Use of a bicistronic GFP-expression vector to characterise ion channels after transfection in mammalian cells.
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.
Trouet
D
D
Laboratorium voor Fysiologie, K.U. Leuven, Belgium.
Nilius
B
B
Voets
T
T
Droogmans
G
G
Eggermont
J
J
D000818:Animals; D018801:CD2 Antigens / Q000378:metabolism; D019556:COS Cells / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D004546:Elapid Venoms / Q000494:pharmacology; D015870:Gene Expression; D005796:Genes / Q000235:genetics*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D007473:Ion Channels / Q000235:genetics / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics* / Q000378:metabolism; D015221:Potassium Channels / Q000378:metabolism; D051381:Rats; D019789:Tetraethylammonium; D013757:Tetraethylammonium Compounds / Q000494:pharmacology; D014162:Transfection / Q000235:genetics*
D018801:CD2 Antigens; D004546:Elapid Venoms; D007473:Ion Channels; D008164:Luminescent Proteins; D015221:Potassium Channels; D013757:Tetraethylammonium Compounds; D049452:Green Fluorescent Proteins; D019789:Tetraethylammonium; C020273:dendrotoxin