Sivasubbu Sridhar, Balciunas Darius, Davidson Ann E, Pickart Michael A, Hermanson Spencer B, Wangensteen Kirk J, Wolbrink Daniel C, Ekker Stephen C
University of Minnesota, Department of Genetics, Cell Biology and Development, Arnold and Mabel Beckman Center for Transposon Research, 321 Church St SE, 6-160 Jackson Hall, Minneapolis, MN 55455, USA.
Mech Dev. 2006 Jul;123(7):513-29. doi: 10.1016/j.mod.2006.06.002. Epub 2006 Jun 9.
We report a novel gene tagging, identification and mutagenicity ('gene-breaking') method for the zebrafish, Danio rerio. This modular approach consists of two distinct and separable molecular cassettes. The first is a gene-finding cassette. In this study, we employed a 3' gene-tagging approach that selectively 'traps' transcripts regardless of expression status, and we show that this cassette identifies both known and novel endogenous transcripts in transgenic zebrafish. The second is a transcriptional termination mutagenicity cassette assembled from a combination of a splice acceptor and polyadenylation signal to disrupt tagged transcripts upon integration into intronic sequence. We identified both novel and conserved loci as linked phenotypic mutations using this gene-breaking strategy, generating molecularly null mutations in both larval lethal and adult viable loci. We show that the Histone 2a family member z (H2afza) variant is essential for larval development through the generation of a lethal locus with a truncation of conserved carboxy-terminal residues in the protein. In principle this gene-breaking strategy is scalable for functional genomics screens and can be used in Sleeping Beauty transposon and other gene delivery systems in the zebrafish.
我们报道了一种针对斑马鱼（Danio rerio）的新型基因标记、鉴定和致突变性（“基因破坏”）方法。这种模块化方法由两个不同且可分离的分子盒组成。第一个是基因发现盒。在本研究中，我们采用了一种3'基因标记方法，该方法可选择性地“捕获”转录本，而不考虑其表达状态，并且我们表明该盒可识别转基因斑马鱼中的已知和新型内源性转录本。第二个是转录终止致突变盒，它由剪接受体和聚腺苷酸化信号组合而成，用于在整合到内含子序列时破坏标记的转录本。我们使用这种基因破坏策略将新型和保守位点鉴定为连锁表型突变，在幼虫致死和成年存活位点均产生了分子水平的无效突变。我们表明，组蛋白2a家族成员z（H2afza）变体通过产生一个在蛋白质中保守的羧基末端残基被截断的致死位点，对幼虫发育至关重要。原则上，这种基因破坏策略可扩展用于功能基因组学筛选，并可用于斑马鱼的睡美人转座子和其他基因递送系统。
