Figure 2

Luciferase expression in double transgenic CaMKIIα-tTA/EGFP-Ptetbi-luc rats. (A) In vivo imaging of luciferase gene activity. Representative example of brain bioluminescence after injection of the luciferase substrate D-luciferin. The rats were anaesthetized during the imaging process using 2.5% isoflurane. Shortly before the measurement, the fur was shaved on top of the head to facilitate the external detection of internally generated photons. Luciferase bioluminescence was restricted to the head region, which demonstrates brain-specific expression in CaMKIIα-tTA/EGFP-Ptetbi-luc rats. Photons were scattered by the skull. (B,C) Quantification of luciferase activity in different brain regions. Animals of the CaMKIIα-tTA lines 4.5 and 4.7 were crossed to the reporter lines EGFP-Ptetbi-luc 66.1 (B) and 71.1 (C), respectively, to obtain double transgenic animals of all four combinations. Luciferase activity in lysates of double transgenic and single transgenic rats (line 66.1 and 71.1) is reported as relative light units (RLU) per μg protein and presented on a log scale. Luciferase activity conveyed by both CaMKIIα-tTA lines 4.5 and 4.7 in combination with the Ptet-reporter 66.1 (B) was significantly higher (P < 0.001 for all measured brain regions) than single transgenic reporter activity (olfactory bulb: F (2,15) = 304, P ≤ 0.001; cortex: F (2,14) = 118, P ≤ 0.001; hippocampus: F (2,14) = 184, P ≤ 0.001; cerebellum: F (2,14) = 37.3, P ≤ 0.001). Identical highly significant results (P < 0.001 for all subregions versus single transgenic reporter rats of line 71.1) were also found for both tTA driver lines in combination with line EGFP-Ptetbi-luc 71.1 (olfactory bulb: F (2,17) = 174, P ≤ 0.001; cortex: F (2,17) = 155, P ≤ 0.001; hippocampus: F (2,16) = 205, P ≤ 0.001; cerebellum: F (2,16) = 28.3, P ≤ 0.001) (C). When the luciferase activity mediated by the two transgenic tTA driver lines was compared between the two, line 4.5 showed a significantly stronger luciferase activity in the olfactory bulb (dF = 12, P ≤ 0.01 in combination with reporter 66.1 in (B); dF = 13, P ≤ 0.05 with reporter 71.1 in (C)), whereas no significant differences were found in the other brain regions (B,C). Moreover, luciferase activity of Ptet-reporter lines 66.1 and 71.1 with both tTA driver lines (4.5/66.1 versus 4.5/77.1 and 4.7/66.1 versus 4.7/71.1) was not significantly different (B,C). All data are presented as mean values + standard error of the mean. Logarithmic data transformation was performed prior to statistical analysis. Stars represent P-values obtained by one-way analysis of variance followed by Bonferroni post hoc test: ***P < 0.001. RLU: relative light units; tTA: tetracycline-controlled transactivator