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Gene discovery and gene therapy call for advanced technologies to reliably assess gene expression; efficient coupling of gene expression to the expression of reporter genes is critical. Various noninvasive molecular imaging modalities have emerged to track biological processes in animal models. Here, we evaluate various strategies to link transgene expression with that of an (imaging) reporter gene. Using lentiviral vectors containing internal ribosomal entry sites (IRES), 2A-like peptides, or a bidirectional promoter, we compared their ability to ensure efficient coexpression of multiple reporter genes. Although the encephalomyocarditis virus (EMCV) IRES yielded functional bicistronic vectors, the expression level of the reporter downstream of IRES was consistently lower than that of the upstream transgene. Interestingly, peptide 2A constructs performed best in vitro and in vivo, providing effective noninvasive …