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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：
8534228
负责人：
DAVID J. GRUNWALD
金额：
$ 17.68万
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.

描述（由申请人提供）：拟议工作的目标是开发有效精确改变斑马鱼基因组的新方法。这里开发的技术将大大扩展并可能彻底改变可以进行的实验类型以及可以用斑马鱼提出的问题类型。我们的方法使用同源重组来替换宿主基因组中的特定序列与引入的外源序列。近期目标是：1）证明靶基因可以通过体细胞组织中的同源重组而改变，2）优化利用单链或双链供体DNA分子实现同源重组的条件，3）测量通过非同源重组机制在非预期基因座处供体DNA序列的基因组整合的发生，（4）证明可遗传的改变可以在后代中产生和恢复。最近已经开发了允许使用工程化核酸酶在特定基因座处非常有效地诱导双链DNA断裂的新技术。染色体DNA断裂刺激病变部位的重组，并已用于启动果蝇和培养的哺乳动物细胞的基因靶向。我们的初步研究结果表明，同时引入靶特异性核酸酶和单链供体DNA分子促进斑马鱼胚胎体细胞组织中的同源重组，导致靶向宿主基因的设计序列变化。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。