USE OF A BICISTRONIC GFP-EXPRESSION VECTOR TO CHARACTERIZE ION CHANNELS AFTER TRANSFECTION IN MAMMALIAN-CELLS

Transient transfection of ion channels into mammalian cells is a usefu l method with which to study ion channel properties. However, a genera l problem in transient transfection procedures is how to select cells that express the transfected cDNA. We have constructed a bicistronic v ector, 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 t he ion channel and Green Fluorescent Protein. After transient transfec tion of COS cells with pCINeo/IRES-GFP containing a rat delayed rectif ier K+ channel cDNA (RCK1, Kv1.1), all green cells (n = 32) expressed the RCK1 channel as identified by the well known kinetics, K+ selectiv ity and pharmacology of Kv1.1. In contrast, non-fluorescent cells (n = 24) were negative with respect to RCK1 expression. It is concluded th at the bicistronic pCINeo/IRES-GFP vector provides an efficient and no n-invasive way of identifying cells which express ion channels after t ransfection. This novel method should greatly facilitate functional st udies of ion channels transfected into mammalian cells.