Enhancing Technologies for Mouse Mutagenesis Screens

这项研究的目的是开发技术，以增强和扩大在小鼠中进行诱变筛选的可用资源。诱变筛选提供了鉴定基因功能和生成小鼠模型以研究人类疾病和残疾的遗传基础的机会。从人类和小鼠基因组计划中获得的信息的好处取决于将基因与生物功能联系起来。为了达到这个目的，已经在老鼠身上启动了几个大规模的诱变项目。预计这些项目将产生数百种新的鼠标模型。然而，仍然需要改进筛选方法，使诱变在小鼠中的应用充分发挥其潜力。我们提出了一种产生标记染色体的策略，允许所有类别的突变筛选后代在整个妊娠期和产后生活中被识别。该标记系统具有全基因组应用的潜力，并已被设计用于整合当前的突变筛选方法和最近的进展，即使用生殖系胜任突变的ES细胞来创建具有大缺失或单基因突变的小鼠。这项技术的成功开发将使集中突变筛选成为可能，可以发现两代或最终一代的隐性突变。这些进步增加了进行筛选的机会，以确定用于研究发育的小鼠模型，或恢复需要衰老小鼠的模型，例如癌症。该系统利用了最近的技术，使用增强的绿色荧光蛋白（EGFP）作为一种普遍表达的转基因，允许在整个妊娠期的小鼠和胚胎中轻松识别。我们计划设计和测试一个单一的载体，驱动EGFP和红色荧光（RFP）变体的普遍表达，其中每个颜色盒可以使用位点特异性Cre或Flp重组酶独立去除。使用该系统产生全基因组资源的可行性将通过产生至少5个来自稳定的随机整合的红/绿胚胎干细胞的小鼠系，并处理50个这些胚胎干细胞克隆以确定其在基因组中的位置来进行测试。这种技术和资源的可获得性和便利的转移到大型中心和个人实验室，应该增加使用诱变进行哺乳动物基因组功能分析的范围和效率。

The objective of this research is to develop technologies that will enhance and expand the resources available for performing mutagenesis screens in the mouse. Mutagenesis screens provide the opportunity to identify gene function and generate mouse models to study the genetic basis of human disease and disability. The benefits of the information obtained from The Human and Mouse Genome Projects depend on linking genes with biological function. Toward this end several large scale mutagenesis projects have been initiated in the mouse. It is anticipated that these projects will generate hundreds of new mouse models. However, there is still the need for improved screening methods that will enable mutagenesis applications in the mouse to reach their full potential. We propose a strategy for generating marked chromosomes that permits all classes of mutagenesis screen progeny to be identified throughout gestation and post-natal life. The marker system has the potential for genome-wide application and has been designed to integrate with current mutagenesis screen approaches and recent advances for using germline competent mutagenized ES cells to create mice that harbor either large deletions or single gene mutations. The successful development of this technology will make possible focused mutagenesis screens that can uncover recessive mutations in either two or ultimately one generation. These advances increase opportunities for conducting screens to identify mouse models for the study of development or to recover models that require aging mice, such as cancer. The system takes advantage of recent technologies using the enhanced green fluorescence protein (EGFP) as a ubiquitously expressed transgene, permitting the easy identification of mice and embryos throughout gestation. We plan to design and test a single vector that drives the ubiquitous expression of both EGFP and the red fluorescent (RFP) variant, where each color cassette can be removed independently using site-specific Cre or Flp recombinases. The feasibility of generating a genome-wide resource using this system will be tested through the generation of at least 5 mouse lines derived from stable, randomly integrated red/green ES cells and processing 50 of these ES cell clones to identify their map position in the genome. The availability and facilitated transfer of this technology and these resources to both large centers and individual labs should enhance the range and efficiency of using mutagenesis for functional analysis of the mammalian genome.