A novel technology to generate conditionally inactivated alleles in mice

一种在小鼠体内产生条件失活等位基因的新技术

• 批准号: 7471453
• 负责人: Guo-Hua Fong
• 金额: $ 21.76万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-01 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7471453
• 关键词: 3' Splice Site; Adult; Alleles; Architecture; Back; Biomedical Research; Blood Vessels; Breeding; Bypass; Cardiovascular Diseases; Cloning; Defect; Development; Disruption; Drug Formulations; Elements; Embryo; Ensure; Evaluation; Excision; Exons; Facility Construction Funding Category; Figs - dietary; Frequencies; Gene Expression; Gene Targeting; Generic Drugs; Genes; Genetic Recombination; Goals; Government; Hybrids; Indium; Individual; Internal Ribosome Entry Site; Introns; Kanamycin Kinase; Knock-out; Knockout Mice; Lead; Mediating; Methods; Mission; Mus; Mutation; Neomycin; Open Reading Frames; Persons; Phenotype; Plasmid Cloning Vector; Plasmids; Poly A; Polyadenylation; Positioning Attribute; Procedures; Process; Processed Genes; Production; Purpose; RNA Processing; RNA Splicing; Relative (related person); Reporter; Reporter Genes; Research; Resistance; Role; Site; Speed; Standards of Weights and Measures; Tamoxifen; Technology; Terminator Codon; Testing; Time; Tissues; Transcript; Transfection; Transgenic Mice; United States National Institutes of Health; Upper arm; Vascular System; Work; angiogenesis; cost; design; embryonic stem cell; expression vector; gene function; genetic manipulation; homologous recombination; human disease; innovation; interest; mutant; mutant mouse model; new technology; novel; protein-tyrosine kinase c-src; success; transmission process; vector

DESCRIPTION (provided by applicant): The long term objective of this project is to develop a method that will lead to high throughput production of conditional knock out mice. The immediate goal of this 2 year project is to develop and optimize a novel procedure that may save 6 - 8 months in each conditional knock out project while cutting cost to about half. This technology has two main features: (1) the development of a latent targeting cassette, which can be applied to any gene and activated by Cre to disrupt the target gene. This use of this cassette will eliminate the tedious process of individually inserting loxP sites; (2) the formulation of a novel cassette that allows highly efficient removal of the neomycin resistance marker without ES cell transfection/single colony selection or breeding with Flpe transgenic mice. The project has two specific aims: (1) Test and optimize different components that will contribute to the disruption of a target gene, and construct a conditional gene disruption cassette that is generally applicable to all genes of known structural organizations; (2) Test the utility of the technology by constructing a Csk (C- terminal Src kinase) conditional knock out allele, and analyze vascular defects due to conditional Csk disruption in early embryos and adult tissues. Csk is chosen to test the technology because it has been found previously that Csk-/- (null) embryos display severe vascular defects. By comparing mutant phenotypes in Csk-/- and Csk conditional knock out embryos, it can be concluded if the proposed gene targeting cassette can indeed effectively disrupt gene function. Furthermore, the Csk conditional knock out mice will also be used to investigate the role of Csk in the adult vascular system and to determine whether endothelial Csk is directly required for angiogenesis. This research will enhance the pace of biomedical research in many fields by accelerating the speed of generating conditional knock out alleles, and directly contribute to angiogenesis research by Csk conditional knock out. Thus, the proposed work is relevant to the mission of NIH, and in particular the NHLBI. Studies of human diseases (such as cardiovascular diseases) are heavily reliant on inducible mutant mouse models generated by genetic manipulation. However, current technologies for the introduction of inducible mutations into mice are very time consuming and expensive. This project aims at developing a novel technology that will significantly simplify this procedure and therefore accelerate biomedical research.

描述（由申请人提供）：本项目的长期目标是开发一种高通量生产条件性基因敲除小鼠的方法。这个为期2年的项目的近期目标是开发和优化一种新的程序，在每个有条件淘汰项目中可以节省6 - 8个月，同时将成本降低一半左右。该技术有两个主要特点：（1）开发了一种潜在靶向盒，它可以应用于任何基因，并被Cre激活以破坏靶基因。这种盒的使用将消除单独插入loxP位点的繁琐过程;（2）配制新的盒，其允许在没有ES细胞转染/单菌落选择或用Flpe转基因小鼠繁殖的情况下高效去除新霉素抗性标记。该项目有两个具体目标：（1）测试和优化有助于破坏靶基因的不同组分，并构建普遍适用于已知结构组织的所有基因的条件基因破坏盒;（2）通过构建一个Csk来测试该技术的实用性（C-末端Src激酶）条件性敲除等位基因，并分析早期胚胎和成体组织中由于条件性Csk破坏而导致的血管缺陷。之所以选择Csk来测试这项技术，是因为之前已经发现Csk-/-（空）胚胎显示出严重的血管缺陷。通过比较Csk-/-和Csk条件敲除胚胎中的突变体表型，可以得出结论，所提出的基因靶向盒是否确实可以有效地破坏基因功能。此外，Csk条件性敲除小鼠也将用于研究Csk在成人血管系统中的作用，并确定内皮Csk是否是血管生成所直接需要的。该研究将加快条件敲除等位基因的产生速度，从而推动生物医学多领域的研究步伐，并直接为Csk条件敲除的血管生成研究做出贡献。因此，拟议的工作与NIH的使命有关，特别是与NHLBI有关。人类疾病（如心血管疾病）的研究在很大程度上依赖于通过遗传操作产生的诱导突变小鼠模型。然而，目前用于将诱导型突变引入小鼠的技术非常耗时且昂贵。该项目旨在开发一种新技术，大大简化这一过程，从而加速生物医学研究。