Enhancing Technologies for Mouse Mutagenesis Screens

这项研究的目的是开发技术，以增强和扩大可用于在小鼠中进行突变筛选的资源。突变筛选提供了识别基因功能和产生小鼠模型的机会，以研究人类疾病和残疾的遗传基础。从人类和老鼠基因组项目中获得的信息的好处取决于将基因与生物功能联系起来。为此，已经在小鼠身上启动了几个大规模的突变项目。预计这些项目将产生数百个新的鼠标型号。然而，仍然需要改进的筛选方法，使小鼠的诱变应用能够充分发挥其潜力。我们提出了一种产生标记染色体的策略，允许所有类别的诱变筛选后代在整个妊娠和出生后的生命中被识别。该标记系统具有在全基因组范围内应用的潜力，并已被设计为与当前的突变筛选方法和最新进展相结合，使用具有生殖系能力的突变的ES细胞来创造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突变筛选成为可能，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查的机会，以识别用于发育研究的小鼠模型，或恢复需要老化小鼠的模型，如癌症。该系统利用了使用增强型绿色荧光蛋白(EGFP)作为普遍表达的转基因的最新技术，使得在整个怀孕过程中很容易识别小鼠和胚胎。我们计划设计和测试一个单一的载体，它驱动EGFP和红色荧光(RFP)变体的无处不在的表达，其中每个色盒可以使用位点特定的Cre或FLP重组酶独立移除。使用该系统产生全基因组资源的可行性将通过从稳定的、随机整合的红/绿ES细胞产生至少5个小鼠品系并处理50个这些ES细胞克隆来确定它们在基因组中的图谱位置来测试。这项技术和这些资源的可获得性和便利转移到大型中心和单独的实验室，应该会扩大利用突变技术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.