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，气道对支气管收缩激动剂的反应性。在没有特定致敏或挑战的情况下，受遗传控制。例如，在人类中，已经在染色体5q31-q33和11q13上鉴定出与气道高反应性相关的遗传基因座。虽然已经确定了与气道高反应性相关的遗传基因座，但尚未确定负责赋予该性状的人类基因。在人类中鉴定这样的基因的两个困难是：1）不能在人类受试者中提供对环境暴露如抗原或环境污染物的严格控制，这可能潜在地改变气道反应性。2)人类基因组的多样性，这使得在远交种群中识别负责特定试验的基因非常困难。由于确定气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确定与在没有环境干预的情况下发生的气道反应性程度相关的数量性状位点（QTL）。在本申请提出的研究中，这些QTL将首先在携带Kit/W或Mgf/Sl基因座突变的C57 BL/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基因的作用的特定信息，所述基因需要肥大细胞的表型表达来在不存在环境或过敏性致敏的情况下传递气道高反应性。这些交叉分析之后将应用一种新的方法来鉴定气道反应性、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进行气道反应性表型分析。该方法将使用两个杂交种A/JxC57/BL/6J和A/JxC3H/HeJ;在两种情况下，A/J是高应答菌株，而C57BL/6J或C3H/HeJ是低应答菌株。在两个杂交即A/JxC57BL/6J和A/JxC3H/HeJ中通过标准QTL方法和通过重复回交方法鉴定的基因座将被搜索候选基因。将检测潜在候选基因在A/J和C47 BL/6J或C3 H/HeJ之间的序列多样性，如果存在，则通过适当的互补和缺失实验确定这些基因的致病作用。在鉴定的基因座处不存在候选基因的情况下，将在BAC和YAC中搜索鉴定的区域中的新基因。

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.