DEVELOPING A RAPID SCREEN TO DETECT OLFACTORY MUTANTS

开发快速筛选来检测嗅觉突变体

基本信息

批准号：
6379550
负责人：
STEVEN L. YOUNGENTOB
金额：
$ 21.33万
依托单位：
UPSTATE MEDICAL UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1999
资助国家：
美国
起止时间：
1999-09-27 至 2003-08-31
项目状态：
已结题

项目摘要

The major objectives of this proposal are: (1) to develop a behavioral assay for screening large numbers of mutagenized mice in a high-throughput environment; and (2) following protocol development and validation, to begin acquiring reference data on standard inbred mouse strains. Our approach will be to develop a technique for estimating the threshold of stimulus-induced sniffing (i.e., respiratory changes) in response to odors, as a method for the rapid quantification of odorant sensitivity. We propose to combine elements of the rigorous psychophysical techniques that are standard in our laboratory with elements from simpler techniques used in olfactory behavioral research. Specifically, we will: (1) apply our experience in measuring an animal's sniffing behavior in response to the presentation of odorant stimuli; (2) apply taste-potentiated odor aversion conditioning to improve stimulus control over the reflexive or unconditioned sniffing response to the presentation of odorant, and thereby increase the sensitivity and reliability of the measure; (3) apply our experience with computer-controlled olfactometry to provide precise control over stimulus generation and delivery; and (4) apply our automated tracking paradigm to rapidly acquire, in a single brief testing session, a threshold estimate of stimulus-induced sniffing. Using our OMP knockout mice as a "gold standard" (i.e., an animal in which a specific gene defect results in an alteration in function without anosmia), we will directly test the efficacy of the procedure to identify a mutant phenotype. Moreover, applying epidemiologic screening principles to the distributions of thresholds for stimulus-induced sniffing in OMP-null and congenic controls, we will establish an operational procedure for the evaluation of unknown animals. We have divided the assay development into five phases designed to: (1) define an appropriate measure of sniffing behavior that is altered between air and odorant stimuli; (2) directly evaluate whether taste-potentiated odor aversion conditioning significantly increases the precision and lowers the threshold for stimulus-induced sniffing, as compared to non-aversion conditioned animals; (3) evaluate the sensitivity, specificity and predictive value of the screening procedure to identify OMP-null mutants, establish comparative reference data for OMP-null and congenic control animals and, on the basis of these data, establish an operational procedure for the future screening of unknown animals; (4) evaluate additional odorants for their utility in the screening procedure; and (5) initiate collection of reference data on inbred strains of mice. For each phase, we will provide detailed information via a Web-based database linked to centralized resources.

该提案的主要目标是：（1）开发一种行为测定，以在高通量环境中筛选大量诱变小鼠；（2）在协议开发和验证之后，开始获取有关标准近交小鼠菌株的参考数据。我们的方法是开发一种技术来估计刺激引起的嗅探（即呼吸变化）的阈值，以响应气味，作为一种快速量化气味敏感性的方法。我们建议将严格的心理物理技术的元素与实验室中标准化的元素与嗅觉行为研究中使用的简单技术的元素结合在一起。具体来说，我们将：（1）运用我们的经验，以响应气味刺激的表现来测量动物的嗅觉行为；（2）采用味觉赋予的气味厌恶条件来改善对反射性或无条件的嗅探反应的刺激控制，从而提高了度量的敏感性和可靠性；（3）运用我们在计算机控制的嗅觉法上的经验，以提供对刺激产生和传递的精确控制；（4）在一次简短的测试会议中，将自动跟踪范式应用于迅速获取，这是刺激引起的嗅探的阈值估计值。使用我们的OMP基因敲除小鼠作为“黄金标准”（即，特定基因缺陷会导致功能变化的动物，而没有厌氧），我们将直接测试该程序的效果以识别突变体表型。此外，将流行病学筛查原理应用于刺激诱导的刺激嗅觉的阈值分布，我们将建立一个操作程序，以评估未知动物。我们已经将测定的开发分为五个阶段，该阶段旨在：（1）定义一种适当的嗅探行为量度，这种措施在空气和气味刺激之间发生了改变；（2）与非抑制条件的动物相比，直接评估味觉受到的气味厌恶条件是否显着提高了精度并降低了刺激引起的嗅探的阈值；（3）评估筛选程序的敏感性，特异性和预测价值，以识别无效的突变体，为无效和先天对照动物建立比较参考数据，并在这些数据的基础上为未知动物的未来筛查建立操作程序；（4）评估其在筛选程序中效用的其他气味；（5）启动有关小鼠近交菌株的参考数据的收集。对于每个阶段，我们将通过链接到集中资源的基于Web的数据库来提供详细的信息。