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. 2011 May 17;19(9):1645–1655. doi: 10.1038/mt.2011.90

Figure 3.

Figure 3

In vivo regulation of hGLuc expression in a human xenogeneic transplantation model with infected human embryonic kidney 293 (HEK293) cells. (a) Noninvasive bioluminescence imaging of G418 induced hGLuc expression from subcutaneously (s.c.) implanted HEK293 xenograft mice. The top panels show the first “switch-on” induction (day 4) while the bottom panels show the second “switch-on” induction (day 10) of the in vivo system. Control and mutant represent NOD-scid IL2Rgammanull (NSG) mice injected s.c. with transduced HEK293 cells harboring the mutant hGLuc construct exposed to phosphate buffered saline and G418, respectively. Wild type (WT) represent NSG mice injected s.c. with transduced HEK293 cells harboring the wild-type hGLuc construct exposed to G418. Experiments were repeated twice and each time involved the use of at least three mice per experimental group. (b,c) The “On” and “Off” regulation of hGLuc expression in HEK293 xenograft model. Luciferase activities were measured in relative light units (RLU) from blood serum. The graph is represented in RLU/mm3 by correlating the luciferase activity to tumor size. The “ON” and “OFF” arrow on the x-axis indicate when the G418 drug was administered and discontinued, respectively. Blood was collected and tumor size was measured, as described in the Materials and Methods, prior to every injection of G418. (d) Two rounds of regulation on the isolated hGLuc mutant xenograft tumor cells, grown in vitro. 2.5 × 105 cells were seeded in 6-wells and exposed to 150 µg/ml of G418 and 1 mg/ml of gentamicin for 48 hours. Half of the cells were lysed and measured for luciferase activities while the other half were kept in culture without any exposure to aminoglycosides. A week later, 2.5 × 105 cells were seeded and exposed to the aminoglycosides for 48 hours before luciferase activities were measured. Xenograft tumor cells were isolated from three mice from two independent experiments. All luciferase readings were normalized to total protein. All luciferase activity experiments were done in triplicate and repeated at least twice. Error bars represent standard deviation.