POSITIONAL CLONING OF MURINE AIRWAY RESPONSIVENESS GENES

在过去的十年中，从许多来源积累的数据表明，气道对支气管收缩剂的反应性。在没有特异性致敏或挑战的情况下，受遗传控制。例如，在人类中，与气道高反应性相关的基因位点已在染色体5q31-q33和11q13上被鉴定出来。虽然已经确定了与气道高反应性相关的基因位点，但尚未确定负责赋予这一特征的人类基因。在人类中识别这些基因的两个困难是：1)无法为人类受试者提供严格的环境暴露控制，如抗原或环境污染物，这可能会改变气道反应。2)人类基因组的多样性，使得在近亲繁殖的种群中鉴定负责特定试验的基因相当困难。由于鉴定气道反应性基因的潜在重要性，我们和其他人已经使用近亲繁殖的小鼠来鉴定在没有环境干预的情况下与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位点突变的C57BL/6J小鼠与A/J小鼠之间的其他杂交中得到证实。这些杂交将提供在没有环境或过敏性致敏的情况下，需要肥大细胞表型表达的基因在传递气道高反应性中的作用的具体信息。对这些杂交进行分析之后，将应用一种新的方法来识别气道反应性、loc、表型驱动的重复回交。这种方法利用了最近引入的气压容积描记技术，该技术允许小鼠无创地进行气道反应性表型分析。这种方法将使用两个交叉A/JxC57/BL/6J和A/JxC3H/HeJ；A/J为高响应菌株，C57BL/6J或C3H/HeJ为低响应菌株。在两个杂交组合A/JxC57BL/6J和A/JxC3H/HeJ中通过标准QTL法和重复回交法鉴定的位点进行候选基因搜索。潜在的候选基因将测试A/J与C47BL/6J或C3H/HeJ之间的序列多样性，如果存在这些基因的致病作用，则通过适当的互补和缺失实验来断言。如果在已鉴定的位点上没有候选基因，则将在已鉴定的区域中搜索BACs和YACs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.