Region Based Functional Annotation of the Mouse Genome

描述(申请人提供)：本研究项目的目标是确定哺乳动物发育所需的基因，并调查花斑缺失复合体所定义的染色体区域的功能内容。随着基因组序列信息的汇编，所确定的基因的结构和调控组织将需要与生物功能联系起来。突变筛选依赖于表型驱动的遗传变异检测，为建立这种联系提供了机会。在这项提案中，我们将结合遗传研究与计算和比较基因组序列分析来对定义的基因组区域进行功能注释。 花斑缺失复合体是一组重叠的缺失，包括小鼠14号染色体远端15-18.5 cM/-39 Mb的区间。在这个区域内已经定位了几个对发育重要的基因座，导致了理解原肠形成、骨骼图案和颅面部缺陷、脊髓畸形和出生时呼吸窘迫的小鼠模型的特征。识别基因和其他保守序列特征的计算和比较基因组分析将与功能研究相结合。传统的两代区域定向ENU突变筛选，利用花椒缺失和毛色斑点，将用于恢复对发育至关重要的隐性突变。此外，我们将开发和使用一种基于绿色荧光蛋白(EGFP)的新型报告系统，该系统利用增强型绿色荧光蛋白(EGFP)和颜色变体进行一代遗传筛选，其中整合了最近开发的胚胎干细胞突变方案。这种方法提供了更高的突变频率，使用了更广泛的诱变剂，并对所有屏幕衍生的后代(小鼠和胚胎)进行了即时视觉分类，便于进行深入的表型分析。在筛选中恢复的突变将使用缺失面板进行精细定位，以便识别与突变表型相关的基因。总之，这些方法将丰富小鼠突变资源，并提供将生物功能与染色体内容和组织联系起来的新见解。

DESCRIPTION (provided by applicant): The goals of this research project are to identify genes required for mammalian development and to investigate the functional content of the chromosomal region defined by the piebald deletion complex. As genomic sequence information is compiled, the structural and regulatory organization of the genes identified will need to be linked with biological function. Mutagenesis screens, relying on phenotype-driven detection of genetic variation, provide an opportunity to establish this connection. In this proposal we will combine genetic studies with computational and comparative genomic sequence analysis to functionally annotate a defined genomic region. The piebald deletion complex is a collection of overlapping deficiencies that encompass a 15-18.5 cM/-39 Mb interval of distal mouse chromosome 14. Several loci important for development have been mapped within this region, resulting in the characterization of mouse models for understanding gastrulation, skeletal patterning and crarnofacial defects, spinal cord malformation and respiratory distress at birth. Computational and comparative genomic analysis to identify genes and additional conserved sequence features will be merged with functional studies. A traditional two-generation regionally directed ENU mutagenesis screen that takes advantage of the piebald deletions and coat color spotting will be used to recover recessive mutations that are essential for development. In addition, we will develop and employ a novel EGFP-based reporter system that takes advantage of the enhanced green fluorescence protein (EGFP) and color variants to conduct a one generation genetic screen that incorporates the recently developed protocols for embryonic stem cell mutagenesis. This approach offers a higher mutation frequency, the use of a wider variety mutagens, and instant visual classification of all screen-derived progeny (mice and embryos) facilitating in depth phenotype analysis. The mutations recovered in the screen will be fine mapped using the deletion panel in order to identify the gene associated with the mutant phenotype. Together, these approaches will enrich the mouse mutant resource and provide new insights relating biological function with chromosome content and organization.