Generation of Transactivator and Target Transgenic Lines

The tTA (also known as Tet-Off) system requires two independent lines of transgenic mice (Figure 1A): one line expresses tTA under the control of a specific promoter, and a second line carries a tTA-responsive tetO promoter linked to the target gene of interest [31–34]. When two TGs are introduced into a single mouse through mating, the tetO-linked gene is activated but only in those cells that express tTA. Expression of the target TG can be suppressed by Dox.

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Figure 1. Regulation of the Clock^Δ19 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 Clock^Δ19 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 Clock^Δ19 TG from Scg2::tTA × tetO::Clock^Δ19-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::Clock^Δ19-HA mice; and (g and h) double transgenic Scg2::tTA/tetO::Clock^Δ19-HA mice.

(D) Western blot analysis of transgenically induced CLOCK^Δ19 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 CLOCK^Δ19.

(E) Western blot analysis of HA-tagged CLOCK^Δ19 of various tissues from the double transgenic mice.

(F–L) Immunocytochemical analysis of Scg2::tTA/tetO::Clock^Δ19-HA mice. (F) Nuclear location of the CLOCK^Δ19-HA. SCN/coronal sections were labeled with anti-HA antibody to detect transgenically induced CLOCK^Δ19-HA (×100 magnification). (G–I) SCN/coronal sections were double labeled to detect transgenically induced CLOCK^Δ19-HA (green, G) and endogenous vasopressin (AVP) (red, H). (I) Overlay of CLOCK^Δ19-HA and AVP expression. (J–L) Double labeling of transgenically induced CLOCK^Δ19-HA (green, J) and endogenous VIP (red, K). (L) Overlay of CLOCK^Δ19-HA and VIP expression. Cells that express both the CLOCK^Δ19-HA and VP/VIP are shown in yellow in overlaid figures. (G–L) Captured with a ×20 objective.

https://doi.org/10.1371/journal.pgen.0030033.g001

To generate an SCN/brain-enriched transactivator line, we first searched for SCN-enriched transcripts that have expression restricted to the brain from the comprehensive mouse tissue expression databases [50,51]. This analysis revealed about 35 candidate genes that were either very highly expressed in the SCN or SCN/brain enriched. We then analyzed these candidate genes for SCN expression using in situ hybridization. From this screen, we found one gene, Secretogranin II (Scg2), that has very high constitutive expression in the SCN and has an expression pattern limited to brain, pituitary, and adrenal glands [52]. Scg2 is a member of the neuroendocrine/neuronal secretory proteins, which are widely distributed in endocrine, neuroendocrine, and neuronal cells [53,54]. Although the exact role of Scg2 is not well understood, research suggests that Scg2 plays biologic roles in neurotransmission and paracrine regulation of central and peripheral actions of the nervous and neuroendocrine systems [54,55]. Because of its brain-enriched pattern of expression and, in particular, its strong expression in the SCN, we explored Scg2 as a tissue-specific activator for the tTA system. We generated an SCN/brain-enriched transactivator line using a 9.8-kb promoter region of Scg2. We examined the pattern of expression using an oligo probe specific to the tTA transcript and showed distinct and strong tTA expression in the SCN but also throughout the brain (Figure 1Ba and 1Bb). To characterize tTA expression further, we crossed the Scg2::tTA line to mice carrying a tetO promoter-lacZ reporter construct [35]. We tested two independent tetO promoter-lacZ reporter lines, namely lines lac1 and lac2 (kindly provided by Mark Mayford, Scripps Research Institute, San Diego, California, United States). Both reporter lines showed similar SCN/brain-enriched expression patterns as demonstrated by β-galactosidase staining (Figure 1Bc and 1Bd). We used the Scg2::tTA line for all subsequent experiments described in this report.

Next, we generated the target line, which carries a tetO promoter fused to a dominant-negative Clock^Δ19 mutant allele [56,57]. In order to clearly assess the inducible expression of the TG, we also fused a hemagglutin (HA) epitope tag on the 3′ end of the cDNA, which does not interfere with CLOCK transcriptional activation [58]. We produced eight independent tetO::Clock^Δ19-HA transgenic lines, and all lines were crossed to Scg2::tTA mice to evaluate whether we could control the expression of the TG in a temporal manner.

