A novel technology to generate conditionally inactivated alleles in mice

描述(申请人提供)：这个项目的长期目标是开发一种方法，将导致条件基因敲除小鼠的高通量生产。这个为期两年的项目的近期目标是开发和优化一种新的程序，在每个有条件的淘汰项目中可以节省6-8个月，同时将成本削减约一半。这项技术有两个主要特点：(1)开发了一种潜在的靶向盒，它可以应用于任何基因，并被Cre激活以干扰靶基因。这种盒的使用将消除单独插入loxP位点的繁琐过程；(2)研制出一种新型盒，它能够高效去除新霉素耐药标记，而无需ES细胞转染/单克隆选择或与Flpe转基因小鼠繁殖。该项目有两个具体目标：(1)测试和优化导致靶基因中断的不同成分，并构建一个普遍适用于所有已知结构组织基因的条件性基因干扰盒；(2)通过构建CSK(C-末端Src激酶)条件敲除等位基因来测试该技术的实用性，并分析早期胚胎和成人组织中因条件CSK中断而导致的血管缺陷。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条件敲除胚胎的突变表型，可以得出结论，所提出的基因打靶盒是否真的能有效地干扰基因功能。此外，CSK条件性基因敲除小鼠也将被用来研究CSK在成人血管系统中的作用，并确定内皮CSK是否直接需要血管生成。本研究将通过加快条件敲除等位基因的生成速度，加快生物医学在多个领域的研究步伐，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的任务有关，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基因操作产生的可诱导突变小鼠模型。然而，目前将可诱导突变引入老鼠体内的技术非常耗时和昂贵。该项目旨在开发一种新技术，大大简化这一过程，从而加快生物医学研究。

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.