Comparison of bidirectional and bicistronic inducible systems for coexpression of connexin genes and fluorescent reporters

Gene expression studies often require inducible coexpression of both a gene of interest and a reporter gene. Fusion of fluorescent reporters can, however, modify protein structure and function. We have generated inducible expression systems for two connexin genes: Cx30 and Cx43. It has been reported recently that reporter fusion to connexins can modify their function. Therefore, we compared two methods of independent reporter coexpression and examined colocalization with induced connexin expression. Identical levels of connexin expression were observed for both the bidirectional and bicistronic expression systems. In contrast, however, reporter gene expression by the bidirectional promoter provided brighter average fluorescent pixel intensity than expression of a reporter gene in a bicistronic transcript. Moreover, as a result of this difference in reporter expression, bidirectional expression systems provided equal or better colocalization between the connexins and reporter gene fluorescence. The results of our study indicate that bidirectional reporter expression provides a robust indicator of transfection and gene expression and, therefore, may favor the use of bidirectional over bicistronic reporters in the design of expression systems where the gene of interest, such as a connexin gene, contains translational motifs or long intronic regions.