Multiplex bioluminescence in vivo imaging employing red and green luciferases for quantitative monitoring of multiple events

Bioluminescence in vivo imaging (BLI) is a powerful tool in preclinical research allowing the real time monitoring of different physiological and pathological conditions in living intact animals using bioluminescent reporter gene technology. To improve the BLI performance, different native or mutant luciferases with different emission spectra or that are secreted have been widely exploited for in vitro bioluminescence-based cellular assays and they are now being applied for in vivo applications. Besides, spectral separation allows to unmix and quantify signals from proteins with different emission spectra. Here we investigated the combined use of green click beetle luciferase (CBG99, λ max emission 537nm) and a red codon-optimized mutant of P pyralis (Ppy-RE8, λ max emission 618nm) for quantitative analysis of red and green bioluminescence signals in vivo. Lentiviruses constitutively expressing Ppy-RE8 and CBG99 luciferase reporter genes were generated and human embryonic kidney cells (Hek293T) were subsequently transfected to express luciferases. Validity of spectral unmixing for the quantitation of the different luciferases was assessed using both a luminometer with bandpass filters and a bioluminescence imager equipped with 18 narrow band 20 nm bandwidth emission filters, covering the range 490 -850 nm. Then, confirmative results were obtained when the cells, expressing Ppy-RE8 or Green CBG99, were injected subcutaneously into immunodeficient mice and BLI imaging was performed in vivo using the same system. The minimum number of detectable cells emitting red or green light in mixed population was 2x10E4. Preliminary in vivo data envisage the future application of these couple of luciferases for monitoring of multiple events simultaneously by means of BLI.