Induction and Selection for Loss of function Mutations

描述(由申请人提供)： 这个拟议项目的目标是开发一种技术来培育生殖系修饰的大鼠，这种修饰会导致特定选定基因(基因敲除)功能的丧失。将开发有效地用ENU诱变雄性大鼠的方法，然后将这些大鼠饲养到未诱变的雌性大鼠以产生F1幼鼠。然后，这些幼崽将在断奶前使用高效的基于酵母的检测方法进行筛选，以确定选定基因的功能丧失突变。这将通过以下目标来实现：将建立用ENU诱变大鼠品系的方案。突变方案将通过量化具有可见表型突变(表型变异)的幼崽的频率和使用大鼠大蓝试验量化雄性生殖细胞中的特定基因突变来进行评估。将建立检测的阳性对照模型。最初的模型将是大鼠基因敲除株(例如，BRCA2基因敲除)，它具有功能突变但适度的无意义介导的RNA衰退(NMD)。第二个大鼠模型将用于评估将NMD降至最低的策略。具有刺鼠基因突变的大鼠，具有高水平的突变型刺鼠RNA的NMD，将用于这一目的。减少NMD的策略包括：酵母检测优化、药物干预和遗传方法。将使用一系列三种基于酵母的检测方法来有效和经济地识别在选定目标基因中携带功能突变的诱变大鼠。第一种被称为“功能蛋白分析”。在这项测试中，研究人员将通过筛选具有全部或部分蛋白质功能丧失的基因产物来选择功能突变。第二种分析方法称为“cdna截断”法，第三种是“基因组DNA(GDNA)截断法”。这些截断分析筛选干扰蛋白质翻译的突变。基因敲除方案将持续评估其提供基因敲除率的能力。每周将对90只大鼠进行大约10个基因或基因片段的敲除筛查。这项检测将在特定的时间间隔进行修改，以反映研究人员不断改进的技术。在大鼠身上建立基因敲除技术将提供开发独特模型的手段，这些模型将扩展人类疾病背后的遗传学知识，并有助于开发预防和治疗这些疾病的新药。

DESCRIPTION (provided by applicant): The goal of this proposed project is to develop a technology to produce rats with germ line modifications which result in the loss of function of specific selected genes (knockouts). Methods will be developed to efficiently mutagenize male rats with ENU and then breed these rats to non-mutagenized females to produce F1 pups. These pups will then be screened before weaning for loss-of-function mutations in selected genes using efficient yeast-based assays. This will be accomplished through the following aims: protocols for mutagenizing rat strains with ENU will be established. Mutagenesis protocols will be evaluated by quantifying the frequency of pups with visible phenotypic mutations (phenodeviants) and by quantifying specific locus mutations in male germ cells using the rat Big Blue assay. Positive control models for the assays will be established. The initial models will be rat knockout strains (e.g., Brca2 knockout) which have a functional mutation but modest nonsense-mediated RNA decay (NMD). A second rat model will be used to evaluate strategies to minimize NMD. Rats with mutations in the agouti gene, having a high level of NMD of mutant agouti RNA, will be used for this purpose. Strategies to minimize NMD include: yeast assay optimization, pharmacological interventions, and genetic approaches. A series of three yeast-based assays to efficiently and economically identify mutagenized rats carrying functional mutations in selected target genes will be used. The first is referred to as the "functional protein assay." In this assay, the investigators will select for functional mutations by screening for a gene product that has full or partial loss of protein function. The second assay type is referred to as the "cDNA truncation" assay, and the third is the "genomic DNA (gDNA) truncation" assay. These truncation assays screen for mutations that interfere with protein translation. Knockout protocols will continuously be evaluated for their ability to provide knockout rats. Ninety rats per week will be screened for knockouts of approximately ten genes or gene fragments. This assay will be modified at specific intervals to reflect the investigator's improving technologies. Establishing knockout technologies in the rat will provide the means to develop unique models that will extend knowledge of the genetics underlying human diseases and aid in the development of novel drugs to both prevent and treat these diseases.