Gene Targeting by Homologous Recombination in the Zebrafish

描述(由申请人提供)：拟议工作的目标是开发新的方法来准确有效地改变斑马鱼的基因组。将在这里开发的技术将极大地扩展并可能彻底改变可以进行的各种实验，以及可以用斑马鱼提出的各种问题。我们的方法使用同源重组来用引入的外源序列替换宿主基因组中的特定序列。其直接目的是：1)证明靶基因可以通过在体细胞组织中的同源重组来改变；2)优化条件以利用单链或双链供体DNA分子完成同源重组；3)测量供体DNA序列在非预期位置通过非同源重组机制发生的基因组整合；以及4)证明可遗传的改变可以产生并在随后的世代中恢复。最近开发的新技术允许使用工程核酸酶非常有效地诱导特定位置的双链DNA断裂。染色体DNA断裂刺激病变部位的重组，并已被用于启动果蝇和培养的哺乳动物细胞的基因靶向。我们的初步结果表明，同时导入靶向特异性核酸酶和单链供体DNA分子可以促进斑马鱼胚胎体细胞组织中的同源重组，从而导致目标宿主基因的设计序列改变。我们预计，拟议的实验将确定斑马鱼基因组常规操作所需的条件。 与公共健康相关：拟议的研究将开发新的、创新的遗传方法，通过同源重组精确操纵斑马鱼基因组。在过去的十年里，斑马鱼已经成为一种主要的模式生物，用于阐明脊椎动物之间保守的遗传、发育、生理和行为过程。将在这里开发的新技术将极大地扩大(可能是革命性的)可以进行的实验的种类，以及可以用斑马鱼提出的问题的种类。

DESCRIPTION (provided by applicant): The goal of the proposed work is to develop new methods for precisely altering the genome of zebrafish efficiently. The technology to be developed here will dramatically expand and likely revolutionize the kinds of experiments that can be performed and the kinds of questions that can be asked with the zebrafish. Our approach uses homologous recombination to replace specific sequences in the host genome with introduced exogenous sequences. The immediate aims are: 1) to demonstrate targeted genes can be altered by homologous recombination in somatic tissues, 2) to optimize conditions to accomplish homologous recombination utilizing single strand or double strand donor DNA molecules, 3) to measure the occurrence of genome integration of donor DNA sequences at unintended loci by non-homologous recombination mechanisms, and 4) to demonstrate that heritable alterations can be produced and recovered in subsequent generations. New technologies have recently been developed that permit the very efficient induction of double strand DNA breaks at specific loci using engineered nucleases. The chromosomal DNA breaks stimulate recombination at the site of the lesion and have been used to initiate gene targeting in fruit flies and cultured mammalian cells. Our preliminary results indicate that simultaneous introduction of target-specific nucleases and single strand donor DNA molecules promotes homologous recombination in the somatic tissues of zebrafish embryos resulting in designed sequence changes at a targeted host gene. We anticipate the proposed experiments will identify conditions needed for the routine manipulation of the zebrafish genome. PUBLIC HEALTH RELEVANCE: The proposed research will develop new, innovative genetic methods for precise manipulation of the zebrafish genome by homologous recombination. Over the last decade the zebrafish has emerged as a major model organism used for elucidating genetic, developmental, physiological, and behavioral processes conserved among vertebrates. The new technology to be developed here will dramatically expand (likely revolutionize) the kinds of experiments that can be performed and the kinds of questions that can be asked with the zebrafish.