A toolkit for gene-targeting in zebrafish

描述（由申请人提供）：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，从而彻底改变如何使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的种类和可以用斑马鱼提出的问题的种类。我们通过“基因靶向”来修饰基因组：在目标位点上由工程核酸酶诱导的双链断裂（DSB）用于刺激目标位点与dsDNA供体分子之间的同源重组/同源定向修复，dsDNA供体分子含有内源位点的修饰版本。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1 / 6的治疗动物将精确修饰的位点传递给后代。50-100个碱基对的改变以最高的频率精确发生，而更大的修改，如引入1-2 kbp的外源序列，以较低的速率恢复。在此，我们利用现有方法和新方法开发基因靶向工具，以提高斑马鱼基因靶向的效率和保真度。我们的第一个目标是开发和测试一套工具和试剂，使任何研究者都可以轻松地在斑马鱼中产生许多标准类型的修饰位点。我们创建的工具包将使研究人员能够常规生产：i)“肽敲入等位基因”，其中肽编码序列已与正常编码序列整合在框架中，因此抗原标记的内源性蛋白质版本得以表达；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)表达内源性产物和报告基因的“双子/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连接的报告基因识别；以及vi)“floxed conditional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系基因功能的新方法。许多利用斑马鱼突变胚胎研究信号通路、染色质重塑或多能性控制的研究都被母体提供的基因产物所混淆。我们将制造试剂来完成有条件地切除生殖系中的基因，从而生产出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靶向的效率和特异性。我们的实验将旨在i)刺激靶向事件，增加进入种系的可能性；Ii)提高将较长的序列（包括整个基因）引入基因组的功效；(3)提高絮状等位基因的恢复频率，其中两个位点需要协调引入，通常被bbb1kbp分隔。

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.