Gene Manipulation of Cephalopod Development

描述(申请人提供)：理解正常的人类发育依赖于对潜在生物学机制的实验洞察的动物模型系统。该项目的长期目标是利用头足类生物学来了解人类器官发生，包括大脑发育。在动物中，人类是拥有不成比例的大大脑的大型有机体。头足类包括鱿鱼、章鱼和墨鱼，是最大的非哺乳动物海洋生物，在所有无脊椎动物中拥有最大的大脑。要在机械学水平上研究头足类的发育，需要鉴定一种易于获得、易于饲养的头足类动物，以及一种允许进行实验操作的胚胎学。调查人员已经确定了这样一种制剂，加利福尼亚州的泥滩章鱼O.bimaculoides。到目前为止，他们已经开发出了从胚泡到孵化的胚胎阶段的体外培养和整体原位杂交的方法。研究人员还分离出了一大批关键发育控制基因的cDNA。为了完成这个模型系统，他们需要开发在这些头足类胚胎中进行基因操作的可靠方法。具体目标1是构建基于章鱼EF1-a和泛素C基因的头足类动物特异性表达载体。具体目标2是开发将质粒输送到章鱼胚胎的方法，研究人员将测试作为方法的电穿孔和使用阳离子聚合物的化学转基因。基因操作资源和方法的发展将使以假说为导向的头足类动物发育的分子和细胞机制研究成为可能。

DESCRIPTION (provided by applicant): Understanding normal human development depends on animal model systems for experimental insight into underlying biological mechanisms. The long-term goal of this project is to use cephalopod biology to understand human organogenesis, including brain development. Among animals, human beings are large organisms with disproportionately large brains. Cephalopods, which include squids, octopuses, and cuttlefish, are the largest non-mammalian marine organisms and have the largest brains among all invertebrates. To study cephalopod development at a mechanistic level requires the identification of a cephalopod with ready availability, easy husbandry, and an embryology permitting experimental manipulation. The investigators have identified such a preparation, the Californian mud-flat octopus, O. bimaculoides. To date, they have developed methods for ex ovo culturing and for whole mount in situ hybridization for embryonic stages from the blastoderm to hatching. The investigators have also isolated a large panel of cDNAs for key developmental control genes. To complete this model system, they need to develop reliable methods of gene manipulation in these cephalopod embryos. Specific Aim 1 is to generate cephalopod-specific expression vectors based on octopus EF1-a and ubiquitin C genes. Specific Aim 2 is to develop methods of plasmid delivery to octopus embryos, for which the investigators will test as methods electroporation and chemical transgenesis using cationic polymers. Development of gene manipulation resources and methods will permit hypothesis-driven research of the molecular and cellular mechanisms of cephalopod development.