Inducible and Reversible Clock Gene Expression in Brain Using the tTA System for the Study of Circadian Behavior (original) (raw)
Figure 1
Regulation of the Clock_Δ_19 TG Using the tTA System
(A) Schematic diagram showing the Tet-Off system and constructs used for generating the tTA transactivator and target tetO transgenic lines. The Scg2 promoter drives the expression of tTA, which binds to an array of cognate operator sequences in the absence of Dox but not in its presence, resulting in transcriptional activation or repression of the HA-tagged ClockΔ19 TG, respectively.
(B) Analysis of the Scg2::tTA line: (a) in situ hybridization of the tTA transcript, using an antisense tTA oligo probe, on coronal brain slices; (b) in situ hybridization of a control sense tTA oligo probe shows absence of specific hybridization and indicates that the antisense tTA oligo probe hybridization is specific; and (c and d) β-galactosidase staining of SCN/brain-specific induction of the LacZ TG in Scg2::tTA/tetO-lacZ mice, using two different tetO-lacZ reporter lines (lac1 and lac2).
(C) In situ hybridization of endogenous WT Clock and HA-tagged ClockΔ19 TG from Scg2::tTA × tetO::_ClockΔ19_-HA matings: representative coronal brain slices of (a and b) WT mice; (c and d) single transgenic Scg2::tTA mice; (e and f) single transgenic tetO::_ClockΔ19_-HA mice; and (g and h) double transgenic Scg2::tTA/tetO::_ClockΔ19_-HA mice.
(D) Western blot analysis of transgenically induced CLOCKΔ19 and endogenous WT CLOCK in cerebellar lysates from all four possible genotypes. For the double transgenic mice, total brain lysates from five of the independent lines are shown with their line identity numbers indicated. Two independent mice from line 5 are also shown. The red asterisk indicates the WT protein, while the green asterisk denotes the HA-tagged CLOCKΔ19.
(E) Western blot analysis of HA-tagged CLOCKΔ19 of various tissues from the double transgenic mice.
(F–L) Immunocytochemical analysis of Scg2::tTA/tetO::Clock_Δ_19 _-_HA mice. (F) Nuclear location of the CLOCKΔ19-HA. SCN/coronal sections were labeled with anti-HA antibody to detect transgenically induced CLOCKΔ19-HA (×100 magnification). (G–I) SCN/coronal sections were double labeled to detect transgenically induced CLOCKΔ19-HA (green, G) and endogenous vasopressin (AVP) (red, H). (I) Overlay of CLOCKΔ19-HA and AVP expression. (J–L) Double labeling of transgenically induced CLOCKΔ19-HA (green, J) and endogenous VIP (red, K). (L) Overlay of CLOCKΔ19-HA and VIP expression. Cells that express both the CLOCKΔ19_-_HA and VP/VIP are shown in yellow in overlaid figures. (G–L) Captured with a ×20 objective.