Tagging Ion Channels with the Green Fluorescent Protein (GFP) as a Method for Studying Ion Channel Function in Transgenic Mouse Models

Abstract

In the past ten years, a multitude of cDNAs encoding ion channels have been cloned and have led to a greater understanding of the biological aspects of ion-channel function. Relevant information on structure, function, and pharmacology has come primarily from expressing reconstituted wild-type or mutant ion channels in heterologous expression systems and assaying channel activity using standard biochemical and electrophysiological techniques. In particular, Xenopus oocytes have proven to be a useful expression system owing to the high level of expression of functional ion channels at the cell surface and the large size of the oocyte, which makes it particularly amenable to ion channel analysis using various electrophysiological techniques. However, interpretation of the functional data can be complicated by the presence of several endogenous oocyte currents that can be induced upon exogenous channel expression (1,2). Expressing cloned ion channels in cultured mammalian cells using the transfection technique can often overcome the problem of superimposed endogenous currents while providing an appropriate model of the mammalian cell. This is especially important when addressing questions of channel protein-processing, pharmacology, and roles of ion channels in signal transduction. Ultimately, however, questions of ion-channel physiology and a potential role of altered channel activity in the disease state requires expression in the whole animal model. Numerous ion channels have now been expressed in a tissue-specific fashion in genetically defined strains of mice using the transgenic expression system (3–8). With a rise in the number of core facilities at most research institutions to generate and house transgenic mouse colonies, as well the increased affordability, the transgenic mouse model has become an increasingly important and informative tool for many researchers. In brief, a linearized DNA construct containing the ion-channel cDNA under control of a tissuespecific promoter, the “transgene,” is microinjected into the pronuclei of mouse zygotes. Generally, the transgene integrates into the mouse chromosomal DNA at the one cell-stage, so it is present in every cell of the animal and is capable of being transmitted through the germ line of the adult mouse. Although integration of the transgene usually occurs at a single site in the genome, the site itself is usually random and may contain several copies of the transgene. Theoretically, each founder mouse may express the transgene differently based on the number of transgene copies present and the site of the integration event (however, copy number in itself is not an adequate predictor of the level of transgene expression [9]). Moreover, analysis of the transgene can be further complicated owing to intrafamily variation of expression (10) within a founder line as well as variegated expression within the target tissue itself (11) (i.e., not every cell within the target tissue expresses the transgene or expression levels vary from cell to cell despite the fact that every cell contains the transgene, see Figs. 2 and Figs. 3). For these reasons, determining the level of transgene expression is critical when comparing phenotypes of different transgenic mouse lines and analyzing the physiology of individual cells within the target tissue. In this regard, we now describe a detection method for transgenic mice that uses variant of the green fluorescent protein (GFP) to tag a transgenically expressed ion channel. As demonstrated below, addition of the GFP-tag has no deleterious effect on channel activity and allows for easy and efficient detection of the GFP-tagged ion channels in individual cells of the transgenic animal using UV illumination. Moreover, as fluorescence intensity is related to the level of transgene expression, this method allows for a quick and qualitative assessment of transgene expression on a cell to cell basis.