Barolo Scott, Castro Brian, Posakony James W
University of California San Diego, La Jolla, CA, USA.
Biotechniques. 2004 Mar;36(3):436-40, 442. doi: 10.2144/04363ST03.
In vivo green fluorescent protein (GFP)/red fluorescent protein (RFP) double-labeling studies have been hampered by several inconvenient properties of DsRed, the first described RFP. These disadvantages include a very slow (> 24 h) maturation time, emission of contaminating green light, and low solubility. A recently developed variant of DsRed, called DsRed.T4, has a much shorter maturation time, no significant green emission, and improved solubility. We have constructed Drosophila P-element transformation vectors encoding DsRed.T4 for promoter/enhancer analysis, labeling of living cells, or RFP tagging of proteins. These new vectors have all of the features of the widely used Pelican/Stinger GFP vectors, including insulator sequences to reduce position effects, an extensive polylinker, and both cytoplasmic and nuclear-localized forms of the reporter. We have also constructed an upstream activating sequence (UAS)-DsRed.T4 vector, for GAL4 activation of the reporter. We find that DsRed.T4 is very easily detected in transgenic flies without contamination of the GFP signal and that it matures to its fluorescent form nearly simultaneously with GFP. This advance in Drosophila reporter technology makes timed double-labeling experiments in developing transgenic animals possible for the first time.
体内绿色荧光蛋白（GFP）/红色荧光蛋白（RFP）双标记研究一直受到首个被描述的RFP——DsRed的几个不利特性的阻碍。这些缺点包括成熟时间非常缓慢（>24小时）、发出污染性的绿光以及溶解度低。最近开发的一种DsRed变体，称为DsRed.T4，其成熟时间要短得多，没有明显的绿色荧光发射，并且溶解度有所提高。我们构建了果蝇P元素转化载体，用于编码DsRed.T4，以进行启动子/增强子分析、活细胞标记或蛋白质的RFP标记。这些新载体具有广泛使用的鹈鹕/刺尾鱼GFP载体的所有特征，包括用于减少位置效应的绝缘子序列、一个广泛的多克隆位点，以及报告基因的细胞质和细胞核定位形式。我们还构建了一个上游激活序列（UAS）-DsRed.T4载体，用于通过GAL4激活报告基因。我们发现，在转基因果蝇中很容易检测到DsRed.T4，且不会污染GFP信号，并且它几乎与GFP同时成熟为荧光形式。果蝇报告技术的这一进展首次使得在发育中的转基因动物中进行定时双标记实验成为可能。
