Gene Manipulation of Cephalopod Development

描述（由申请人提供）：了解正常的人类发育依赖于动物模型系统，用于对潜在生物学机制的实验洞察。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，人类是具有不成比例的大脑的大型生物。包括鱿鱼、章鱼和乌贼在内的鲸类是最大的非哺乳类海洋生物，在所有无脊椎动物中拥有最大的大脑。要在机械水平上研究头足类动物的发育，需要鉴定一种容易获得、易于饲养和胚胎学允许实验操作的头足类动物。研究人员已经确定了这样一种制剂，加利福尼亚泥滩章鱼，O。双棘突。到目前为止，他们已经开发出了从胚盘到孵化的胚胎阶段的卵外培养和整装原位杂交的方法。研究人员还分离出了一大组关键发育控制基因的cDNA。为了完成这个模型系统，他们需要在这些头足类动物胚胎中开发可靠的基因操作方法。具体目的1是构建章鱼EF 1-a和泛素C基因的头足类特异性表达载体。具体目标2是开发向章鱼胚胎递送质粒的方法，为此研究人员将测试电穿孔和使用阳离子聚合物的化学转基因方法。基因操作资源和方法的发展将允许假说驱动的头足类动物发育的分子和细胞机制的研究。

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.