When Two Rreporter Genes Are Better Than One

Sometimes choosing the right reporter gene is a hard matter. Let choose fluorescent proteins: they are very bright, but they need also an external source of light for excitation and because of that, some specimen suffer from ligth toxicity or results in autofluorescence; do you choose luciferase enzymes? they are less bright - so you need more sophisticated instruments - and if you want to record endpoint (RLU) proportional to gene expression, your have to give saturating amounts of ATP and luciferin to your sample. Although with cell cultures it is quite simple to fulfill the requirements of reporter gene, or eventually move to another reporter system to integrate intrisinc limitations of a single reporter measurement, this is not true for reporter animals. Once you knock your GFP mouse, your EGFP zebrafish, or your GUS Arabidobsis, you have to deal forever with specific limitation typical of the selected reporter.

Multimodality technologies effort to fill that gap by coupling one promoter with two or three different reporter genes. Up to date, there are three strategies in plasmid construction that claim for multimodality: bicistronic vectors, fusion proteins and bidirectional promoters. Recently, the bicistronic vector was the strategy choosed by Luisa Ottobrini and colleagues from Milan University to develop multimodality imaging of estrogen receptor transcriptional activity, as reporterd in the latest print issue of the European Journal of Nuclear Medicine and Molecular Imaging (a forum for the exchange of clinical and scientific information for the community involved in molecular investigation of diseases).

In the developed construct, a promoter activated by the estrogen receptor, drive the expression of two reporter genes, the firefly luciferase - for in vivo bioluminescent imaging (BLI) -, and a mutated form of the domaminergic D2 receptor (D2R80A) for positron emission tomography (PET). Thanks to the internal ribosome entry site (IRES) of the encephalomyocarditis virus, the two reporter genes can be translated from a single RNA transcript. Finally, insulator sequences (MAR) flanks the construct and prevents enhancer-mediated activation, or repression of transcription by chromatin, assuring the reporter to be transcriptionally accessible in every cell in which estrogen receptors are expressed.

According to the authors,

The coupling of a nuclear with an optical reporter gene yields highly informative data.

So... two is better than one, and now it remains to wait for the first reporter animal generated with such kind of construct.

Ottobrini, L., Ciana, P., Moresco, R., Lecchi, M., Belloli, S., Martelli, C., Todde, S., Fazio, F., Gambhir, S.S., Maggi, A., Lucignani, G. (2008). Development of a bicistronic vector for multimodality imaging of estrogen receptor activity in a breast cancer model: preliminary application. European Journal of Nuclear Medicine and Molecular Imaging, 35(2), 365-378. DOI: 10.1007/s00259-007-0578-z