Zebrafish ES cell lines for targeted mutagenesis

描述（由申请人提供）：斑马鱼具有使其成为脊椎动物发育和人类疾病遗传研究的理想模型的特征。尽管大规模的正向诱变筛选已经成功地应用于斑马鱼，以识别调节早期发育的基因，但目前还没有通过插入诱变来实现靶向基因失活的方法。本研究的目的是利用斑马鱼胚胎干（ES）细胞系开发基因靶向方法。为了实现这一目标，现有的斑马鱼ES细胞培养系统将被优化用于生产鱼类的基因敲除系。将建立支持生殖系胚胎干细胞在培养中最佳生长和存活的条件，并开发提高同源重组菌落分离效率的方法。为了证明这种基因靶向方法的可行性，胚胎干细胞细胞系将被用来靶向一个对正常发育很重要的基因的失活，产生一个具有明显且特征明确的表型的敲除突变体。本研究的具体目的是：1)确定使用晚期斑马鱼ES细胞系生产生殖系嵌合体的频率；2)建立高频率产生生殖系嵌合体的克隆来源ES细胞系；3)从斑马鱼脾脏中获得一种比目前使用的鳟鱼脾脏细胞系更有效地维持斑马鱼ES细胞的种系能力的饲养细胞系；4)确定在胚胎干细胞培养中分离同源重组菌落的最有效方法；5)利用胚胎干细胞培养物靶向无尾基因失活，验证该基因靶向方法的可行性。这项工作开发的基于胚胎干细胞的基因靶向方法将补充目前应用于斑马鱼的其他遗传方法，并增加斑马鱼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.