Biases in Drosophila melanogaster protein trap screens
Biases in Drosophila melanogaster protein trap screens
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DOI：
10.1186/1471-2164-10-249
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发表时间：
2009-05-28
期刊：
影响因子：
4.4
通讯作者：
Adryan, Boris
中科院分区：
文献类型：
--
作者：
Aleksic, Jelena;Lazic, Ranko;Adryan, Boris
背景资料：在体内真实的时间内定位或跟踪内源性蛋白质的能力对于细胞生物学或系统生物学研究具有巨大的实用性。蛋白质陷阱筛选利用转座子携带的人工报告外显子的随机基因组插入（例如，G.编码绿色荧光蛋白，GFP）插入内源基因的内含子中，以产生荧光融合蛋白。尽管最近的努力，旨在实现全面覆盖的果蝇基因组中编码的基因，产生蛋白质traps的基因库仍然small.Results：我们分析了收集可用的蛋白质陷阱线在果蝇和确定潜在的偏见，可能会限制基因组覆盖率在蛋白质陷阱屏幕。这里研究的蛋白质陷阱屏幕主要使用P元件载体，因此表现出一些与该转座子相关的相同的位置偏差，这在全面的果蝇基因破坏项目中是显而易见的。我们进一步发现，蛋白陷阱靶基因通常在胚胎发育过程中表现出广泛和持久的表达，这可能有助于更好地检测。此外，我们研究了GFP外显子对宿主蛋白质结构的可能影响，发现蛋白质陷阱插入对于编码无序蛋白质区域的外显子-外显子边界具有显著的偏倚。38.8%的GFP插入落在无序蛋白质区域，而非捕获P元件插入落在编码序列内含子的情况下仅为23.4%（p < 10（-4））。有趣的是，即使在预测蛋白质结构域的情况下，蛋白质陷阱插入也经常发生在编码表面暴露区域的区域中，这些区域可能是功能中性的。考虑到所观察到的各种偏见，我们预测，不到三分之一的含内含子的基因可能是适合捕获由现有的methods.Conclusion：我们的分析表明，该实用程序的P-元件载体蛋白陷阱屏幕已在很大程度上耗尽，大约2,800个基因可能仍然适合使用piggyBac载体。因此，基于当前方法的蛋白质陷阱策略不太可能提供真正的全基因组覆盖。我们认为，无论是转座子减少插入偏见或重组为基础的靶向技术将需要在果蝇的全面基因组覆盖。
Background: The ability to localise or follow endogenous proteins in real time in vivo is of tremendous utility for cell biology or systems biology studies. Protein trap screens utilise the random genomic insertion of a transposon-borne artificial reporter exon (e. g. encoding the green fluorescent protein, GFP) into an intron of an endogenous gene to generate a fluorescent fusion protein. Despite recent efforts aimed at achieving comprehensive coverage of the genes encoded in the Drosophila genome, the repertoire of genes that yield protein traps is still small.Results: We analysed the collection of available protein trap lines in Drosophila melanogaster and identified potential biases that are likely to restrict genome coverage in protein trap screens. The protein trap screens investigated here primarily used P-element vectors and thus exhibit some of the same positional biases associated with this transposon that are evident from the comprehensive Drosophila Gene Disruption Project. We further found that protein trap target genes usually exhibit broad and persistent expression during embryonic development, which is likely to facilitate better detection. In addition, we investigated the likely influence of the GFP exon on host protein structure and found that protein trap insertions have a significant bias for exon-exon boundaries that encode disordered protein regions. 38.8% of GFP insertions land in disordered protein regions compared with only 23.4% in the case of non-trapping P-element insertions landing in coding sequence introns (p < 10(-4)). Interestingly, even in cases where protein domains are predicted, protein trap insertions frequently occur in regions encoding surface exposed areas that are likely to be functionally neutral. Considering the various biases observed, we predict that less than one third of intron-containing genes are likely to be amenable to trapping by the existing methods.Conclusion: Our analyses suggest that the utility of P-element vectors for protein trap screens has largely been exhausted, and that approximately 2,800 genes may still be amenable using piggyBac vectors. Thus protein trap strategies based on current approaches are unlikely to offer true genome-wide coverage. We suggest that either transposons with reduced insertion bias or recombineering-based targeting techniques will be required for comprehensive genome coverage in Drosophila.