Functional Genomics in a Simple Model Metazoan

描述(申请人提供)：这项申请将开发强大的技术来操纵基因表达和研究一个重要的新后生动物模型系统的胚胎中的基因功能，线虫载体。媒介线虫全基因组测序和EST分析的惊人新结果表明，脊椎动物与媒介线虫等捕食性动物的基因组内容比苍蝇或线虫有更大的相似性。这一发现使人们对派生的遗传模型系统可能对人类生物学产生的意义产生了怀疑，从而为理解遗传变异和形态复杂性之间的祖先关系提供了机会。维氏微孢子虫的重要系统发育地位将有助于理解脊椎动物发育模式的分子控制差异，但在衍生的蜕皮虫遗传模型系统中没有，这为了解脊椎动物谱系辐射之前部署的古老分子模式机制提供了重要的见解。能够在实验中解剖这些动物的分子通路，对于理解正常人类健康和疾病所涉及的基因调控网络变异的起源至关重要。这项工作利用了联合基因组研究所(能源部)对媒介新城疫进行的完整基因组测序，并将为未来的现场实验提供额外的技术和方法背景。这一应用将大大增强我们启动长期研究计划的能力，该计划旨在发现基因并对所有活着的动物共同的基因调控途径进行功能研究。这一应用的目的是：具体目标1：优化转基因应用的电穿孔传递技术。具体目的2：利用转座子介导的插入技术创造稳定的转基因动物。具体目的3：通过BAC克隆的同源重组获得表达标记基因的转基因动物。已经生成了表达结构，坚实的初步数据以及具有经过验证的成就历史的人员确保了快速的进展。这些数据将为利用增强子陷阱技术发现原肠发育过程中胚层的形成、两侧对称性的起源、神经发育/组织和体细胞/生殖细胞生物学等重要发育事件的基因，以及通过插入突变进行基因网络进化的功能分析提供技术基础。

DESCRIPTION (provided by applicant): This application will develop robust techniques to manipulate gene expression and investigate gene function in embryos of an important new metazoan model system, Nematostella vectensis. Surprising new results from total genome sequencing and EST analyses of N. vectensis indicate that vertebrates share greater similarities in genome content with cnidarians like N. vectensis than either do with flies or nematodes. This revelation casts doubt on the significance that derived genetic model systems may have on human biology and thus provides the opportunity to understand the ancestral relationships between genetic variation and morphological complexity. The crucial phylogenetic position of N. vectensis will allow an understanding of the differences in the molecular control of developmental patterning seen in vertebrates, but absent in derived ecdysozoan genetic models systems, providing important insight into the ancient molecular patterning mechanism deployed before the radiation of vertebrate lineages. Being able to experimentally dissect molecular pathways in these animals is crucial to understanding the origin of variation in genetic regulatory networks involved in normal human health and disease. This work takes advantage of the complete genome sequencing of N. vectensis by the Joint Genome Institute (Department of Energy) and will provide additional technical and methodological background for future experiments in the field. This application will substantially enhance our ability to launch a long term research program for gene discovery and functional investigations of genetic regulatory pathways common to all living animals. The aims of this application are: Specific Aim 1: to optimize electroporation delivery techniques for transgene applications. Specific Aim 2: to create stable transgenic animals using transposon mediated insertion technology. Specific Aim 3: generate transgenic animals expressing loci marked through homologous recombination of BAC clones. Expression constructs have already been generated and solid preliminary data along with personnel with proven history of accomplishment insure that rapid progress will be made. These data will provide the technical basis for gene discovery for important developmental events such as germ layer formation during gastrulation, origin of bilateral symmetry, neural development/organization, and somatic/germ stem cell biology by enhancer trap techniques and for the functional analysis of gene network evolution by insertional mutagenesis.