A novel technology to generate conditionally inactivated alleles in mice

描述（由申请人提供）：该项目的长期目标是开发一种能够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颖的程序，可以在每个条件淘汰项目中节省 6 - 8 个月的时间，同时将成本削减约一半。该技术有两个主要特点：（1）开发了一个潜在的靶向盒，它可以应用于任何基因并被Cre激活以破坏目标基因。使用该盒将消除单独插入 loxP 位点的繁琐过程； (2) 新型盒的配制，可以高效去除新霉素抗性标记，无需 ES 细胞转染/单菌落选择或用 Flpe 转基因小鼠进行育种。该项目有两个具体目标：（1）测试和优化有助于破坏目标基因的不同组件，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Csk（C端Src激酶）条件性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体组织中条件性Csk破坏引起的血管缺陷。选择 Csk 来测试该技术是因为之前发现 Csk-/-（无效）胚胎表现出严重的血管缺陷。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破坏基因功能。此外，Csk条件敲除小鼠还将用于研究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。该研究将通过加快条件敲除等位基因的生成速度，提升多个领域生物医学研究的步伐，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，特别是 NHLBI 的使命相关。人类疾病（例如心血管疾病）的研究严重依赖于通过基因操作产生的诱导突变小鼠模型。然而，目前将诱导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，该技术将显着简化该程序，从而加速生物医学研究。

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.