POSITIONAL CLONING OF MURINE AIRWAY RESPONSIVENESS GENES

在过去的十年中，从许多来源获得的数据表明，呼吸道对支气管收缩激动剂的反应性。在没有特定敏感度或挑战的情况下，处于基因控制之下。例如，在人类中，已在染色体5q31-q33和11q13上确定了与呼吸道高反应性相关的遗传位点。虽然已经确定了与呼吸道高反应性有关的遗传基因，但还没有发现与这一特征有关的人类基因。在人类中识别此类基因的两个困难是：1)无法在人类受试者中提供对环境暴露的严格控制，如抗原或环境污染物，这可能会改变呼吸道的反应性。2)人类基因组的多样性，这使得在近交种群中识别负责特定试验的基因变得非常困难。由于识别呼吸道反应性基因的潜在重要性，我们和其他人使用近交系小鼠来识别数量性状基因座S，该基因座与在没有环境干预的情况下发生的呼吸道反应性程度有关。在这项申请中提出的研究中，这些QTL将首先在携带Kit/W或MGF/S1基因座突变的C57BL/6J小鼠与A/J小鼠之间的额外杂交中得到确认。这些交叉将提供关于在没有环境或过敏性敏化的情况下，需要肥大细胞表型表达的基因在传递呼吸道高反应性方面所起的作用的具体信息。在分析这些交叉之后，将应用一种新的方法来识别呼吸道反应性、LOC、表型驱动的重复回交。这种方法利用了最近引入的气压体积描记技术，该技术允许非侵入性地对小鼠的呼吸道反应进行表型鉴定。这种方法将使用A/JxC57/BL/6J和A/JxC3H/Hej两个杂交组合；在这两种情况下，A/J都是高应答毒株，而C57BL/6J或C3H/Hej是低应答毒株。在A/JxC57BL/6J和A/JxC3H/HeJ两个杂交组合中，用标准QTL方法和重复回交方法确定的基因座将被寻找候选基因。将测试A/J和C47BL/6J或C3H/HeJ之间的潜在候选基因的序列多样性，如果存在，则通过适当的互补和缺失实验来断言这些基因的致病作用。在已识别的基因座上没有候选基因的情况下，将在已识别的区域中搜索BAC和YAC以寻找新的基因。

In the past ten years data have accrued from a number of sources which indicate that airway responsiveness to bronchoconstrictor agonists. In the absence of specific sensitization or challenge, is under genetic control. For example, in humans genetic loci linked to airway hyperresponsiveness have been identified on chromosomes 5q31-q33 and 11q13. Although genetic loci linked to airway hyperresponsiveness have been identified, no human genes responsible for conferring this trait have been identified. Two of the difficulties in identifying such genes in humans are: 1) the inability to provide in human subjects tight control of environmental exposures such as to antigens or environmental pollutants, which could potentially modify airway responsiveness. 2) The diversity of the human genome, which makes identification of genes responsible for given trials quite difficult in outbred populations. Because of the potential importance of identifying genes for airway responsiveness, we, and others, have used inbred mice to identify quantitative trait loci (QTL)s linked to the degree for airway responsiveness that occurs in the absence of environmental intervention. In the research proposed in this application, these QTLs will first be confirmed in additional crosses, between C57BL/6J mice carrying mutations at the Kit/W or the Mgf/Sl loci, and A/J mice. These crosses will provide specific information about the role of genes which require mast cells for their phenotypic expression in conveying airway hyperresponsiveness in the absence of environmental or allergic sensitization. The analysis of these crosses will be followed by the application of a novel approach to identifying airway responsiveness, loc, phenotypically driven repetitive backcrossing. This approach takes advantage of the recently introduced technique of barometric plethysmography which allows mice to be phenotyped for airway resp9onsivess non-invasively. This approach will use two crosses A/JxC57/BL/6J and A/JxC3H/HeJ; in both cases A/J i the high responder strain while C57BL/6J or C3H/HeJ is the low responder strain. Loci which are identified by both the standard QTL approach and by the repetitive backcrossing approach in both the crosses, i.e. A/JxC57BL/6J and A/JxC3H/HeJ, will be searched for candidate genes. Potential candidate genes will be tested for sequence diversity between A/J and C47BL/6J or C3H/HeJ and if this exists the causative role of these genes asserted through appropriate complementation and deletion experiments. In the absence of candidate genes at the loci identified, BACs and YACs will be searched for novel genes in the regions identified.