A toolkit for gene-targeting in zebrafish

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，从而彻底改变如何使用斑马鱼研究基因功能。这里开发的技术将大大扩展可以进行的实验种类和可以用斑马鱼提出的问题种类。我们通过“基因靶向”修饰基因组：在靶向基因座处由工程化核酸酶诱导的双链断裂（DSB）用于刺激靶向基因座与含有内源基因座的修饰形式的dsDNA供体分子之间的同源重组/同源性定向修复。我们目前的方法以非常高的效率对斑马鱼基因组进行了靶向修饰：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。大约50-100个碱基对的改变以最高频率精确地发生，而较大的修饰，例如引入1- 2kbp的外源序列段，以较低的速率恢复。在这里，我们使用我们现有的方法和新的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一套工具和试剂，使任何研究人员都可以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者能够常规地产生：i）“肽敲入等位基因”，其中肽编码序列已与正常编码序列整合在框内，因此表达内源蛋白的抗原标记形式; 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creERT 2敲入等位基因; iv）“双顺反子/双产物等位基因”，从其表达内源产物以及报道基因; v）“标记的”等位基因，其中靶向变化可通过共引入的紧密连接的报道基因鉴定;和vi）“floxed条件等位基因”，其中必需基因序列侧翼为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的存在所混淆。我们将生产试剂来完成生殖系基因的条件性切除，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打靶的效率和特异性。我们的实验将旨在i）刺激具有增加的进入生殖系的概率的靶向事件; ii）提高可以将更长的序列（包括整个基因）引入基因组中的功效;以及iii）提高回收floxed等位基因的频率，其中需要协调地引入两个lox位点（通常相隔>1 kbp）。

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.