Core--Embryonic Stem Cell Gene Manipulation

核心--胚胎干细胞基因操控

• 批准号: 7312581
• 负责人: Gerald W. Dorn
• 金额: $ 43.29万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-01-01 至 2009-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7312581
• 关键词: artificial chromosomes; biomedical facility; biotechnology; disease /disorder model; electroporation; embryonic stem cell; experimental designs; gene expression; gene targeting; genetic manipulation; genetically modified animals; heart failure; laboratory mouse; model design /development; transfection /expression vector

Among the themes of this SCCOR proposal, one that has played a major role in its development is the delineation of critical cardiac signaling pathways and identification, in the components of these pathways, of human genetic polymorphisms that modify the susceptibility to, or progression of, heart failure. Our group of investigators has applied a systematic vertically-integrated analysis for identifying novel polymorphisms in cardiac signaling and calcium-handling genes, defining associations with specific clinical features or outcomes, determining the functional effects of these polymorphisms using in vitro expression systems, and establishing the pathophysiological effects of these polymorphisms in intact biological systems, i.e. genetically manipulated mice and rabbits. Although associative and case-control human studies drive these investigations, and will clearly provide the biological framework for mechanistic studies, the development of suitable mouse models will permit experiments to be performed on individual gene variants that are simply not attainable with human subjects. To facilitate this vertical scientific approach, Core D will perform complex manipulations of the mouse genome in order to generate optimal mouse models for the SCCOR investigators' mechanistic studies, including conditional and conventional knock-outs, knock-ins, multi-gene substitutions, and targeted transgenesis. In support of the SCCOR projects, the Core will assist in the design of targeting strategies, will engineer targeting vectors, will electroporate, select for and identify targeted ES cells, and will perform blastocyst injection for generation of chimeric mice.

在这项SCCOR提案的主题中，在其发展过程中发挥了重要作用的一个主题是描绘关键的心脏信号通路，并在这些通路的组成部分中识别改变心力衰竭易感性或进展的人类基因多态。我们的研究小组应用了系统的垂直整合分析来确定心脏信号和钙处理基因的新的多态，定义与特定临床特征或结果的关联，使用体外表达系统确定这些多态的功能影响，并在完整的生物系统中建立这些多态的病理生理效应，即基因操纵的小鼠和兔子。尽管联合和病例对照人体研究推动了这些研究，并将明确提供生物学框架 机理研究，合适的小鼠模型的开发将允许实验 在人类受试者无法获得的单个基因变异上进行研究。为了促进这一垂直的科学方法，Core D将对小鼠基因组进行复杂的操作，以便为SCCOR研究人员的机制研究生成最佳的小鼠模型，包括条件和常规的敲除、敲入、多基因替换和靶向转基因。为支持SCCOR项目，该中心将协助设计靶向策略，设计靶向载体，电穿孔，选择和识别目标ES细胞，并将进行囊胚注射以产生嵌合体小鼠。