Use of a bicistronic GFP-expression vector to characterise ion channels after transfection in mammalian cells
Use of a bicistronic GFP-expression vector to characterise ion channels after transfection in mammalian cells
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使用双顺反子 GFP 表达载体表征哺乳动物细胞转染后的离子通道
DOI：
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发表时间：
1997
期刊：
影响因子：
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通讯作者：
J. Eggermont
中科院分区：
文献类型：
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作者：
D. Trouet;B. Nilius;T. Voets;G. Droogmans;J. Eggermont
摘要离子通道瞬时转染哺乳动物细胞是研究离子通道特性的有效方法。 然而，瞬时转染程序中的一般问题是如何选择表达转染的cDNA的细胞。我们已经构建了一个双顺反子载体，pCINeo/IRES-GFP，它利用一个红移的变体的绿色荧光蛋白作为体内细胞标记。将离子通道cDNA掺入双顺反子单元中允许离子通道和绿色荧光蛋白的偶联表达。用含有大鼠延迟整流钾通道cDNA（RCK 1，Kv1.1）的pCINeo/IRES-GFP瞬时转染COS细胞后，所有绿色细胞（n = 32）表达RCK 1通道，如通过Kv1.1的众所周知的动力学、K+选择性和药理学所鉴定的。相反，非荧光细胞（n = 24）在RCK 1表达方面为阴性。结论是双顺反子pCINeo/IRES-GFP载体提供了一种有效和非侵入性的方法来鉴定转染后表达离子通道的细胞。这种新方法将极大地促进转染到哺乳动物细胞中的离子通道的功能研究。
Abstract Transient transfection of ion channels into mammalian cells is a useful method with which to study ion channel properties. However, a general problem in transient transfection procedures is how to select cells that express the transfected cDNA. We have constructed a bicistronic vector, pCINeo/IRES-GFP, which utilises a red-shifted variant of Green Fluorescent Protein as an in vivo cell marker. Incorporation of an ion channel cDNA into the bicistronic unit allows coupled expression of the ion channel and Green Fluorescent Protein. After transient transfection of COS cells with pCINeo/IRES-GFP containing a rat delayed rectifier K+ channel cDNA (RCK1, Kv1.1), all green cells (n = 32) expressed the RCK1 channel as identified by the well known kinetics, K+ selectivity and pharmacology of Kv1.1. In contrast, non-fluorescent cells (n = 24) were negative with respect to RCK1 expression. It is concluded that the bicistronic pCINeo/IRES-GFP vector provides an efficient and non-invasive way of identifying cells which express ion channels after transfection. This novel method should greatly facilitate functional studies of ion channels transfected into mammalian cells.
影响因子：
13.8
作者：
CUBITT, AB;HEIM, R;TSIEN, RY
通讯作者：
TSIEN, RY
4-氨基吡啶结合和缓慢失活在大鼠 Kv1.1 和 Shaker 钾通道中是相互排斥的。
影响因子：
3.6
作者：
Castle,NA;Fadous,SR;Logothetis,DE;Wang,GK
通讯作者：
Wang,GK