FORWARD AND REVERSE SCREENS FOR TEMPERATURE-SENSITIVE MUTATIONS IN ZEBRAFISH

描述(申请人提供)：尽管斑马鱼领域快速增长，但一个重要的差距是能够轻松评估基因功能，而不是对基因的第一合子要求。条件性突变，例如在高温或限制性温度(例如33摄氏度)下表达表型，但在低温或允许温度(24摄氏度)下是正常的温度敏感突变，允许在不同的发育时期剥夺或恢复基因功能，以探索基因功能在动物整个生命周期的不同过程中的贡献。斑马鱼是采用温度敏感策略的理想选择，因为它在一生中在24到33摄氏度的温度下茁壮成长。在这个项目中，我们将使用正向和反向遗传筛选技术来评估识别斑马鱼温度敏感突变的策略，组装一个由100个温度敏感突变组成的小组，并采用二次筛选，其中一次是针对母体效应造成的表型，另一次是针对后期幼虫在维持成体干细胞方面的缺陷。 与公共卫生相关：我们将使用正向和反向基因筛查来识别100个温度敏感突变。我们将使用单倍体和二倍体筛查来鉴定在33度表达的突变，但在24度时表现正常。二次筛选将包括在小组中评估突变体对母体的贡献，以及维持循环或静止的成人干细胞。

DESCRIPTION (provided by applicant): Despite the rapid growth of the zebrafish field, an important gap is the ability to easily assess gene function beyond the first zygotic requirement for the gene. Conditional mutations, such as temperature-sensitive mutations that express phenotypes at a high or restrictive temperature (for instance, 33 degrees) but are normal at a low or permissive temperature (24 degrees) allow gene function to be taken away or restored at different times of development, to explore the contribution of gene function in different processes across the animal's life cycle. The zebrafish is ideal for employing temperature-sensitive strategies, because it grows robustly at temperatures from 24 to 33 degrees throughout its life. In this project, we will use forward and reverse genetic screening techniques to evaluate strategies for identifying temperature-sensitive mutations in zebrafish, assemble a panel of 100 temperature-sensitive mutations, and employ secondary screens, including one for phenotypes caused by maternal effects and a second for late larval defects in maintaining adult stem cells. PUBLIC HEALTH RELEVANCE: We will employ forward and reverse genetic screens to identify a panel of 100 temperature-sensitive mutations. We will employ both haploids and diploid screens to identify mutations expressed at 33 degrees, but phenotypically normal at 24 degrees. Secondary screens will include assessing mutants in panel for maternal contribution and for maintaining cycling or quiescent adult stem cells.