all current efforts are focused), but also in transcriptional regulation, transport, secretion and degradation. The specific aims of this study are to (1) identify changes in gene expression in Taxus suspension cultures with enhanced accumulation of paclitaxel through the identification of mRNAs that accumulate differentially, (2) construct high quality cDNA libraries and isolate full-length clones of these differentially expressed mRNAs, (3) predict putative gene function using publicly available bioinformatics resources, and (4) functionally characterize differentially expressed genes using both stable transformation technologies and assaying of relevant enzymatic activities. The results of the proposed research will identify genes involved in global control of paclitaxel metabolism which can then be used in the design of strategies to optimize the plant cell culture process for paclitaxel production and help alleviate the paclitaxel supply problem for both patient treatments and clinical trials. It is important to emphasize that the outcomes of the proposed research, particularly the work centering around methods development, has the potential to significantly impact research on the production of other valuable medicinals from plant cell culture systems.

描述（由申请人提供）：植物细胞培养物可用于减轻对供应重要植物源药物的收获的需求。由于经济限制，目前商业上用于制造此类产品的植物细胞培养方法很少。通过更好地了解全局代谢控制，可以克服低体积生产率和生产率变异性的限制。植物细胞培养作为抗癌剂紫杉醇的供应方法受到了极大的关注，紫杉醇已被 FDA 批准用于治疗乳腺癌、卵巢癌和肺癌以及艾滋病相关的卡波西肉瘤。拟议的研究项目集中于利用分子方法来描述红豆杉细胞培养物中紫杉醇积累的全局代谢控制。我们提出了一种与其他研究小组显着不同的方法，即使用转录分析来识别参与全局代谢控制的基因。这些基因不仅涉及紫杉醇生物合成（当前所有努力的重点），而且还涉及转录调控、运输、分泌和降解。本研究的具体目的是 (1) 通过鉴定差异积累的 mRNA，确定紫杉醇积累增加的红豆杉悬浮培养物中基因表达的变化，(2) 构建高质量 cDNA 文库并分离这些的全长克隆差异表达的 mRNA，(3) 使用公开的生物信息学资源预测假定的基因功能，以及 (4) 使用稳定转化技术和相关酶活性测定来功能表征差异表达的基因。拟议研究的结果将鉴定参与紫杉醇代谢全局控制的基因，然后可用于设计优化紫杉醇生产的植物细胞培养过程的策略，并有助于缓解患者治疗和临床试验的紫杉醇供应问题。需要强调的是，拟议研究的成果，特别是围绕方法开发的工作，有可能对利用植物细胞培养系统生产其他有价值药物的研究产生重大影响。