Core B - Mutagenesis and cell expression core

核心 B - 诱变和细胞表达核心

• 批准号: 10247555
• 负责人: Jen Qian Pan
• 金额: $ 36.23万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-30 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10247555
• 关键词: Adopted; Alternative Splicing; Brain; CRISPR/Cas technology; Cell Line; Cells; Communities; Complementary DNA; Cryopreservation; Cultured Cells; Electrophysiology (science); Electroporation; Engineering; Ensure; Epilepsy; Evaluation; Exhibits; Functional disorder; Future; Generations; Genome; Human; Institutes; Ion Channel; Laboratories; Methods; Molecular; Mus; Mutagenesis; Mutant Strains Mice; Mutation; Neonatal; Neurobiology; Neurons; Pattern; Pharmacology; Pharmacology Study; Phenotype; Plasmids; Point Mutation; Potassium; Protein Isoforms; Proteins; RNA Splicing; Recombinant DNA; Recombinants; Research; Services; Site; Site-Directed Mutagenesis; Sodium; Standardization; System; Technology; Time; Transfection; Universities; Variant; Vendor; Work; cellular engineering; cost; cost effective; design and construction; early onset; genetic variant; genome editing; human model; innovative technologies; interest; mouse model; novel; patch clamp; programs; promoter; stable cell line; success; vector; voltage

PROJECT SUMMARY – Core B Our Center proposes to study a large number of previously uncharacterized epilepsy-associated genetic variants in human ion channels using a high-throughput strategy (Project 1). We also propose to generate novel mouse models of prototypical variants after we demonstrate that mutant mouse ion channels exhibit patterns of dysfunction similar to the corresponding human variant (Project 3). Both of these research endeavors will rely heavily upon the engineering of variants in recombinant ion channels and the generation of expression systems in heterologous cultured cells. In addition, Project 3 requires the design and construction of targeting vectors for generation of novel mouse models using genome editing technologies. The Mutagenesis and Cell Expression Core (Core B) will be responsible for these two activities. To accomplish this work in an efficient, cost-effective, and rigorous manner, Core B will exploit standardized high-throughput workflows for site-directed mutagenesis and heterologous cell transfection that unite innovative technologies with economies of scale. The specific variants to be generated will be determined by the Variant Prioritization and Curation Core (Core A). Service activities will be distributed between the Translational Neurobiology program at the Broad Institute of Harvard University and MIT and the Department of Pharmacology at Northwestern University to take advantage of advanced recombinant DNA and cell engineering expertise on these two campuses. Core B will provide the following services: 1) to engineer recombinant human ion channel plasmids and variants for functional evaluation; 2) to generate and distribute heterologous cells transiently or stably expressing human ion channel variants; and 3) to generate and heterologously express prototypical mouse ion channel variants for comparison with the corresponding human variants and to construct murine targeting vectors.

项目概要-核心B 我们的中心建议研究大量以前未表征的癫痫相关的遗传学， 使用高通量策略（项目1）在人类离子通道中的变异。我们还建议生成 在我们证明突变小鼠离子通道表现出 类似于相应的人类变异的功能障碍模式（项目3）。这两项研究 这些努力将严重依赖于重组离子通道中变体的工程改造和 在异源培养细胞中的表达系统。此外，项目3要求设计和施工 使用基因组编辑技术生成新型小鼠模型的靶向载体。的 诱变和细胞表达核心（核心B）将负责这两项活动。 为了以高效、经济和严格的方式完成这项工作，核心B将利用标准化的 用于定点诱变和异源细胞转染的高通量工作流程 具有规模经济的创新技术。将通过以下方式确定要生成的特定变体： 变体优先级和固化核心（Core A）。服务活动将在 哈佛大学和麻省理工学院布罗德研究所的转化神经生物学项目 利用先进的重组DNA和细胞技术， 这两个园区的工程专业知识。核心B将提供以下服务：1）工程师 用于功能评价的重组人离子通道质粒和变体; 2）产生和分布 瞬时或稳定表达人离子通道变体的异源细胞;和3）产生和 异源表达的原型小鼠离子通道变体，用于与相应的人比较 变体和构建鼠靶向载体。