Restricted Expression of the tetO-Linked Clock^Δ19 TG

We assessed whether the Clock^Δ19-HA TG is expressed in Scg2::tTA/tetO::Clock^Δ19-HA double transgenic mice by in situ hybridization using oligo probes targeted to exon 19 of the Clock gene and to the HA tag (Figure 1C). The original ENU-induced Clock^Δ19 mutation is an A-to-T transversion in a splice donor site, causing skipping of exon 19 [57]; thus, the exon 19-specific probe detects only the endogenous WT Clock transcript. Conversely, the HA tag-specific probe detects only the Clock^Δ19-HA TG transcript. All mice showed endogenous WT Clock transcript expression as expected. However, while only double transgenic Scg2::tTA/tetO::Clock^Δ19-HA mice showed clear hybridization with the HA tag-specific probe, single transgenic (Scg2::tTA or tetO::Clock^Δ19-HA) and WT mice did not show any expression of the HA tag. Thus, expression of the Clock^Δ19-HA TG, which is evident in the SCN and throughout the brain, is induced by tTA in the double transgenic mice only. Similarly, Western blot analyses showed that only the double transgenic mice expressed the mutant TG protein (Figures 1D and S1A). Anti-CLOCK antibody detects both the mutant and WT proteins, which can be discriminated by their size difference. The lack of exon 19 in the mutant allele results in the deletion of 51 amino acids and subsequently produces a shorter protein for the Clock^Δ19-HA TG compared to the WT allele. All eight independent tetO::Clock^Δ19-HA transgenic lines expressed the mutant protein when crossed to Scg2::tTA mice (Figures 1D and S1A). In addition, the mean density ratio measurement of the WT and mutant proteins in Western blot analyses revealed that expression of the mutant protein is 0.51- to 1.05-fold relative to the WT protein in these lines. This was also demonstrated by Southern analysis (unpublished data), which showed that target lines carried approximately one or two copies of the TG. In order to test for leakiness of the tetO promoter, we inspected whether the tetO::Clock^Δ19-HA single transgenic mice from each independent line expressed the TG using the anti-HA antibody against the brain lysates (Figure S1B). Of the eight independent target lines, only one (line 12) showed activation of the TG (likely due to a position effect on the TG), and therefore this line was excluded from further analysis. Thus, we obtained seven independent tetO target lines that showed tightly regulated inducible expression.

We next assessed tissue specificity and spatial expression of the Clock^Δ19-HA TG in double transgenic mice. Western blot analyses indicated that expression of the TG is brain and SCN enriched (Figure 1E). Because Scg2 participates in the neuroendocrine system [54,55], as expected, the TG was also expressed in the pituitary gland (Figure 1E); however, there was no expression in other peripheral tissues, such as the kidney, lung, liver, and spleen. Using fluorescence microscopy and an HA tag antibody, we explored the spatial expression of the TG within the SCN and found that Clock^Δ19-HA TG expression is nuclear (Figure 1F). In WT mice, CLOCK has been shown to be nuclear and is expressed constitutively in the mouse SCN [59]. In contrast, rhythmically expressed negative factors of the circadian gene, such as PER1, PER2, and CRY1, accumulate in the nuclei at the end of circadian night [59,60]. To delineate further the spatial organization of the HA-containing cells within the SCN, we compared HA expression with the distribution of neuropeptide markers (Figure 1G–1L). Classically, the SCN is organized into two major subdivisions: a core that lies adjacent to the optic chiasm and is composed of neurons that predominantly produce vasoactive intestinal polypeptide (VIP) and a shell that surrounds the core and contain a large population of neurons that produce arginine vasopressin (AVP) [61–64]. Examination of the HA-containing cells showed that more than 90% of the cells in the SCN express the TG and that expression occurs throughout both the core and the shell. Serial sections through the rostral-caudal extent of the SCN showed that HA-expressing cells colocalized with AVP- and VIP-expressing cells (Figures 1F–1L, S2, and S3). Indeed, all AVP- and VIP-positive cell bodies in the SCN were also HA positive. Taken together, the detailed evaluation using Western blot and immunocytochemical analyses demonstrates that Scg2-driven Clock^Δ19-HA TG expression is tissue specific and enriched in the brain and in the majority of cells in the SCN, including the core and the shell.

Regulation of Conditionally Expressed Clock^Δ19 Allele by Doxycycline: Changes in Behavioral Circadian Rhythms

