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控因子之一。在简单的生物体中，这些基因的突变会导致身体部位的剧烈变化，例如，腿从头上伸出来代替触角。然而，在哺乳动物中，突变通常会产生相当温和的影响。这可能是由于哺乳动物中有大量的Hox基因及其广泛的重叠功能。在这一建议中，我们描述了一种新的重组策略，该策略允许我们同时将移码突变引入连续的Hox基因集，同时保持分散的共享增强子完整。我们建议完成一个丰富的Hox突变小鼠资源的创建，这将允许解剖两侧和旁系Hox基因功能关系。虽然31个Hox基因在肾脏发育过程中表现出强烈的表达，但只有6个基因具有发育功能。我们建议通过创建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HoxD、3、4、8和HoxA9、10、11、HoxC9、10、11和HoxD9、10、11突变的小鼠进行的初步研究发现了有趣的表型，包括肾细胞数量急剧减少、肾小球增大、肾积水和不能形成肾小泡，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下游靶点重叠集的表征和定义其分子功能和生物过程的基因本体分析来定义。利用新的微流体/RNA-Seq方法，对Hox突变体中受干扰的肾脏基因表达模式的研究将扩展到单细胞水平。此外，Chip-Seq将用于确定Hox DNA结合位点的重叠集和区分直接靶标。该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.