Gene Targeting by Homologous Recombination in the Zebrafish

斑马鱼中同源重组的基因打靶

基本信息

批准号：
8364774
负责人：
DAVID J. GRUNWALD
金额：
$ 22.42万
依托单位：
UNIVERSITY OF UTAH
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-08-20 至 2014-07-31
项目状态：
已结题

项目摘要

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.

描述（由申请人提供）：拟议的工作的目的是开发有效改变斑马鱼基因组的新方法。这里要开发的技术将大大扩展并可能彻底改变可以执行的实验的种类以及斑马鱼可以提出的各种问题。我们的方法使用同源重组来用引入的外源序列代替宿主基因组中的特定序列。直接目的是：1）证明可以通过在躯体组织中的同源重组来改变靶向基因，2）优化条件，以实现单一链或双股供体DNA分子的同源重组，3）通过不间断的供体DNA序列的发生，以衡量供体DNA序列的发生，并衡量供体DNA序列的出现，并既不意外地任何型的发生率，供应均不在供体的发生。在随后的世代中生产和回收。最近已经开发了新技术，可以使用工程核酸酶在特定基因座上非常有效地诱导双链DNA断裂。染色体DNA断裂刺激病变部位的重组，并已用于在果蝇和培养的哺乳动物细胞中启动基因靶向。我们的初步结果表明，同时引入靶标特异性核酸酶和单链供体DNA分子会促进斑马鱼胚胎的体细胞组织中的同源重组，从而导致靶向宿主基因的序列变化。我们预计所提出的实验将确定斑马鱼基因组常规操纵所需的条件。公共卫生相关性：拟议的研究将开发新的创新遗传方法，以通过同源重组来精确操纵斑马鱼基因组。在过去的十年中，斑马鱼已成为用于阐明脊椎动物中遗传，发育，生理和行为过程的主要模型生物。这里要开发的新技术将大大扩展（可能彻底改变）可以执行的实验类型，以及可以用斑马鱼提出的各种问题。