Inducible and Reversible Clock Gene Expression in Brain Using the tTA System for the Study of Circadian Behavior (original) (raw)
Figure 4
Rapid Inhibition and Reversal of tTA Mediated tetO::_Clock_Δ_19_-HA Transcription in Scg2::tTA/tetO::_Clock_Δ_19_-HA Double Transgenic Mice to a Lower Concentration of Dox
Representative actograms of (A) a single transgenic tetO::_Clock_Δ_19_-HA mice and (B–D) double transgenic Scg2::tTA/tetO::_Clock_Δ_19_-HA mice on 10 μg/ml Dox treatment. Administration of 10 μg/ml Dox was just as effective as 2 mg/ml Dox on the free-running wheel period; all double transgenic mice showed a shortening of circadian period. Withdrawal of Dox (−Dox, second arrow) led to reversal back to the previous long circadian period by next day.
(E) Comparison of free-running period estimates for all four genotypes. Data are presented as mean with corresponding 95% confidence interval. Numbers on the bottom of the bars indicate the number of animals in each group that were wheel tested for their locomotor activity rhythm. Overall, there were significant differences in mean circadian period among genotypes (F3,78 = 118.77, p < 0.00005). Pairwise comparisons indicated that there were no significant differences on circadian period in single transgenic lines (Scg2::tTA or tetO::_Clock_Δ_19_-HA) compared to the WT littermates; however, a significantly longer free-running period was observed in double transgenic mice compared to WT, Scg2::tTA, and tetO::_Clock_Δ_19_-HA (*p < 0.0005 for each comparison), indicating that the transgenically induced _Clock_Δ_19_-HA TG expression causes this period difference. With administration of 10 μg/ml Dox, no difference was observed among the genotypes on circadian period (F3,62 = 1.93, p = 0.1339), thus the transgenically induced _Clock_Δ_19_-HA TG expression was “turned off.” Rapid reversal was achieved when the double transgenic mice were returned to water treatment (−Dox); their free-running period reverted back to the previous circadian period, lengthening about 1 h (**paired _t_-test, t 22 = −17.2177, p < 0.00005).
(F) In situ hybridization of the _Clock_Δ_19_-HA transcript on coronal brain slices from the single tetO::_Clock_Δ_19_-HA and double transgenic mice (Scg2::tTA/tetO::_Clock_Δ_19_-HA) on water (−Dox, left side) and 10 μg/ml Dox (+Dox, right side) treatments, respectively. TG transcript was detected using an antisense HA oligo probe. The transgenically induced _Clock_Δ_19_-HA transcript is “turned off” by day 3 on 10 μg/ml Dox treatment.