Zebrafish ES cell lines for targeted mutagenesis

用于靶向诱变的斑马鱼 ES 细胞系

基本信息

批准号：
8055654
负责人：
PAUL COLLODI
金额：
$ 16.77万
依托单位：
PURDUE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-05-21 至 2012-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8055654
关键词：
Address Alleles Antibodies Cell Culture System Cell Culture Techniques Cell Line Cells Characteristics Chimera organism Chimerism Complement Cultured Cells Derivation procedure Development Diploidy Disease ES Cell Line Electroporation Embryo Embryonic Development Engineering Event Fishes Fluorescence-Activated Cell Sorting Funding Gene Silencing Gene Targeting Generations Genes Genetic Genetic Models Germ Cells Germ Lines Goals Grant Growth Human Development In Vitro Insertional Mutagenesis Karyotype Knock-out Methods Microinjections Mutagenesis Organism Other Genetics Pattern Formation Peptides Phenotype Pigmentation physiologic function Plasmids Population Probability Production Proteins Recombinants Research Screening procedure Staging Stem Cell Factor Stem cells Stromal Cell-Derived Factor 1 Stromal Cells Structure of primordial sex cell Study models Testing Time Transgenic Organisms Transplantation Work Writing Zebrafish base chemokine embryonic stem cell hatching homologous recombination human disease in vivo stem transmission process

项目摘要

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.

描述（由申请人提供）：斑马鱼所具有的特性使其成为脊椎动物发育和人类疾病遗传研究的理想模型。尽管大规模正向诱变筛选已成功应用于斑马鱼，以鉴定调节早期发育和人类疾病的基因，但尚无通过插入诱变使目标基因失活的方法。本研究的目标是开发利用斑马鱼胚胎干（ES）细胞和原始生殖细胞（PGC）系的基因靶向方法。为了实现这一目标，现有的斑马鱼 ES 细胞培养系统将进行优化，用于生产鱼类基因敲除系。体外抗体筛选将用于鉴定 ES 细胞集落，这些细胞在经历基因靶向事件后仍然有能力为宿主胚胎的种系做出贡献。此外，将建立和评估应更有效地促进宿主种系传播的 PGC 培养物。为了证明这种基因靶向方法的可行性，ES细胞系将用于靶向对正常发育和模式形成重要的基因的失活，产生具有明显且特征良好的表型的基因敲除鱼。本研究的具体目标是：1) 鉴定特异性识别多能和种系活性斑马鱼 ES 细胞集落的抗体。 2) 使用先前建立的方法来分离具有被破坏的ntl和hag基因拷贝的ES细胞集落，并使用特定目标1中鉴定的抗体结合核型分析来选择那些具有种系感受态和二倍体的集落。 3) 使用携带失活ntl或hag基因的二倍体、种系感受态ES细胞培养物来产生鱼的敲除系。 4) 使用先前开发的方法从晚期胚胎建立 PGS 培养物，并确定培养的 PC 是否比 ES 细胞更有效地产生种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，并增加这种生物体作为研究人类发育和疾病中重要基因的模型的价值。