A common question is "which reporters to choose" for dual reporter assays. Let's say you want to monitor and assess tumor growth and track lymphocyte infiltration towards the tumor. As a rule of thumb we will use the strongest reporter for the weakest signal. The strongest reporter to date is Firefly Luciferase - in particular Promega's pGL4-luc2. As the T-lymphocytes will distribute in circulation and may home in locations other than the tumor, we will label the T-lymphocytes with firefly luciferase. For the tumor we then need to choose an alternative reporter. Obvious choices are Renilla luciferase, and the new red shifted fluorescent proteins.
Links to Reporter Reagent Resources
Dual Reporter Optical In Vivo Imaging Assays
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Enhanced Firefly Luciferase - Rabinovich et al., 2008
Visualizing fewer than 10 mouse T cells with an enhanced firefly luciferase in immunocompetent mouse models of cancer.
M. D. Anderson Cancer Center, 7455 Fannin Street, Houston, TX 77054, USA.
Antigen specific T cell migration to sites of infection or cancer is critical for an effective immune response. In mouse models of cancer, the number of lymphocytes reaching the tumor is typically only a few hundred, yet technology capable of imaging these cells using bioluminescence has yet to be achieved. A combination of codon optimization, removal of cryptic splice sites and retroviral modification was used to engineer an enhanced firefly luciferase (ffLuc) vector. Compared with ffLuc, T cells expressing our construct generated >100 times more light, permitting detection of as few as three cells implanted s.c. while maintaining long term coexpression of a reporter gene (Thy1.1). Expression of enhanced ffLuc in mouse T cells permitted the tracking of <3 x 10(4) adoptively transferred T cells infiltrating sites of vaccination and preestablished tumors. Penetration of light through deep tissues, including the liver and spleen, was also observed. Finally, we were able to enumerate infiltrating mouse lymphocytes constituting <0.3% of total tumor cellularity, representing a significant improvement over standard methods of quantitation including flow cytometry.