A protein trap strategy to detect GFP-tagged proteins expressed from their endogenous loci in Drosophila
A protein trap strategy to detect GFP-tagged proteins expressed from their endogenous loci in Drosophila
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DOI：
10.1073/pnas.261408198
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发表时间：
2001-12-18
影响因子：
11.1
通讯作者：
Chia, W
中科院分区：
文献类型：
--
作者：
Morin, X;Daneman, R;Chia, W
在果蝇中，增强子陷阱策略允许快速获得表达模式、分子数据和捕获基因的突变。然而，它们没有在蛋白质水平上提供任何信息，例如关于蛋白质亚细胞定位的信息。利用绿色荧光蛋白(GFP)作为可转座的P元件携带的可移动人工外显子，我们建立了一种蛋白质捕获系统。我们筛选了个体苍蝇，在这些个体中，GFP标记了从它们的内源性基因位点表达的全长内源蛋白，使我们能够观察它们的细胞和亚细胞分布。GFP融合几乎针对细胞的任何一个隔室。在插入已知基因的情况下，我们观察到融合蛋白的亚细胞定位与所描述的内源性蛋白的分布相对应。人工GFP外显子不干扰上游和下游剪接事件。许多插入片段对应于果蝇基因组计划没有预测到的基因。我们的结果表明，在果蝇体内构建蛋白质陷阱是可行的。GFP实时揭示了蛋白质在整个活体中分布的动态，并为许多细胞结构和隔间提供了有用的标记。
In Drosophila, enhancer trap strategies allow rapid access to expression patterns, molecular data, and mutations in trapped genes. However, they do not give any information at the protein level, e.g., about the protein subcellular localization. Using the green fluorescent protein (GFP) as a mobile artificial exon carried by a transposable P-element, we have developed a protein trap system. We screened for individual flies, in which GFP tags full-length endogenous proteins expressed from their endogenous locus, allowing us to observe their cellular and subcellular distribution. GFP fusions are targeted to virtually any compartment of the cell. In the case of insertions in previously known genes, we observe that the subcellular localization of the fusion protein corresponds to the described distribution of the endogenous protein. The artificial GFP exon does not disturb upstream and downstream splicing events. Many insertions correspond to genes not predicted by the Drosophila Genome Project. Our results show the feasibility of a protein trap in Drosophila. GFP reveals in real time the dynamics of protein's distribution in the whole, live organism and provides useful markers for a number of cellular structures and compartments.