Zebrafish ES cell lines for targeted mutagenesis

描述(申请人提供)：斑马鱼具有的特征使其成为脊椎动物发育和人类疾病遗传学研究的理想模型。虽然大规模的正向诱变筛选已经成功地应用于斑马鱼以确定调控早期发育的基因，但还没有通过插入突变来靶向基因失活的方法。这项研究的目标是开发利用斑马鱼胚胎干细胞系进行基因打靶的方法。为了实现这一目标，现有的斑马鱼胚胎干细胞培养系统将进行优化，以用于生产鱼类的淘汰线。将建立支持生殖系有能力的ES细胞在培养中最佳生长和存活的条件，并将开发提高同源重组克隆分离效率的方法。为了证明这种基因打靶方法的可行性，将利用ES细胞系来靶向对正常发育至关重要的基因的失活，产生具有明显和良好特征的表型的敲除突变体。本研究的具体目的是：1)确定利用后期传代的斑马鱼ES细胞系产生生殖系嵌合体的频率；2)建立克隆衍生的ES细胞系，高频率地产生生殖系嵌合体；3)从斑马鱼脾细胞系中获得一种比目前使用的鲑鱼脾细胞系更有效的饲养细胞系，它在维持斑马鱼细胞的生殖系能力方面比目前使用的鲑鱼脾细胞系更有效；4)确定在ES细胞培养中分离同源重组体的最有效方法；5)使用ES细胞培养物靶向失活NO尾部基因，以证明这种基因打靶方法的可行性。这项工作开发的基于ES细胞的基因打靶方法将补充目前应用于斑马鱼的其他遗传方法，并增加这种生物作为研究人类发育和疾病重要基因的模型的价值。

DESCRIPTION (provided by applicant): The zebrafish possesses characteristics that make it an ideal model for genetic studies of vertebrate development and human disease. Although large-scale forward mutagenesis screens have been successfully applied to the zebrafish to identify genes that regulate early development, methods are not available for targeted gene inactivation by insertional mutagenesis. The goal of this research is to develop gene-targeting methods using zebrafish embryonic stem (ES) cell lines. To accomplish this goal, the existing zebrafish ES cell culture system will be optimized for use in the production of knockout lines of fish. Conditions will be established that support the optimal growth and survival of germ-line competent ES cells in culture and methods will be developed to improve the efficiency of homologous recombinant colony isolation. To demonstrate the feasibility of this gene targeting approach, the ES cell lines will be utilized to target the inactivation of a gene that is important for normal development, producing a knockout mutant possessing an obvious and well-characterized phenotype. The specific aims of this research are: 1) determine the frequency of germ-line chimera production using late-passage zebrafish ES cell lines; 2) establish clonally-derived ES cell lines that produce germ-line chimeras at a high frequency; 3) derive a feeder cell line from zebrafish spleen that is more effective than the currently used trout spleen cell line at maintaining germ-line competency of the zebrafish ES cells; 4) identify the most efficient method to isolate colonies of homologous recombinants in the ES cell cultures; 5) use ES cell cultures to target the inactivation of the no tail gene to demonstrate the feasibility of this gene-targeting approach. The ES cell-based gene targeting approach developed from this work will complement other genetic methods currently applied to zebrafish and increase the value of this organism as a model for the study of genes important in human development and disease.