An enhanced green fluorescent protein allows sensitive detection of gene transfer in mammalian cells
An enhanced green fluorescent protein allows sensitive detection of gene transfer in mammalian cells
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DOI：
10.1006/bbrc.1996.1573
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发表时间：
1996-10-23
影响因子：
3.1
通讯作者：
Kain, SR
中科院分区：
文献类型：
--
作者：
Zhang, GH;Gurtu, V;Kain, SR
维多利亚Aequorea victoria水母的绿色荧光蛋白（GFP）已成为一种重要的基因表达标记。然而，野生型GFP的敏感性一直低于标准报告蛋白，如利用酶扩增的β -半乳糖苷酶。为了提高转染的哺乳动物细胞中GFP的检测，我们构建了一种独特的GFP变体，该变体包含发色团突变，使蛋白质的亮度是野生型GFP的35倍，并对密码子进行了优化，以便在哺乳动物细胞中更高的表达。GFP编码序列的这些变化提供了增强的GFP (EGFP)，大大提高了报告蛋白的敏感性。我们发现，将EGFP表达载体传递到哺乳动物细胞中，在16-24小时的转染间隔后，可以很容易地检测到明亮的荧光。用EGFP对转染细胞的目视检测似乎比用β -半乳糖苷酶催化X-gal底物转化的等效测量更敏感。我们的结论是，EGFP允许敏感和方便的检测基因转移在哺乳动物细胞。(C) 1996学术出版社，Inc.
The green fluorescent protein (GFP) from the jellyfish Aequorea victoria has become an important marker of gene expression. However, the sensitivity of wild-type GFP has been below that of standard reporter proteins, such as beta-galactosidase, which utilize enzymatic amplification. To improve the detection of GFP in transfected mammalian cells, we have constructed a unique GFP variant which contains chromophore mutations that make the protein 35 times brighter than wild-type GFP, and is codon-optimized for higher expression in mammalian cells. These changes in the GFP coding sequence provide an enhanced GFP (EGFP) that greatly increases the sensitivity of the reporter protein. We show that the EGFP expression vector delivered into mammalian cells gives rise to bright fluorescence that is readily detectable following a 16-24 hr transfection interval. Visual detection of transfected cells with EGFP appears to be more sensitive than equivalent measurements with beta-galactosidase catalyzed conversion of the X-gal substrate. We conclude that EGFP allows sensitive and convenient detection of gene transfer in mammalian cells. (C) 1996 Academic Press, Inc.