SBIR Phase II: High throughput screening for splice variant cDNAs

SBIR II 期：剪接变体 cDNA 的高通量筛选

• 批准号: 7327517
• 负责人: JEAN-MICHEL A LELIAS
• 金额: $ 43.14万
• 依托单位: GENOFI, LLC
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-03-01 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7327517
• 关键词: Alternative Splicing; Breast; Cancer Etiology; Cancer cell line; Cataloging; Catalogs; Cessation of life; Cloning; Code; Collection; Colon; Communities; Complementary DNA; Data; Data Set; Databases; Development; Diagnostic; Disease; Equipment and supply inventories; Evaluation; Event; Exons; Experimental Designs; Facility Construction Funding Category; Fractionation; Future; Genbank; Gene Expression Regulation; Gene Targeting; Genes; Genetic Polymorphism; Genomics; Genus Cola; Goals; Health; Hereditary Disease; Human; International; Investigation; Libraries; Lung; Malignant Neoplasms; Maps; Messenger RNA; Methodology; Methods; Molecular; Mus; Normal tissue morphology; Numbers; Open Reading Frames; Personal Satisfaction; Phase; Play; Polymerase Chain Reaction; Process; Prostate; Protein Isoforms; Proteins; Proteome; Protocols documentation; Publications; RNA; RNA Splicing; Range; Rattus; Research; Research Personnel; Role; Scientist; Screening procedure; Small Business Funding Mechanisms; Small Business Innovation Research Grant; Source; System; Time; Tissues; Transcript; Variant; Work; cDNA Library; computerized tools; cost; design; direct application; drug development; genome sequencing; high throughput screening; human Numb protein; human tissue; interest; mutant; novel; research study; size; tool; tumor; web interface

DESCRIPTION (provided by applicant): The catalog of human splice variant mRNAs is believed to be far from complete with many undiscovered isoforms likely to play an important role in health and disease. The current practice of hunting for splice variants by screening cDNA libraries one at a time is both time consuming and expensive. Higher throughput and more cost-effective methods of screening would be helpful in accelerating this important enterprise. The aim of this project is to create a valuable research tool for the rapid identification of alternative splice isoforms. The Library Sampler described in this proposal represents a collection of high-quality cDNA libraries organized in two panels of 384 low-complexity pools that can be used to identify splice variants, polymorphisms and mutants from a broad range of normal tissues, tumors and cancer cell lines. The main advantage of this proposed work is to allow a simple PCR approach to efficiently screen a large diversity of human sources represented by over 9 million cDNA clones. Due to the low complexity of the cDNA pools the PCR products can be sequenced directly and used in sub-cloning experiments, giving the researcher an immediate answer on a set of mRNA isoforms corresponding to the gene of interest. A graphical web interface will be developed using a comprehensive eukaryotic splice variant database to assist scientists with their experimental design, and a set of novel splice isoforms from a list of selected genes will be identified to demonstrate the value of this new discovery tool. The Library Sampler will provide the researcher community with a powerful system to quickly identify all variant transcripts expressed from a single gene. Cataloging all the mRNA isoforms will be fundamental for understanding the complexity of the human proteome, and since many of these variants have been associated with genetic diseases, their discovery will offer new potentials for drug development and molecular diagnostics. In October 2004 the International Human Genome Sequencing Consortium reduced the estimated number of human protein-coding genes to only 20,000 to 25,000 genes, a surprisingly low number for our species, suggesting that gene regulation is far more important than gene number. It is well known that most of our genes produce more than one protein by alternative splicing of their primary transcript, but the identification of all these splice variant forms is far from complete. The proposed Library Sampler provides a powerful research tool to rapidly identify these splice isoforms from a broad range of normal tissues, tumors and cancer cell lines, and because many splice isoforms have been associated with human genetic diseases, their discovery will offer new potentials for drug development and molecular diagnostics.

描述（由申请人提供）：据信人类剪接变体mRNA的目录远不完整，其中许多未发现的同种型可能在健康和疾病中起重要作用。目前通过一次筛选一个cDNA文库来寻找剪接变体的做法既耗时又昂贵。更高的通量和更经济有效的筛选方法将有助于加速这一重要事业。本项目的目的是建立一个有价值的研究工具，快速识别的选择性剪接异构体。本提案中描述的文库取样器代表了由两组384个低复杂性库组成的高质量cDNA文库的集合，其可用于从广泛的正常组织、肿瘤和癌细胞系中鉴定剪接变体、多态性和突变体。这项工作的主要优点是允许一个简单的PCR方法，以有效地筛选超过900万个cDNA克隆代表的人类来源的大的多样性。由于cDNA库的复杂性低，PCR产物可以直接测序并用于亚克隆实验，为研究人员提供了一组对应于感兴趣基因的mRNA亚型的即时答案。将使用一个全面的真核生物剪接变体数据库开发一个图形化的网络界面，以帮助科学家进行实验设计，并从一系列选定的基因中确定一组新的剪接异构体，以证明这种新的发现工具的价值。文库取样器将为研究人员提供一个强大的系统，以快速识别从单个基因表达的所有变体转录本。对所有mRNA异构体进行分类将是理解人类蛋白质组复杂性的基础，由于这些变体中的许多与遗传疾病有关，它们的发现将为药物开发和分子诊断提供新的潜力。2004年10月，国际人类基因组测序联盟（International Human Genome Sequencing Consortium）将人类蛋白质编码基因的估计数量减少到只有20，000到25，000个，这对我们这个物种来说是一个令人惊讶的低数字，这表明基因调控比基因数量重要得多。众所周知，我们的大多数基因通过选择性剪接它们的初级转录物产生一种以上的蛋白质，但是所有这些剪接变体形式的鉴定还远未完成。拟议的文库采样器提供了一个强大的研究工具，以快速识别这些剪接异构体从广泛的正常组织，肿瘤和癌细胞系，因为许多剪接异构体已与人类遗传疾病，他们的发现将提供新的药物开发和分子诊断的潜力。