Validation of a triple reporter lentiviral vector encoding eGFP, firefly luciferase and HSV-thymidine kinase in vitro and in vivo

Background: Lentiviral vectors are a popular tool for contemporary researchers. Despite the versatility for use both in vitro and in vivo, its limited packaging capacity can impair the use when one wants to combine a therapeutic and a re-porter gene. We have constructed a lentiviral vector (LV) encoding eGFP, firefly luciferase (fLuc) and HSV truncated thymidine kinase (HSV-tTK) connected with viral 2A-pep-tide sequences. This triple reporter vector (LV-3R) expresses all three transgenes in an equivalent ratio and with all the functional capacities. Methods and Results: The LV encoding eGFP, fLuc and HSV-tTK was evaluated in vitro after transduction of 293T cells. eGFP was assessed using FACS analysis, fLuc activity was measured using the luciferase assay and HSV-tTK function was evaluated with gancyclovir toxicity. All three trans-genes were functional in this in vitro testing. GL261 mouse glioma cells were transduced with LV-3R and sorted for stringent eGFP expression. The cells were subsequently injected in NMRI mice. Bioluminscent imaging (BLI) was used to quantify tumor growth non-invasively. One cohort received gancyclovir treatment whereas the other cohort did not. After two weeks, the treated cohort showed a marked decrease in BLI signal and a reduction in tumor volume was noted after sacrifice of the animals. Conclusion: These results show the versatility of a LV encoding several transgenes using 2A-peptide sequences both in vitro and in vivo. Reporter genes can as such be combined with therapeutic genes in animal disease models.