Recombineering based analysis of Hox function in kidney development

描述(由申请人提供)：HOX基因是发育的关键遗传调节基因之一。在简单的生物体中，这些基因的突变可能会导致身体部位的戏剧性变化，例如，腿从头部突出代替了触角。然而，在哺乳动物中，突变通常产生的影响要温和得多。这可能是哺乳动物中HOX基因数量多得多及其广泛重叠功能的结果。在这项提案中，我们描述了一种新颖的重组工程策略，该策略允许我们同时将移码突变引入连续的HOX基因集，同时保持散布的共享增强子不变。我们建议完成丰富的HOX突变小鼠资源的创建，这将允许解剖侧翼和旁系HOX基因的功能关系。虽然31个HOX基因在肾脏发育过程中表现出强劲的表达，但只有6个基因的发育功能被确定。我们建议通过建立HOX侧翼和旁系功能逐步减少的小鼠来揭示其他HOX基因的作用。对已经突变了HoxD，3，4，8和HoxA9，10，11，HoxC9，10，11和HoxD9，10，11的小鼠的初步研究发现了有趣的表型，包括肾单位数量的急剧减少，肾小球肿大，肾积水，以及无法产生肾小泡，发现了额外的HOX基因在肾脏发育中的重要性。我们还建议利用我们的GUDMAP联盟的专业知识和数据库，对所产生的肾脏表型进行广泛的分析。HOX功能的分子机制将通过描述重叠的下游靶标集合和定义其分子功能和生物学过程的基因本体论分析来确定。利用新型微流控技术/RNA-Seq方法，对HOX突变型受扰肾脏基因表达模式的研究将扩展到单细胞水平。此外，芯片序列将被用来定义重叠的Hox DNA结合位点集，并用于区分直接靶标。这一结果将提高我们对肾脏发育遗传回路的理解，从而增强未来的DNA诊断和iPS介导的病变或畸形肾脏的再生和/或替换。

DESCRIPTION (provided by applicant): The Hox genes are among the key genetic regulators of development. Mutations of these genes in simple organisms can cause dramatic transformations of body parts, with legs for example protruding from the head in place of antennae. In mammals, however, mutations generally give considerably milder effects. This is likely the result of the much greater number of Hox genes in mammals and their extensive overlapping functions. In this proposal we describe a novel recombineering strategy that allows us to simultaneously introduce frameshift mutations into sets of contiguous Hox genes, while leaving interspersed shared enhancers intact. We propose to complete the creation of a rich resource of Hox mutant mice that will allow the dissection of both flanking and paralogous Hox gene functional relationships. While 31 Hox genes show robust expression during kidney development, developmental functions have only been identified for six. We propose to unveil the roles of the other Hox genes by creating mice with stepwise reductions in Hox flanking and paralogous function. Preliminary studies of mice already made with mutations in HoxD, 3, 4, 8 and HoxA9, 10, 11, HoxC9, 10, 11 and HoxD9, 10, 11, have identified interesting phenotypes, including dramatic reductions in nephron number, glomerulomegaly, hydronephrosis, and failure to make renal vesicles, discovering the importance of additional Hox genes in kidney development. We also propose an extensive analysis of resulting kidney phenotypes, leveraging our GUDMAP consortium expertise and database. The moleular mechanisms of Hox function will be defined through characterization of overlapping sets of downstream targets and Gene Ontology analysis defining their molecular functions and biological processes. The study of disturbed kidney gene expression patterns in Hox mutants will extend to the single cell level, using novel microfluidics/RNA-Seq approaches. In addition Chip-Seq will be used to define the overlapping sets of Hox DNA binding sites and to distinguish direct targets. The results will improve our understanding of the genetic circuitry of kidney development and will thereby augment future DNA diagnosis and iPS mediated regeneration and/or replacement of diseased or malformed kidneys.