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。虽然大规模的正向突变筛选已经成功地应用于斑马鱼，以确定调控早期发育和人类疾病的基因，但还没有通过插入突变来靶向基因失活的方法。本研究的目标是利用斑马鱼胚胎干细胞(ES细胞)和原始生殖细胞(PGC)系开发基因打靶方法。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。体外抗体筛查将用于识别ES细胞的克隆，这些细胞在经历了基因靶向事件后，仍有能力为宿主胚胎的生殖系做出贡献。此外，还将建立对宿主胚系有更高贡献的PGC培养物，并对其进行种系传播评估。为了证明这种基因打靶方法的可行性，ES细胞系将被用来靶向对正常发育和模式形成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鱼。这项研究的具体目的是：1)识别特异性识别多能性和生殖系有能力的斑马鱼ES细胞克隆的抗体。2)使用先前建立的方法分离具有NTL和HAG基因中断拷贝的ES细胞克隆，并利用特异性目的1中鉴定的抗体结合核型分析筛选出具有生殖系能力和二倍体的克隆。3)使用携带灭活的NTL或HAG基因的二倍体、生殖系能力强的ES细胞培养物来产生鱼类的敲除系。4)使用先前开发的方法建立晚期胚胎的PGSS培养，并确定培养的PC是否比ES细胞更有效地产生生殖系嵌合体和可存活的F1胚胎。斑马鱼是胚胎发育和人类疾病遗传学研究的重要模型。这项工作开发的基于干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 and human disease, 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 and primordial germ cell (PGC) 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. An in vitro antibody screen will be used to identify colonies of ES cells that remain competent to contribute to the germ line of a host embryo after the cells have undergone a gene targeting event. Also, PGC cultures that should contribute more efficiently to the host germ line will be established and evaluated for germ line transmission. To demonstrate the feasibility of this gene targeting approach, the ES cell lines will be utilized to target the inactivation of genes that are important for normal development and pattern formation, producing knockout fish possessing obvious and well-characterized phenotypes. The specific aims of this research are: 1) Identify antibodies that specifically recognize pluripotent and germ line competent zebrafish ES cell colonies. 2) Use previously established methods to isolate colonies of ES cells that possess disrupted copies of the ntl and hag genes and select those colonies that are germ line competent and diploid using the antibodies identified in specific aim 1 combined with karyotype analysis. 3) Use the diploid, germ line competent ES cell cultures that carry an inactivated ntl or hag gene to generate knockout lines of fish. 4) Use previously developed methods to establish cultures of PGSs from late-stage embryos and determine if the cultured PCs generate germ line chimeras and viable F1 embryos more efficiently than ES cells. The zebrafish is an important model for genetic studies of embryo development and human disease. The stem 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.