Green fluorescent protein and its derivatives as versatile markers for gene expression in living Drosophila melanogaster, plant and mammalian cells
Green fluorescent protein and its derivatives as versatile markers for gene expression in living Drosophila melanogaster, plant and mammalian cells
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DOI：
10.1016/0378-1119(95)00700-8
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发表时间：
1996-07-01
期刊：
影响因子：
3.5
通讯作者：
Kay, SA
中科院分区：
文献类型：
--
作者：
Plautz, JD;Day, RN;Kay, SA
我们研究了绿色荧光蛋白（GFP）作为活的成年黑腹果蝇（Drosophilamelanogaster，Dm）和培养的植物和哺乳动物细胞中基因表达的标记物的效用。使用Dm，我们产生了携带玻璃响应性gfp融合基因的转基因果蝇以测试GFP作为空间报告基因的效用。在成年活体果蝇中，GFP在单眼和眼睛中清晰可见。我们已经优化了用于区分GFP信号与活Dm中丰富的自体荧光的过滤器的使用。此外，我们已经使用绿色荧光蛋白，以确定光感受器细胞的蛹眼文化已被固定和染色，根据标准的组织学程序。在瞬时转染大豆悬浮培养物后，在单个活植物细胞中也检测到GFP，证明GFP是植物细胞中有效的转化标记物。类似地，用GFP的修饰形式S65T瞬时转染哺乳动物细胞，允许检测表达报告基因的单个活细胞。然后，我们使用CellScan系统穷举光子再分配（EPR）去卷积算法来生成活细胞中GFP荧光的高分辨率三维图像。
We have investigated the utility of the green fluorescent protein (GFP) as a marker for gene expression in living adult Drosophila melanogaster (Dm) and cultured plant and mammalian cells, Using Dm, we generated transgenic flies bearing a glass-responsive gfp fusion gene to test the utility of GFP as a spatial reporter. In the adult living fly, GFP is clearly visible in the ocelli and the eye. We have optimized the use of filters for distinguishing the GFP signal from abundant autofluorescence in living Dm. In addition, we have used GFP to identify photoreceptor cells in pupal eye cultures that have been fixed and stained according to standard histological procedures. GFP was also detected in individual living plant cells following transient transfection of soybean suspension cultures, demonstrating that GFP is an effective transformation marker in plant cells, Similarly, transient transfection of mammalian cells with a modified form of GFP, S65T, allowed detection of single living cells expressing the reporter, This modified form of GFP gave a robust signal that was resistant to photobleaching. We then used a CellScan system exhaustive photon reassignment (EPR) deconvolution algorithm to generate high-resolution three-dimensional images of GFP fluorescence in the living cell.