A toolkit for gene-targeting in zebrafish

描述(申请人提供)：拟议工作的目的是显著提高精确修改斑马鱼基因组的能力，从而革命性地利用斑马鱼研究基因功能。这里开发的技术将极大地扩展可以进行的实验类型和可以向斑马鱼提出的问题的类型。我们通过‘基因打靶’来修改基因组：在靶基因座上由工程核酸酶诱导的双链断裂(DSB)被用来刺激靶基因座与含有内源基因座修饰版本的dsDNA供体分子之间的同源重组/同源定向修复。我们目前的方法以非常高的效率产生了斑马鱼基因组的靶向修饰：高达六分之一的处理动物将精确修改的基因位点传递给后代。50-100个碱基对的变化以最高的频率精确发生，而较大的修改，如引入1-2kbp的外源序列延伸，则以较低的速率恢复。在这里，我们利用我们现有的方法和新的方法来开发基因打靶工具，以提高斑马鱼基因打靶的效率和保真度。我们的第一个目标是开发和测试一套工具和试剂，使任何研究人员都可以很容易地在斑马鱼中产生许多标准类型的修饰基因。我们创建的工具包将使研究人员能够常规地产生：i)‘多肽敲入等位基因’，其中的多肽编码序列已与正常的编码序列整合在一起，从而表达内源蛋白的抗原标记版本；ii)‘报告敲入/敲除等位基因’，其中该基因座表达的是报告蛋白而不是其正常产物；iii)cre和creERT2敲入等位基因；iv)‘双顺反子/两产物等位基因’，内源产物以及报告从其中表达；V)被标记的等位基因，其中靶向改变可由共同引入的紧密连锁的报告基因识别；以及vi)被标记的条件等位基因，其中基本基因序列两侧是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。许多使用突变的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混乱。我们将生产试剂来完成生殖系中基因的有条件消融，从而生产出缺乏母体提供的基因产品的卵子和胚胎。我们的最终目标是提高基因打靶的效率和特异性。我们的实验将旨在：1)刺激靶向事件，增加进入生殖系的可能性；2)提高将包括整个基因在内的更长序列引入基因组的效率；3)提高恢复花等位基因的频率，在这两个位点上，通常需要协调引入两个通常被&gt；1 kbp分隔的lox位点。

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.