Development of tools for site-directed analysis of gene function

基因功能定点分析工具的开发

• 批准号: 10187374
• 负责人: KARL J CLARK
• 金额: $ 6.23万
• 依托单位: IOWA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-15 至 2021-08-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10187374
• 关键词: AGFG1 gene; Actins; Address; Adhesions; Adopted; Adult; Affect; Alleles; Animal Model; Animals; Annexins; Binding; Biological; Blood Vessels; Blood flow; CRISPR/Cas technology; Carrier Proteins; Catalogs; Cell Communication; Cell Proliferation; Cell membrane; Cells; Complex; Custom; Cytoskeleton; DNA Double Strand Break; Data; Defect; Development; Disease; Disease Progression; Endothelial Cells; Endothelium; Event; Female; Fetal Development; Financial compensation; Gene Delivery; Gene Expression; Gene Family; Gene Mutation; Genes; Genetic; Genetic Polymorphism; Genetic Transcription; Genome; Goals; Growth; Health; Health Promotion; Hour; Human; Hypoxia; KDR gene; Knock-in; Knock-out; Lesion; Malignant Female Reproductive System Neoplasm; Malignant Neoplasms; Malignant neoplasm of ovary; Maternal Mortality; Mediating; Methodology; Methods; Modeling; Molecular; Morphology; Mutation; Nucleotides; Operating System; Organ; Outcome; Ovary; Pathologic Neovascularization; Pathology; Patients; Periodicity; Phenocopy; Phenotype; Physiologic Neovascularization; Placenta; Polycystic Ovary Syndrome; Pregnancy; Process; Publishing; RNA Splicing; Reproductive Health; Role; Scaffolding Protein; Signal Transduction; Signaling Molecule; Site; Site-Directed Mutagenesis; System; Techniques; Technology; Testing; Texas; Time; Tissues; Transcript; Uterine hemorrhage; Variant; Woman; Writing; Zebrafish; angiogenesis; beta catenin; cadherin 5; cancer diagnosis; cell motility; corpus luteum; design; endometriosis; female reproductive system; gene function; human disease; human model; in vivo; insertion/deletion mutation; interest; knock-down; malignant breast neoplasm; membrane activity; mutant; new growth; nuclease; reproductive function; reproductive organ; response; tool; tool development; transcription activator-like effector nucleases; wound healing

The overarching goal of this application is to create tools and efficient methods to define genes that can promote human health. While a tremendous amount of data has been cataloged on gene mutation and changes in gene expression associated with complex human disease, our understanding of those genes that could be co-opted to restore patient health is lacking. To address this need and test for genes that when restored to wild type function promote health, we propose develop mutagenic, revertible and conditional alleles that provide spatial and temporal control of gene expression. The ability to make site-specific, untagged mutant alleles in zebrafish and other models has been greatly advanced by custom nucleases that include TALENs and CRISPR/Cas9 systems. These systems operate on the same principle: they are designed to bind to specific sequences in the genome and create a double strand break. The goals of this proposal leverage the activities of TALEN and CRISPR/Cas9 technologies to make site-specific double strand breaks. These tools and techniques will have direct implications for providing precise gene editing techniques to assess the roles of genes in disease and their ability to promote health following disease progression. While we will develop these methodologies in zebrafish due to their ease of gene delivery, we anticipate these methodologies will not only enhance the efficiency of gene editing but will be readily adaptable for use in other model organisms and large animals. In our opinion, this will have important implications for modeling human disease and health in animal systems by greatly enhancing the ability to make predictable alleles, small nucleotide polymorphisms similar to those associated with human disease, and conditional alleles to test for the ability of a gene to restore health.

这个应用程序的首要目标是创建工具和有效的方法来定义 能促进人类健康的基因。虽然大量的数据已经被编目 与复杂人类疾病相关的基因突变和基因表达变化， 我们对那些可以用来恢复病人健康的基因缺乏了解。到 解决这一需要并测试当恢复到野生型功能时促进健康的基因， 我们建议开发诱变的、可回复的和条件性的等位基因， 基因表达的时间控制。制造位点特异性的无标签突变等位基因的能力 在斑马鱼和其他模型中的应用已经通过定制核酸酶得到了极大的改进， TALEN和CRISPR/Cas9系统。这些系统的工作原理相同： 被设计为与基因组中的特定序列结合并产生双链断裂。的 该提案的目标是利用TALEN和CRISPR/Cas9技术的活动， 位点特异性双链断裂。这些工具和技术将直接影响到 提供精确的基因编辑技术，以评估基因在疾病中的作用及其 在疾病进展后促进健康的能力。当我们开发这些 由于它们易于基因递送，我们预计这些方法在斑马鱼中是可行的。 这些方法不仅可以提高基因编辑的效率， 用于其他模式生物和大型动物。在我们看来，这将具有重要意义。 在动物系统中模拟人类疾病和健康的影响， 能够预测等位基因，小核苷酸多态性类似于那些相关的 和条件等位基因来测试基因恢复健康的能力。