Glycome-Enhanced KnockOut (GEKO) Technology

糖类增强敲除 (GEKO) 技术

• 批准号: 8985066
• 负责人: Derek W Abbott
• 金额: $ 30.01万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-10 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8985066
• 关键词: Ablation; Address; Adoption; American; Awareness; Biochemical; Biology; Cell Line; Cells; Chinese Hamster; Chinese Hamster Ovary Cell; Clustered Regularly Interspaced Short Palindromic Repeats; Collection; Communities; Complex; Defect; Derivation procedure; Development; Disease; Dissection; Enzymes; Equipment; Flow Cytometry; Funding; Genes; Genetic; Grant; Growth; Histocompatibility Testing; Human; Human Biology; Immunity; Immunology; In Vitro; Knock-out; Knowledge; Lectin; Libraries; Malignant Neoplasms; Mass Spectrum Analysis; Methodology; Molecular Biology; Mus; Mutation; Nerve Degeneration; Ovary; Pathway interactions; Phase; Polysaccharides; Population; Protein-Carbohydrate Interaction; Proteomics; Reagent; Recombinant Proteins; Renaissance; Research; Research Personnel; Role; Series; Signal Transduction; Sorting - Cell Movement; Source; Subfamily lentivirinae; System; Techniques; Technology; Time; Tissues; Western Blotting; Work; anticancer research; arm; base; biological systems; chemical synthesis; cost; field study; genetic manipulation; genetic technology; glycosylation; immortalized cell; improved; in vivo; instrumentation; interest; new technology; novel; protein function; public health relevance; sialylation; targeted sequencing; tool

 DESCRIPTION (provided by applicant): The field of glycomics is a technically challenging field which commonly utilizes specialized reagents and equipment, thereby limiting its widespread adoption in secondary fields of impact. Moreover, many of the advances have emphasized chemical synthesis, macromolecular interactions, and improved mass spectroscopy methodology, which has pushed the field forward in terms of our biochemical understanding but has also done little to lower the barrier of entry for outside investigators. Due to the lack of expansion into other fields, the impact of the glycome on in vivo biology and our knowledge of how the glycome fits into disease mechanisms remains in its nascent phase. To address this gap, we must strive to provide a more complete set of tools for biomedical investigators to explore the influence of the glycome in any biological system or pathway, and we believe that the way forward is to provide facile genetic technology applicable to both murine and human biology without the requirements for specialized expertise. We therefore propose to utilize CRISPR-based molecular biology to create Glycome-Enabled KnockOut (GEKO) targeting technology for the selective ablation of glycome- associated genes across multiple species. The primary deliverables from the proposed GEKO technology are: (1) 100 validated CRISPR targeting constructs targeting 50 genetic loci for the selective ablation of glycome- associated genes, (2) validated targeting sequences for agile application of the CRISPR technology to other systems, and (3) 50 human HEK293 GEKO cell lines with validated knockout of each glycome-associated gene. These tools will enable glycobiologists and non-glycobiologists alike to easily manipulate the glycome through genetics in vitro, ex vivo, and in vivo; thereby providing an inexpensive gateway to explore the influence of glycans on the function of proteins, cells, and tissues in a wide variety of normal and disease contexts ranging from cancer and neurodegeneration to intracellular signaling and immunity.

 描述（由适用提供）：糖果学领域是一个技术挑战领域，通常使用专门的试剂和设备，从而限制了其在二级影响领域的宽度采用。此外，许多进步都具有紧急化学合成，大分子相互作用和改进的质谱法，这已经使我们的生化理解推动了领域的前进，但也很少降低外部研究人员进入障碍。到期的 由于缺乏向其他领域的扩展，糖核对体内生物学的影响以及我们对糖核如何适应疾病机制如何保持其新生阶段的了解。为了解决这一差距，我们必须努力为生物医学研究人员提供一组更完整的工具，以探索糖斑点在任何生物系统或途径中的影响，并且我们相信前进的道路是提供适用于鼠类和人类生物学的便捷遗传技术，而无需使用专业专家。因此，我们建议利用基于CRISPR的分子生物学来创建启用基因敲除（GEKO）靶向技术，以选择性消融多种物种的Glyce-相关基因。拟议的Geko技术的主要可交付成果是：（1）100个经过验证的CRISPR靶向构造，靶向50个遗传位置，用于选择性消融基因的选择性消融，（2）验证了将CRISPR技术应用于其他系统敏捷的靶向序列，（3）50个50人HEK293 HEK293 GEKO GEKO细胞与每个glycocied Genee cassiped Genee cassiped Genee cassocied Genee cassocied Genee cassoccied Genee。这些工具将使糖生物学家和非血糖学家能够轻松地通过体外，Ex Vivo和In Vivo来轻松操纵糖蛋白。从而提供了廉价的门户，以探索聚糖对蛋白质，细胞和组织功能的影响，在各种正常和疾病环境中，从癌症和神经变性到细胞内信号传导和免疫力。