A Validated shRNA Vector Set for Protein Kinases

经过验证的蛋白激酶 shRNA 载体集

• 批准号: 6934035
• 负责人: LIANG CAO
• 金额: $ 22.67万
• 依托单位: ORIGENE TECHNOLOGIES, INC.
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-05-13 至 2006-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6934035
• 关键词: RNA interference; antineoplastics; biotechnology; cell line; complementary DNA; drug discovery /isolation; expression cloning; gene expression; genetic library; high throughput technology; kinase inhibitor; neoplasm /cancer genetics; neoplasm /cancer pharmacology; plasmids; polymerase chain reaction; protein kinase; protein tyrosine kinase; technology /technique development; transfection /expression vector

DESCRIPTION (provided by applicant): Protein kinases are key regulators of all aspects of neoplasia, including proliferation, angiogenesis, invasion, and metastasis. The completion of human genome allowed a more comprehensive examination of the entire kinase gene family that was last suggested to have 518 genes. These proteins mediate most of the cellular signal transduction processes in eukaryotic cells. By modification of substrate activities, they control transcription, metabolism, cell movement, cell proliferation, differentiation, and apoptosis. In addition to their critical roles in cancer development, protein kinases are also implicated in a wide range of human diseases in the metabolic system, immune system and central nervous system. Protein kinases also emerge as great targets for drug discovery and development, particularly in the area of cancer. Kinases are often critical in regulating signal transduction in response to external signals. They are enzymes that are good targets for traditional pharmaceutical drug development, particularly for screening chemical inhibitors. Kinase inhibitors can be selective due to structure differences in the active sites. Furthermore, as many receptor tyrosine kinases are critical for cancer development, they are also good targets for antibody therapies. With the success of Glivec, Iressa, Herceptin, and Erbitux, more kinase specific drug candidates are in the race to reach the patients. At OriGene, we are committed to facilitate the research and drug development in the arena of protein kinases by generating critical reagents, both expression cDNA clones and inhibitory clones for the entire family of protein kinases. OriGene has built a collection of 460 human full-length kinase genes in an expression system. In the proposed project, we will develop a complete set of validated RNA interference (RNAi) expression vectors for the entire family of human protein kinases. To reach this end, we adapted and optimized a robust shRNA (small hairpin RNA) expression library approach where the gene specific shRNA vectors are generated directly from the cDNA of kinase genes. With this kinase shRNA library method, one (1) can generate a large number of shRNA expression vectors from their cDNA in a throughput manner. The method is faster, cheaper and better than the commonly used synthetic oligos-based plasmid construction approach. The overall objectives of this Phase I grant are: 1) to generate kinase shRNA expression libraries utilizing the available kinase genes; 2) to sequence validate about 8 unique shRNA vectors per kinase gene; 3) to functionally validate at least one shRNA vector per kinase gene in inhibiting the target gene expression. We believe that these products will be essential for all cancer researchers by allowing long-term studies of kinase knockdown both in vitro and in vivo. Phase II of this project is to develop and validate shRNA expression vectors for 5,000 human genes that are implicated in human health and medicine.

描述（由申请人提供）：蛋白激酶是肿瘤形成各个方面的关键调节因子，包括增殖、血管生成、侵袭和转移。人类基因组的完成使得我们能够对整个激酶基因家族进行更全面的检查，最后认为该家族有 518 个基因。这些蛋白质介导真核细胞中的大部分细胞信号转导过程。通过改变底物活性，它们控制转录、代谢、细胞运动、细胞增殖、分化和凋亡。除了在癌症发展中发挥关键作用外，蛋白激酶还与代谢系统、免疫系统和中枢神经系统等多种人类疾病有关。蛋白激酶也成为药物发现和开发的重要靶点，特别是在癌症领域。激酶在调节响应外部信号的信号转导中通常至关重要。它们是传统药物开发的良好靶点，特别是筛选化学抑制剂的酶。由于活性位点的结构差异，激酶抑制剂可以具有选择性。此外，由于许多受体酪氨酸激酶对于癌症的发展至关重要，因此它们也是抗体治疗的良好靶标。随着格列卫、易瑞沙、赫赛汀和爱必妥的成功，更多激酶特异性候选药物正在竞相进入患者手中。在 OriGene，我们致力于通过生成关键试剂（包括整个蛋白激酶家族的表达 cDNA 克隆和抑制克隆）来促进蛋白激酶领域的研究和药物开发。 OriGene 在表达系统中构建了 460 个人类全长激酶基因的集合。在拟议的项目中，我们将为整个人类蛋白激酶家族开发一整套经过验证的 RNA 干扰 (RNAi) 表达载体。为了实现这一目标，我们采用并优化了强大的 shRNA（小发夹 RNA）表达库方法，其中基因特异性 shRNA 载体直接从激酶基因的 cDNA 生成。通过这种激酶 shRNA 文库方法，一 (1) 可以以通量方式从其 cDNA 生成大量 shRNA 表达载体。该方法比常用的基于合成寡核苷酸的质粒构建方法更快、更便宜且更好。第一阶段资助的总体目标是： 1) 利用可用的激酶基因生成激酶 shRNA 表达文库； 2) 对每个激酶基因大约8个独特的shRNA载体进行测序验证； 3) 功能验证每个激酶基因至少一个shRNA载体抑制靶基因表达的能力。我们相信，这些产品对于所有癌症研究人员来说都是必不可少的，因为它们允许对体外和体内激酶敲低进行长期研究。该项目的第二阶段是开发和验证与人类健康和医学有关的 5,000 个人类基因的 shRNA 表达载体。