Insertional mutagenesis screen for otic genes in Xenopus

描述(由申请人提供)：本研究的总体目标是 非洲爪蛙耳道发育和功能相关基因的鉴定和突变 (青蛙)。青蛙的外部胚胎发育特征 允许通过快速视觉轻松访问特定的发育阶段 检查。我们将在非洲爪哇进行插入突变筛选，使用 包含剪接受体(SA)序列和标记的基因陷阱载体 绿色荧光蛋白基因。由于标记基因缺乏启动子， 只有正确整合到外显子中，它才能被转录和翻译 或内源基因的内含子。明亮的绿色荧光蛋白的后续表达 插入突变在活胚胎中是可见的。此外，由于 捕获的(和可能发生突变的)基因总是被GFP标记，很容易 区分携带突变的胚胎和不携带突变的胚胎。我们会 使用肉眼检查筛选插入绿色荧光蛋白的小蝌蚪- 转基因进入在听觉发育过程中表达的基因。我们提出以下建议 具体目标：(1)我们将使用插入突变产生稳定的GFP- 携带转基因的真正二倍体热带非洲爪哇表达系 在耳区表达。这些稳定的品系将被用于研究 插入产生的突变表型。(2)我们将克隆和鉴定 绿色荧光蛋白转基因插入的外源基因。(3)我们将描述 绿色荧光蛋白转基因表达的组织发育 活杂合子胚胎或固定组织中的荧光视频显微镜 采用免疫组织化学方法(LIGH和EM)。这些调查的结果 将提供与耳聋相关的特定基因的基本知识 以及可用于未来研究的突变系 这些基因在声学或前庭功能中的特定功能。

DESCRIPTION (provided by applicant): The broad goal of this research is to identify and mutate genes involved in otic development and function in Xenopus (frog). The well-characterized external embryological development of the frog allows simple access to specific developmental stages by rapid visual inspection. We will conduct an insertional mutagenesis screen in Xenopus using a gene-trap vector containing a splice acceptor (SA) sequence followed by a marker gene, green fluorescent protein (GFP). Since the marker gene lacks a promoter, it can only be transcribed and translated if it integrates properly into an exon or intron of an endogenous gene. Expression of the bright GFP following insertional mutagenesis is visible in living embryos. In addition, since the trapped (and prospectively mutated) gene is always marked by GFP, one can easily distinguish between embryos carrying the mutation from ones that do not. We will use visual inspection to screen for tadpoles with insertions of the GFP- transgene into genes expressed during otic development. We propose the following specific aims: (1) We will use insertional mutagenesis to generate stable GFP- expressing lines of the true diploid, Xenopus tropicalis, carrying transgenes expressed in the otic regions. These stable lines will be used for studying the mutant phenotype generated by the insertion. (2) We will clone and characterize otic genes into which the GFP transgene has inserted. (3) We will characterize the development of the tissue in which the GFP-transgene is expressed using fluorescence video microscopy in live heterozygotic embryos or in fixed tissues using immunohistochemistry (both light and EM). Results of these investigations will provide essential knowledge of the specific genes involved in otic development as well as mutant lines that can be used for future studies of the specific function of these genes in acoustic or vestibular functions.