Manipulating zebrafish genome--conserved helicases

描述(申请人提供)：尽管斑马鱼作为一种遗传系统具有许多优势，但目前还没有通过定向突变来操纵其基因组的技术。我们的目标是通过控制生殖系中的RecQ解旋酶的活性来克服斑马鱼的这一障碍。有证据表明，在人类、小鼠和鸡中，由Bloom综合征(BLM)和RECQL5基因编码的酶通常在DNA损伤修复过程中调节同源重组。它们正常活性的降低导致自发姐妹染色单体交换和同源重组的增加，也与染色体外同源重组的频率增加有关。我们建议暂时抑制斑马鱼胚胎中的BLM和RECQL5功能，以允许操纵基因组，包括在种系中进行有针对性的突变。我们已经鉴定了BLM和RECQL5的斑马鱼同源基因，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可能的显性负性突变抑制内源基因功能的能力。我们已经证明了将dnblm RNA注射到色素突变金色杂合子的胚胎中可以诱导同源物之间的重组，在发育3天时可以检测到视网膜色素上皮中突变细胞的克隆。我们将使用这项测试来确定注射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，我们建议通过添加纳米3‘非编码区，将dnRNAs靶向于原始生殖细胞，将它们的活性限制在注射胚胎的生殖系。在生殖系中解旋酶功能暂时被抑制的FISH将被提高，并对生殖细胞存活、生育力下降和诱导的染色体重排或其他突变进行筛选。这将确立在下一代恢复靶向突变的可行性。最后，我们进行了靶向构建，引入了2个基因座的突变。第一种是将GFP插入到死端基因，该基因编码一种在生殖细胞中特异表达的假定的RNA解旋酶；阳性靶向事件可以被评分为注射的幼虫中的荧光生殖细胞。第二种方法将在Somitobun基因中引入显性点突变；携带该突变的卵母细胞产生的胚胎将显示出特有的图案缺陷。这些检测将被用于优化基因靶向的方案。一旦建立了这些抑制RecQ解旋酶功能的技术，就可以促进对斑马鱼基因组的许多操作，例如创建遗传马赛克、产生纯合子的生殖细胞以进行母系效应突变，并最终进行种系同源重组。

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.