'Green mice' and their potential usage is biological research
'Green mice' and their potential usage is biological research
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DOI：
10.1016/s0014-5793(98)00593-6
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发表时间：
1998-06-23
期刊：
影响因子：
3.5
通讯作者：
Okabe, M
中科院分区：
文献类型：
--
作者：
Ikawa, M;Yamada, S;Okabe, M
维多利亚水母（Aequorea victoria）的绿色荧光蛋白（GFP）是由238个氨基酸组成的单肽[1]。当Ca 2+与水母发光蛋白结合时，水母发光首先被激活，随后发出蓝色荧光。蓝色荧光和随后从激活的水母发光蛋白的能量转移导致GFP发射绿色荧光。因此，与酶不同，GFP不需要底物来发射荧光。相反，它只需要能量来激发荧光团。GFP的荧光团通过氧化后Ser 65、Tyr 66和Gly 67的翻译后自动环化形成。这种构象变化不需要底物或辅因子。因此，即使在异源物种中表达，GFP也可以是荧光性的。在位于由11个L-片层组成的桶状篮的中心的荧光团形成之后[2]，GFP变得相当稳定，并且在高达65 ℃、pH 11、1%SDS或6 M氯化胍的条件下保持荧光[3^ 5]。自从第一次报道绿色荧光蛋白在秀丽隐杆线虫中的表达以来，这种新颖而简单的报告基因由于其作为基因表达的体内标记物的潜力而引起了人们的极大兴趣，因为经常使用的其他报告基因编码酶，例如L-半乳糖基转移酶（lacZ）、氯霉素乙酰转移酶（CAT）和荧光素酶（luc），并且需要底物来检测基因表达。最近，已经开发了几种穿透质膜并在细胞质中加工以用于活细胞的底物[7]。然而，仍然需要用这些标记物装载基底。在这种情况下，GFP比其他常用的报告基因具有显著的优势。由于GFP在没有任何底物或辅因子的情况下发射绿色荧光，因此可以通过照射活细胞来监测GFP的存在。GFP可能为使用完整细胞和生物体作为实验系统打开大门[4，5，8]。在这里，我们描述了我们在转基因小鼠系统中表达GFP的试验，并概述了GFP作为哺乳动物重要标志物的应用。
Green£ uorescent protein (GFP) from the jelly¢ sh Aequorea victoria is a single peptide of 238 amino acids [1]. Aequorea bioluminescence is¢ rst activated when Ca2+ binds to aequorin following emission of blue£ uorescence. Blue£ uorescence and subsequent energy transfer from activated aequorin cause GFP to emit green£ uorescence. Therefore, unlike enzymes, GFP needs no substrate to emit£ uorescence. Rather, it requires only energy to excite the£ uorophore. The£ uorophore of GFP is formed by post-translational auto-cyclization of Ser65, Tyr66, and Gly67 following oxidation. This conformational change requires no substrate (s) or cofactor (s). Therefore, GFP can be£ uorescent even when expressed in heterologous species. After the formation of£ uorophore that locates in the center of a barrel-like basket composed of 11 L-sheets [2], GFP becomes quite stable and remains£ uorescent up to 65³C, pH 11, 1% SDS or 6 M guanidinium chloride [3^ 5]. Since the¢ rst report of expression of GFP in Caenorhabditis elegans [6], this novel and simple reporter has attracted much interest for its potential as an in vivo marker of gene expression, because other reporter genes that are used frequently encode enzymes, eg L-galactosyltransferase (lacZ), chloramphenicol acetyltransferase (CAT), and¢ re£ y luciferase (luc), and require substrates to detect the gene expression. Recently, several substrates have been developed that penetrate the plasma membrane and are processed in the cytoplasm for use in living cells [7]. However, it is still necessary to load the substrate with these markers. In this context, GFP has a signi¢ cant advantage over other commonly used reporters. Since GFP emits green£ uorescence without any substrates or cofactors, one can monitor the presence of GFP by illuminating living cells. GFP may open the door for the use of intact cells and organisms as experimental systems [4, 5, 8]. Here we describe our trials in expressing GFP in a transgenic mouse system and give an overview of the application of GFP as a vital marker in mammals.