Manipulating zebrafish genome--conserved helicases

操纵斑马鱼基因组——保守解旋酶

• 批准号: 7084837
• 负责人: Shannon Fisher
• 金额: $ 16.95万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-04-12 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7084837
• 关键词: Bloom syndrome; RNA; animal breeding; bioassay; biotechnology; developmental genetics; early embryonic stage; fertility; gene targeting; genetic recombination; genetic regulation; genetically modified animals; germ cells; green fluorescent proteins; helicase; retinal pigment epithelium; zebrafish

DESCRIPTION (provided by applicant): Despite the many advantages of zebrafish as a genetic system, there is currently no technology to manipulate its genome through targeted mutagenesis. We aim to overcome this barrier in zebrafish, through manipulation of the activity of RecQ helicases in the germline. There is evidence in human, mouse, and chicken that the enzymes encoded by the Bloom syndrome (BLM) and RECQL5 genes normally function to regulate homologous recombination during DNA damage repair. Reduction of their normal activities leads to an increase in spontaneous sister chromatid exchange and homologue recombination, and also correlates with increased frequency of extrachromosomal homologous recombination. We propose to transiently repress BLM and RECQL5 functions in the zebrafish embryo to allow manipulations of the genome, including targeted mutagenesis in the germline. We have identified the zebrafish orthologues of BLM and RECQL5 and verified their expression in the early embryo. First, we will measure the ability of putative dominant negative (dn) mutations of blm and recqIS to repress endogenous gene function. We have shown that injection of dnblm RNA into embryos heterozygous for the pigment mutation golden induces recombination between homologues, detectable as clones of mutant cells in the retinal pigment epithelium at 3 days of development. We will use this assay to determine the best combination of injected RNAs to transiently repress helicase function while still allowing normal development. We then propose to target dnRNAs to primordial germ cells through the addition of the nanosl 3'UTR, confining their activity to the germline of injected embryos. Fish in which helicase function in the germline has been transiently suppressed will be raised and screened for germ cell survival, reduced fertility, and induced chromosomal rearrangements or other mutations. This will establish the feasibility of recovering targeted mutations in the next generation. Finally, we have made targeting constructs to introduce mutations in 2 loci. The first will insert GFP into the dead end locus, which encodes a putative RNA helicase expressed specifically in germ cells; positive targeting events can be scored as fluorescent germ cells in injected larvae. The second will introduce a dominant point mutation into the somitobun gene; embryos derived from oocytes carrying the mutation will display a characteristic patterning defect. These assays will be used to optimize the protocol for gene targeting. Once established, these techniques for repressing function of RecQ helicases could facilitate many manipulations of the zebrafish genome, such as creation of genetic mosaics; generation of germ cells homozygous for maternal effect mutations, and ultimately germline homologous recombination.

描述（由申请人提供）：尽管斑马鱼作为遗传系统具有许多优点，但目前还没有通过定向诱变操纵其基因组的技术。我们的目标是通过操纵种系中 RecQ 解旋酶的活性来克服斑马鱼的这一障碍。在人类、小鼠和鸡身上有证据表明，布卢姆综合征 (BLM) 和 RECQL5 基因编码的酶通常在 DNA 损伤修复过程中发挥调节同源重组的作用。其正常活动的减少导致自发姐妹染色单体交换和同源重组的增加，并且还与染色体外同源重组频率的增加相关。我们建议暂时抑制斑马鱼胚胎中的 BLM 和 RECQL5 功能，以允许对基因组进行操作，包括种系中的定向诱变。我们已经鉴定了 BLM 和 RECQL5 的斑马鱼直系同源物，并验证了它们在早期胚胎中的表达。首先，我们将测量 blm 和 recqIS 的推定显性失活 (dn) 突变抑制内源基因功能的能力。我们已经证明，将 dnblm RNA 注射到色素突变金色杂合的胚胎中会诱导同源物之间的重组，在发育 3 天时可检测到视网膜色素上皮中突变细胞的克隆。我们将使用该测定来确定注射 RNA 的最佳组合，以暂时抑制解旋酶功能，同时仍允许正常发育。然后，我们建议通过添加 nanosl 3'UTR 将 dnRNA 靶向原始生殖细胞，将其活性限制在注射胚胎的种系中。种系中解旋酶功能被暂时抑制的鱼将被饲养并筛选其生殖细胞存活、生育力降低以及诱导的染色体重排或其他突变。这将建立在下一代恢复靶向突变的可行性。最后，我们制作了靶向构建体以在 2 个基因座中引入突变。第一个将 GFP 插入死端基因座，该基因座编码在生殖细胞中特异性表达的推定 RNA 解旋酶；阳性靶向事件可以作为注射幼虫中的荧光生殖细胞进行评分。第二个将在 somitobun 基因中引入显性点突变；来自携带突变的卵母细胞的胚胎将表现出特征性的图案缺陷。这些测定将用于优化基因靶向方案。一旦建立，这些抑制 RecQ 解旋酶功能的技术可以促进斑马鱼基因组的许多操作，例如创建遗传嵌合体；产生母体效应突变的纯合生殖细胞，并最终进行种系同源重组。