High throughput screening for splice variant cDNAs

剪接变异 cDNA 的高通量筛选

• 批准号: 7107393
• 负责人: JEAN-MICHEL A LELIAS
• 金额: $ 10万
• 依托单位: GENOFI, LLC
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-01 至 2007-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7107393
• 关键词: genes; high throughput technology; human; library

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 1 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 goal of this proposed work is to develop a consumable research tool that enables rapid and cost- effective screening for splice variant cDNAs. The Library Sampler will be comprised of 15 high-quality cDNA libraries representing 28 human cancer cell lines and 34 human tissues. To evaluate the utility of the Library Sampler, it will be screened by PCR for the known splice variants of 10 genes implicated in various cancers. It is expected that full-length clones representing 70-80% of known isoforms will be identified along with clones representing many novel isoforms. In Phase II, the Library Sampler will be optimized, product application data generated, and a large-scale manufacturing process developed. Relevance to public health: In October 2004 the International Human Genome Sequencing Consortium reduced the estimated number of human protein-coding genes to only 20,000-25,000 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 1 protein by alternative splicing of the primary transcript of the gene but the indentification of all these isoforms is far from complete. The Library Sampler provides a powerful research tool to identify these splice isoforms from a broad range of normal tissues and cancer cell lines, and because many splice isoforms have been associated with human genetic diseases, their discovery offers new potentials for the development of clinical biomarkers and molecular diagnostic testing.

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1个cDNA文库来寻找剪接变体的做法既耗时又昂贵。更高的通量和更具成本效益的筛选方法将有助于加速这一重要事业。这项工作的目标是开发一种可消耗的研究工具，能够快速和成本有效的筛选剪接变异体cDNA。文库取样器将由代表28种人类癌细胞系和34种人类组织的15个高质量cDNA文库组成。为了评估文库取样器的效用，将通过PCR筛选与各种癌症有关的10个基因的已知剪接变体。预期代表已知同种型的70-80%的全长克隆将与代表许多新同种型的克隆一起被鉴定沿着。在第二阶段，将优化库采样器，生成产品应用数据，并开发大规模制造工艺。与公共卫生的相关性：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 - 25，000个基因，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基因的初级转录本产生一种以上的蛋白质，但所有这些异构体的鉴定远未完成。文库取样器提供了一个强大的研究工具，可以从广泛的正常组织和癌细胞系中识别这些剪接异构体，并且由于许多剪接异构体与人类遗传疾病有关，它们的发现为临床生物标志物和分子诊断测试的发展提供了新的潜力。