GFP Affects Human T Cell Activation and Cytokine Production following In Vitro Stimulation
GFP Affects Human T Cell Activation and Cytokine Production following In Vitro Stimulation
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DOI：
10.1371/journal.pone.0050068
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发表时间：
2013-04-05
期刊：
影响因子：
3.7
通讯作者：
Wren, Jonathan D.
中科院分区：
文献类型：
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作者：
Koelsch, Kristi A.;Wang, YuJing;Wren, Jonathan D.
有许多来自不同物种的绿色荧光蛋白（gfp）对细胞生物学家来说是无价的，因为它们能够提供蛋白质表达的实验可视化。自从它们最初被发现以来，它们已经被修改和改进，以提供更稳定的变体，其发射范围跨越各种颜色。由于它们易于在体外和体内表达，它们是分子生物学中用作标记物的一个有吸引力的选择。一般认为，gfp对引入它们的细胞的影响可以忽略不计。然而，越来越多的报告表明，情况并非总是如此。因此，由于绿色荧光蛋白的普遍使用，记录非预期影响的性质和程度是很重要的。在本报告中，我们发现GFP影响T细胞的激活，导致同样表达TurboGFP的人原代T细胞在刺激后聚集缺陷，激活标记CD25和IL-2细胞因子的产生上调。我们使用了一种常规用于检测NF-kappa B通路的报告基因实验，发现在共转染TurboGFP的HEK293和HeLa细胞中，NF-kappa B的激活降低，这表明GFP干扰了NF-kappa B通路的信号传导。这些发现表明，使用gfp标记的载体可能会对T细胞的体外实验产生负面影响，强调对照对于鉴定gfp诱导的任何效应至关重要。
There are many Green Fluorescent Proteins (GFPs) originating from diverse species that are invaluable to cell biologists today because of their ability to provide experimental visualization of protein expression. Since their initial discovery, they have been modified and improved to provide more stable variants with emission ranges spanning a wide array of colors. Due to their ease of expression both in-vitro and in-vivo, they are an attractive choice for use as markers in molecular biology. GFPs are generally assumed to have negligible effects on the cells to which they have been introduced. However, a growing number of reports indicate that this is not always the case. Consequently, because of GFP's ubiquitous use, it is important to document the nature and extent of unintended effects. In this report, we find that GFP affects T cell activation, leading to defects in clustering, upregulation of the activation marker CD25 and IL-2 cytokine production following stimulation in human primary T cells that also express TurboGFP. We utilized a reporter assay which has been routinely used to assay the NF-kappa B pathway and found reduced NF-kappa B activitation in stimulated HEK293 and HeLa cells that were co-transfected with TurboGFP, suggesting that GFP interferes with signaling through the NF-kappa B pathway. These findings indicate that the utilization of GFP-tagged vectors may negatively impact in vitro experiments in T cells, emphasizing the critical importance of controls to identify any GFP-induced effects.