Mammalian genetic analysis of embryonic neural overgrow*

描述(由申请者提供)：此建议书是完成正在进行的 定位与小鼠轴缺陷相关的基因，并继续研究一种新的 并克隆了斑马鱼的神经性突变。一个终极目标是制作一个模型 在老鼠身上。到目前为止，一些模式生物已经被用于 分子发育研究：那些具有非常好的遗传或基因的研究 这些开发阶段很容易访问，但出于技术原因 基因研究不是一种选择。最近，斑马鱼开始使用 因为它同时满足了这两个标准。它的优点之一是它是一种 脊椎动物，一天可以获得数百个胚胎，这些胚胎 透明且易于可视化。因此，早期的新表型 神经发育是显而易见的，而哺乳动物是无法获得的。 在斑马鱼的逆转录病毒突变筛选中，突变具有病毒标签，可以 可以很容易地克隆它。从麻省理工学院的霍普金斯大学屏幕上，我选择了 分析一种引起组织紊乱和肿瘤样的隐性致死基因(Hi904) 神经系统过度生长。该基因已被克隆，是一个新的基因。我们 将在鱼身上做容易完成的实验，其余的在老鼠身上做 我们可以通过同源重组来重建突变。这些实验 斑马鱼的研究将包括：Hi904消息在胚胎中的表达研究， 用特征明确的分子标记分析评估细胞数量 突变胚胎的神经系统，以及模式和状态 差异化。这将通过原位杂交和 抗体染色。在小鼠身上的实验将包括：获得完整的 包括预期可选剪接形式的小鼠cDNA，表达分析 正常组织和肿瘤组织及胚胎的原位杂交。我们将解决 通过将突变的鱼类细胞移植到裸鼠体内的致瘤性。我们会让 用于功能研究的抗体。小鼠的突变将通过使用 同源重组。Hi904的生化和细胞功能 将通过研究蛋白质的定位和相互作用来探索蛋白质。它是 预计这组基因将定义一种新的肿瘤抑制因子 在儿童原始神经外胚层肿瘤发生中的作用。

DESCRIPTION (provided by applicant): This proposal is to complete an ongoing mapping of genes involved in axis defects in mice and move on to study a new and cloned zebrafish neurogenic mutation. One ultimate goal is to make a model of it in mouse. To date, a number of model organisms have been used in the study of molecular development: those that have really good genetics or ones for which developing stages are readily accessible but for technical reasons genetic studies are not an option. Recently, zebrafish have come into use because it satisfies both criteria. Among its advantages are that it is a vertebrate, hundreds of embryos can be obtained in a day, the embryos are transparent and can be easily visualized. Thus, novel phenotypes in early neural development are evident whereas they would be inaccessible in a mammal. In a retroviral mutant screen in zebrafish, a mutation has a viral tag that can be used to easily clone it. From the Hopkins screen at MIT, I have chosen to analyze a recessive lethal (Hi904) that causes disorganization and tumor-like overgrowth of the nervous system. The gene has been cloned and is novel. We will do experiments that are easily accomplished in fish, and the rest in mice where we can recreate the mutation by homologous recombination. The experiments in zebrafish will include: expression studies of Hi904 message in embryos, analysis with well-characterized molecular markers to assess cell number in the nervous system of mutant embryos, and also patterning and state of differentiation. This will be accomplished by in situ hybridization and antibody staining. The experiments in mice will include: obtaining the complete mouse cDNAs including expected alternative splice forms, expression analysis in normal and tumor tissues and in situ hybridization in embryos. We will address tumorigenicity by transplanting mutant fish cells into nude mice. We will make antibody for functional studies. Mutations in mouse will be generated using homologous recombination. The biochemical and cellular function of Hi904 protein will be explored by studying its localization and interactions. It is expected that this set of genes will define a new tumor suppressor that plays-a role in the development of primitive neuroectodermal tumors of children.