Fluorescent fusion protein knockout mediated by anti-GFP nanobody
Fluorescent fusion protein knockout mediated by anti-GFP nanobody
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DOI：
10.1038/nsmb.2180
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发表时间：
2012-01-01
影响因子：
16.8
通讯作者：
Affolter, Markus
中科院分区：
文献类型：
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作者：
Caussinus, Emmanuel;Kanca, Oguz;Affolter, Markus
利用基因突变来研究体内蛋白质功能是现代生物学的一个中心范式。反向遗传学的最新进展，如RNA干扰和吗啉代被广泛用于进一步应用这一范式。然而，这些系统作用于蛋白质水平的上游，并且蛋白质消耗取决于现有靶蛋白的周转率。在这里，我们提出了deGradFP，在任何真核遗传系统中直接和快速耗尽目标绿色荧光蛋白（GFP）融合的遗传编码方法。这种方法是通用的，因为它依赖于进化上高度保守的真核生物功能，泛素途径。它是可追踪的，因为GFP标签可用于监测蛋白质敲除。在许多情况下，它是一种即用型解决方案，因为GFP蛋白诱捕器储备库正在黑腹果蝇和斑马鱼中产生。
The use of genetic mutations to study protein functions in vivo is a central paradigm of modern biology. Recent advances in reverse genetics such as RNA interference and morpholinos are widely used to further apply this paradigm. Nevertheless, such systems act upstream of the proteic level, and protein depletion depends on the turnover rate of the existing target proteins. Here we present deGradFP, a genetically encoded method for direct and fast depletion of target green fluorescent protein (GFP) fusions in any eukaryotic genetic system. This method is universal because it relies on an evolutionarily highly conserved eukaryotic function, the ubiquitin pathway. It is traceable, because the GFP tag can be used to monitor the protein knockout. In many cases, it is a ready-to-use solution, as GFP protein-trap stock collections are being generated in Drosophila melanogaster and in Danio rerio.