Insertional mutagenesis screen for otic genes in Xenopus

描述（由申请人提供）：本研究的主要目标是 非洲爪蟾耳发育和功能相关基因鉴定和突变 （青蛙）。青蛙的外部胚胎发育特征明显 可以通过快速的视觉观察， 检查我们将使用 含有剪接受体（SA）序列和随后的标记的基因诱捕载体 基因、绿色荧光蛋白（GFP）。由于标记基因缺少启动子， 只有当它正确整合到一个外显子中， 或内源基因的内含子。表达明亮的GFP， 插入诱变在活胚胎中是可见的。此外，由于 捕获的（和预期突变的）基因总是用GFP标记，人们可以很容易地 区分携带突变的胚胎和不携带突变的胚胎。我们将 使用视觉检查来筛选带有GFP插入物的蝌蚪， 转基因到在耳发育过程中表达的基因中。我们提议下列 具体目的：（1）我们将利用插入突变产生稳定的GFP- 携带转基因的真二倍体热带爪蟾的表达系 在耳区表达。这些稳定的谱线将用于研究 由插入产生的突变表型。(2)我们将克隆并描述 GFP转基因已插入的耳基因。(3)我们将描述 其中表达GFP转基因的组织的发育， 活杂合子胚胎或固定组织中的荧光视频显微镜 使用免疫组织化学（光和EM）。这些研究结果 将提供有关耳炎相关的特定基因的基本知识， 开发以及突变株系，可用于未来的研究， 这些基因在听觉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.