Derivation of germline competent rabbit embryonic stem cell lines

描述（由申请人提供）：由于兔与人类在解剖学、遗传和生化方面的相似性，它是研究许多人类疾病的良好动物模型。它也用于药物筛选，生产抗体和生产治疗蛋白。兔子足够大，可以为实验工作提供足够数量的组织，产生足够数量的抗体或治疗性蛋白质，但它又足够小，对大多数实验室来说是经济的。迄今为止，原核显微注射仍然是生产转基因家兔的主要方法。目前还没有兔胚干（rbES）细胞系。然而，原核显微注射效率低，导致随机整合，不能产生基因靶向转基因动物。该SBIR项目的目的是建立生殖系胜任的rbES细胞系，用于生产基因靶向转基因家兔。在第一阶段，我们建立了兔胚胎成纤维细胞作为饲养层，合成了重组兔白血病抑制因子（LIF），并获得了推定的rbES细胞。在第二阶段，我们将努力验证这些rbES细胞在囊胚注射后是否可以促进生殖系细胞，我们将努力从这些细胞中生产基因靶向转基因兔子。II期项目的具体目标包括：(1)获得具有分化抑制剂的新型rbES细胞系；(2)评价rbES细胞的种系能力；(3)产生基因靶向的转基因家兔。该项目的成功将在非小鼠物种中建立第一个具有生殖系能力的胚胎干细胞系，提高兔的转基因效率，并使基因靶向兔的生产成为可能。

DESCRIPTION (provided by applicant): The rabbit is a good animal model for the study of many human diseases because of its anatomical, genetic and biochemical similarities to the human. It is also used for drug screening, production of antibody and production of therapeutic proteins. The rabbit is large enough to provide adequate quantities of tissue for experimental work, produce sufficient amount of antibody or therapeutic proteins, and yet it is small enough to be economical for most laboratories. To date, pronuclear microinjection remains the primary method to produce transgenic rabbits. There are no germline competent rabbit embryonic stem (rbES) cell lines available. Pronuclear microinjection, however, is of low efficiency, results in random integration, and cannot produce gene targeted transgenic animals. The purpose of this SBIR project is to establish germline competent rbES cell lines for the production of gene targeted transgenic rabbits. In Phase I we established rabbit embryonic fibroblasts as feeder layers, synthesized recombinant rabbit leukemia inhibitory (LIF) factor, and derived putative rbES cells. In Phase II, we will work to validate if these rbES cells can contribute to germ line cells after blastocyst injection and we will work to produce gene targeted transgenic rabbits from these cells. Specific Aims in Phase II project include: (1) derive new rbES cell lines with inhibitors of differentiation; (2) evaluate germline competency of rbES cells; and (3) produce gene targeted transgenic rabbits. The success of this project will establish the first germline competent ES cell lines in a non-murine species, improve the transgenic efficiency in rabbits, and make the production of gene targeted rabbits possible. PUBLIC HEALTH RELEVANCE: The rabbit is a good animal model for the study of many human diseases because of its anatomical, genetic and biochemical similarities to the human. It is also used for drug screening, production of antibody and production of therapeutic proteins. The main reason that rabbit has not been in these fields is that there have been no established germline competent rabbit embryonic stem (rbES) cell lines. The success of this project will establish the first germline competent ES cell lines in a non-murine species, improve the transgenic efficiency in rabbits, and make the production of gene targeted rabbits possible.