A toolkit for gene-targeting in zebrafish

斑马鱼基因靶向工具包

基本信息

批准号：
9059742
负责人：
DAVID J. GRUNWALD
金额：
$ 39.02万
依托单位：
UNIVERSITY OF UTAH
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-07-11 至 2019-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9059742
关键词：
Ablation Alleles Animal Model Animals Antigens Base Pairing Biological Biological Assay Biomedical Research Chromatin Clustered Regularly Interspaced Short Palindromic Repeats Code Communities Disease Embryo Engineering Essential Genes Event Fertilization Frequencies Gene Expression Gene Expression Regulation Gene Targeting Genes Genetic Recombination Genome Germ Cells Germ Lines Goals Health Human Knock-in Knock-out Laboratories Link LoxP-flanked allele Measures Methods Modification Peptides Physiological Plasmids Positioning Attribute Probability Process Production Proteins Reagent Reporter Reporter Genes Reporting Research Research Personnel Signal Pathway Site Site-Directed Mutagenesis Specificity Staging Stretching Technology Testing Time Transgenes Variant Work Zebrafish base cell type chimeric gene chromatin remodeling drug testing ds-DNA egg gene function gene product homologous recombination human disease improved mutant novel strategies nuclease offspring pluripotency reagent testing recombinase recombinational repair repaired research study tool transcription factor transcription termination zebrafish genome zygote

项目摘要

DESCRIPTION (provided by applicant): The purpose of the proposed work is to significantly advance the ability to precisely modify the zebrafish genome and consequently revolutionize how gene function can be studied using zebrafish. The technologies developed here will dramatically expand the kinds of experiments that can be performed and the kinds of questions that can be asked with the zebrafish. We modify the genome by 'gene targeting': a double strand break (DSB) induced by an engineered nuclease at a targeted locus is used to stimulate homologous recombination / homology directed repair between the targeted locus and a dsDNA donor molecule that harbors a modified version of the endogenous locus. Our current methods yield targeted modification of the zebrafish genome with very high efficiency: up to 1 in 6 of the treated animals transmit a precisely modified locus to offspring. Alterations on the order of 50-100 base pairs occur with precision at the highest frequency, whereas larger modifications, such as the introduction of a 1-2 kbp stretch of exogenous sequence, are recovered at lower rates. Here we develop tools for gene targeting using our current methods and new approaches to improve the efficiency and fidelity of gene targeting in zebrafish. Our first goal is to develop ad test a set of tools and reagents that make it easy for any investigator to generate many standard types of modified loci in zebrafish. The toolkit we create will enable investigators to routinely produce: i) 'peptide knock-in alleles' in which peptide-encoding sequences have been integrated in frame with the normal coding sequence so antigen-tagged versions of the endogenous proteins are expressed; ii) 'reporter knock-in/knock-out alleles' in which the locus expresses a reporter protein instead of its normal product; iii) cre and creERT2 knock-in alleles; iv) 'bicistronic/two-product alleles' from which the endogenous product as well as a reporter are expressed; v) 'tagged' alleles in which a targeted change is identifiable by a co-introduced, tightly linked reporter gene; and vi) 'floxed conditional alleles', in which essential gene sequences are flanked by loxP recombination sites. A second goal is to develop and test a new approach for analyzing maternally supplied gene functions. Many studies using mutant zebrafish embryos to study signaling pathways, chromatin remodeling, or the control of pluripotency are confounded by the presence of maternally supplied gene products. We will generate reagents to accomplish conditional ablation of a gene in the germ line, allowing production of eggs and embryos that lack a maternally supplied gene product. Our final goal is to improve the efficiency and specificity of gene targeting. Our experiments will be aimed at i) stimulating targeting events that have increased probability of entering the germ line; ii) improving the efficacy with which longer sequences, including entire genes, can be introduced into the genome; and iii) improving the frequency of recovering floxed alleles, where two lox sites, often separated by >1 kbp need to be coordinately introduced.

描述（由申请人提供）：拟议工作的目的是显着提高精确修改斑马鱼基因组的能力，从而彻底改变使用斑马鱼研究基因功能的方式。这里开发的技术将极大地扩展可以进行的实验种类以及可以用斑马鱼提出的问题种类。我们通过“基因靶向”修改基因组：由工程核酸酶在目标位点诱导的双链断裂 (DSB) 用于刺激目标位点和包含修改版本的 dsDNA 供体分子之间的同源重组/同源定向修复内源基因座。我们目前的方法以非常高的效率对斑马鱼基因组进行有针对性的修饰：多达六分之一的接受治疗的动物将精确修饰的基因座传递给后代。 50-100 个碱基对数量级的改变以最高频率精确发生，而较大的修改，例如引入 1-2 kbp 的外源序列，则以较低的速率恢复。在这里，我们使用现有方法和新方法开发基因打靶工具，以提高斑马鱼基因打靶的效率和保真度。我们的首要目标是开发一套测试工具和试剂，使任何研究人员都能轻松地在斑马鱼中生成许多标准类型的修饰基因座。我们创建的工具包将使研究人员能够常规地产生： i) “肽敲入等位基因”，其中肽编码序列已与正常编码序列框内整合，以便表达内源蛋白的抗原标记版本； ii)“报告基因敲入/敲除等位基因”，其中基因座表达报告蛋白而不是其正常产物； iii) cre 和 creERT2 敲入等位基因； iv) 表达内源产物和报告基因的“双顺反子/双产物等位基因”； v)“标记”等位基因，其中目标变化可通过共同引入的、紧密相连的报告基因来识别； vi)“floxed 条件等位基因”，其中必需基因序列的两侧是 loxP 重组位点。第二个目标是开发和测试一种分析母体提供的基因功能的新方法。许多使用突变斑马鱼胚胎来研究信号通路、染色质重塑或多能性控制的研究都因母体提供的基因产物的存在而受到干扰。我们将生产试剂来完成种系中基因的有条件消融，从而产生缺乏母体提供的基因产物的卵子和胚胎。我们的最终目标是提高基因打靶的效率和特异性。我们的实验旨在 i) 刺激靶向事件，从而增加进入种系的可能性； ii) 提高将更长序列（包括整个基因）引入基因组的效率； iii) 提高恢复 floxed 等位基因的频率，其中需要协调引入两个 lox 位点，通常相距 >1 kbp。