Enhancer trapping in zebrafish using the Sleeping Beauty transposon.
Enhancer trapping in zebrafish using the Sleeping Beauty transposon.
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DOI：
10.1186/1471-2164-5-62
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发表时间：
2004-09-03
期刊：
影响因子：
4.4
通讯作者：
Ekker SC
中科院分区：
文献类型：
--
作者：
Balciunas D;Davidson AE;Sivasubbu S;Hermanson SB;Welle Z;Ekker SC
在后生动物基因的功能元件中，增强子特别难以发现和注释。在果蝇中的开创性实验已经证明了使用无脊椎动物来解决这个功能基因组学问题的增强子“捕获”的价值。我们调节了基于睡美人转座的转基因盒，以建立一种增强子捕获技术，用于脊椎动物模型系统（斑马鱼）。我们从90个产生GFP表达后代的创始人中建立了9个具有不同组织或器官特异性GFP表达模式的斑马鱼系。我们已经对这些品系进行了分子表征，并表明在每一品系中，特定的GFP表达模式是由于单一的转座事件。许多插入是在目前的基因组组装中预测的斑马鱼基因的内含子中。我们已经确定了以前的特点，以及新的表达模式，从这个屏幕。例如，ET 7系在mkp 3基因座附近含有转座子插入，并在中脑-后脑边界、前脑和脑室中表达GFP，与已知的FGF 8依赖性mkp 3表达结构域的子集相匹配。相反，ET 2系由于插入聚（ADP-核糖）糖水解酶（PARG）位点而在尾侧初级运动神经元中特异性表达GFP。使用内源性PARG mRNA的原位杂交技术证实了这种令人惊讶的表达模式，表明增强子陷阱以良好的保真度复制了这种意想不到的和高度定位的PARG表达。最后，我们表明，这是可能的切除一个睡美人转座子从斑马鱼种系的基因组位置。这种基因组学工具提供了一个机会，大规模的生物学方法相结合的表达和基因组水平的序列分析使用作为模板的整个脊椎动物基因组。
Among functional elements of a metazoan gene, enhancers are particularly difficult to find and annotate. Pioneering experiments in Drosophila have demonstrated the value of enhancer "trapping" using an invertebrate to address this functional genomics problem. We modulated a Sleeping Beauty transposon-based transgenesis cassette to establish an enhancer trapping technique for use in a vertebrate model system, zebrafish Danio rerio. We established 9 lines of zebrafish with distinct tissue- or organ-specific GFP expression patterns from 90 founders that produced GFP-expressing progeny. We have molecularly characterized these lines and show that in each line, a specific GFP expression pattern is due to a single transposition event. Many of the insertions are into introns of zebrafish genes predicted in the current genome assembly. We have identified both previously characterized as well as novel expression patterns from this screen. For example, the ET7 line harbors a transposon insertion near the mkp3 locus and expresses GFP in the midbrain-hindbrain boundary, forebrain and the ventricle, matching a subset of the known FGF8-dependent mkp3 expression domain. The ET2 line, in contrast, expresses GFP specifically in caudal primary motoneurons due to an insertion into the poly(ADP-ribose) glycohydrolase (PARG) locus. This surprising expression pattern was confirmed using in situ hybridization techniques for the endogenous PARG mRNA, indicating the enhancer trap has replicated this unexpected and highly localized PARG expression with good fidelity. Finally, we show that it is possible to excise a Sleeping Beauty transposon from a genomic location in the zebrafish germline. This genomics tool offers the opportunity for large-scale biological approaches combining both expression and genomic-level sequence analysis using as a template an entire vertebrate genome.
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影响因子：
2.7
作者：
Davidson, AE;Balciunas, D;Ekker, SC
通讯作者：
Ekker, SC
影响因子：
16
作者：
Izsvák, Z;Stüwe, EE;Ivics, Z
通讯作者：
Ivics, Z
影响因子：
64.5
作者：
JAENISCH, R;JAHNER, D;GROTKOPP, D
通讯作者：
GROTKOPP, D
影响因子：
64.8
作者：
ALLEN, ND;CRAN, DG;SURANI, MA
通讯作者：
SURANI, MA
DOI：
10.1073/pnas.87.16.6331
发表时间：
1990-08-01
影响因子：
11.1
作者：
BONNEROT, C;GRIMBER, G;NICOLAS, JF
通讯作者：
NICOLAS, JF