Enhancing Technologies for Mouse Mutagenesis Screens

这项研究的目的是开发技术，将加强和扩大可用于在小鼠中进行诱变筛选的资源。突变筛选提供了鉴定基因功能和产生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益处取决于将基因与生物功能联系起来。为此，已经在小鼠中启动了几个大规模诱变项目。预计这些项目将产生数百种新的小鼠模型。然而，仍然需要改进的筛选方法，以使小鼠中的诱变应用能够充分发挥其潜力。我们提出了一种策略，用于产生标记的染色体，允许所有类别的诱变筛选后代，以确定整个妊娠期和出生后的生活。该标记系统具有全基因组应用的潜力，并已被设计为与当前的诱变筛选方法和最近的进展，使用种系感受态诱变ES细胞，以创建具有大的缺失或单基因突变的小鼠。这项技术的成功开发将使集中诱变筛选成为可能，这种筛选可以在两代或最终一代中发现隐性突变。这些进展增加了进行筛选的机会，以确定用于发育研究的小鼠模型或恢复需要衰老小鼠的模型，如癌症。该系统利用了最新的技术，使用增强型绿色荧光蛋白（EGFP）作为普遍表达的转基因，允许在整个妊娠期间容易地识别小鼠和胚胎。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重组酶独立地去除。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个源自稳定的随机整合的红/绿色ES细胞的小鼠品系并处理50个这些ES细胞克隆以鉴定它们在基因组中的图谱位置来测试。这种技术和这些资源向大型中心和单个实验室的可获得性和便利的转移应提高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.