The original N-ethyl-N-nitrosourea (ENU)-induced Clock^Δ19 mutant mice exhibit several behavioral alterations compared to WT mice, one of which is the lengthened free-running period of wheel-running activity. Heterozygous Clock^Δ19/+ mice show a lengthening of the circadian period of approximately 1 h, while homozygous Clock^Δ19/Clock^Δ19 mice exhibit periods about 4 h longer compared to WT mice and fail to express persistent circadian rhythms when maintained in constant darkness [65]. Because the Clock^Δ19 mutant allele behaves as an antimorph (one type of dominant-negative mutation), it competes with the WT allele in the generation of circadian rhythms [56,57,66]. In this study, because the Scg2::tTA/tetO::Clock^Δ19-HA double transgenic mice express the Clock^Δ19-HA TG as well as both copies of the endogenous WT CLOCK, we hypothesized that these mice would exhibit a free-running period of wheel activity rhythm similar to that of the heterozygous Clock^Δ19/+ mice. To test this prediction, running-wheel behavior of all four genotypes from the Scg2::tTA x tetO::Clock^Δ19-HA matings was recorded (Figure 2). All mice entrained to a 12/12-h light–dark cycle, initiating their nightly bouts of activity at the beginning of the dark phase with the majority of locomotor activity restricted to the night. Upon release into constant darkness (DD), however, only the double transgenic mice displayed a lengthened circadian period approximately 1 h greater than that of WT mice (23.9 to 24.8 h versus 23.4 to 23.7 h, respectively). Pairwise comparisons indicated that there were no significant differences in circadian period among the single transgenic lines (Scg2::tTA or tetO::Clock^Δ19-HA) compared to WT littermates, while the period observed in double transgenic mice was significantly longer compared to the WT, Scg2::tTA, and tetO::Clock^Δ19-HA mice (mean DD comparison among all genotypes, F_3,78 = 118.77, p < 0.00005; each pairwise comparison [double transgenic versus others], p < 0.0005). Double transgenic mice from all seven independent tetO lines showed lengthening of the circadian period similar to the circadian period exhibited in the heterozygous Clock mutant mice. Therefore, conditional expression of the dominant-negative mutant allele TG in the SCN and brain is sufficient to drive altered running wheel activity behavior. As described above, these independent lines bear one or two copies of the TG, and each line displays slightly different expression levels (0.51- to 1.05-fold; Figure 1D). It is noteworthy that those double transgenic mice, which carry one or two copies of the Clock^Δ19 TG and two endogenous WT copies of Clock, display a free-running period similar to that of the Clock heterozygote mice (Clock^Δ19/+), which carry one copy of each allele (mutant and WT Clock). (There was a trend toward longer circadian period in lines that expressed higher levels; however, the differences were subtle.) It is not surprising, therefore, that we do not observe a circadian rhythm phenotype similar to that of Clock homozygous-like behavior (i.e., 4-h lengthening and arrhythmicity) in double transgenic mice, given the relative magnitude of TG expression in relation to the expression of WT CLOCK.

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Figure 2. Locomotor Activity Records of Progeny from Scg2::tTA x tetO::Clock^Δ19-HA Matings: Representative Actograms

Actograms are of (A) WT, (B) single transgenic Scg2::tTA, (C) single transgenic tetO::Clock^Δ19-HA, and (D) double transgenic Scg2::tTA/tetO::Clock^Δ19-HA mice. Activity records were double plotted so that 48 h is represented horizontally, with each day presented beneath and to the right of the preceding day. Wheel-running activity is indicated by black markings. The initial light cycle is depicted at the top of the record. All animals were maintained on a 12/12-h light–dark cycle for the first 12 to 14 d shown and then transferred to DD, as indicated by the bar next to each record. Numbers on the left indicate days of study. Six of the seven independent lines are shown. All double transgenic mice showed a lengthening of free-running period of about 1 h. Mice that carry a single TG only (Scg2::tTA or tetO::Clock^Δ19-HA) showed a free-running period similar to WT littermates. At the indicated red circle, all animals were given Dox (2 mg/ml) to “turn off” transgenically induced Clock^Δ19-HA TG. Only the double transgenic mice showed a shortening of free-running period, indicating suppression of the Clock^Δ19-HA TG expression. Period estimates during the Dox treatment showed wheel activity rhythm similar to WT mice. Administration of Dox in the single transgenic and WT mice showed no significant effects on the free-running period. Missing activity data (blank lines) on several concurrent days was due to computer malfunction.

https://doi.org/10.1371/journal.pgen.0030033.g002

One advantage of tetracycline controlled gene expression is that a gene of interest can be repeatedly turned on and off noninvasively. To examine Dox-dependent transactivation, we treated all mice initially with 2 mg/ml Dox in their drinking water (Figure 2). Upon treatment, the Scg2::tTA/tetO::Clock^Δ19-HA double transgenic mice showed a shortening of circadian period in about a day, indicating a rapid suppression of Clock^Δ19-HA TG expression. Period estimates during the Dox treatment showed a mean circadian period of 23.7 h (SD = 0.196, n = 16) in double transgenic mice, similar to their WT littermates (23.6 h, SD = 0.108, n = 22). Next, we examined whether the effect was reversible by removing Dox from the drinking water after 3 wk of treatment. The circadian period of double transgenic mice, however, did not immediately return to the previous long circadian period; rather, it took longer than 3 mo for reversal (Figure 3). One possibility for this long and gradual reversal of the circadian period may be due to period “after effects”, a form of behavioral plasticity in which the free-running period of circadian behavior undergoes experience-dependent changes, such as, in this case, exposure to Dox [67,68]. However, Dox is a potent effector substance for the tetracycline-controlled gene expression system and it is also lipophilic in nature; thus, a likely possibility is that the dosage used may have been too high to achieve rapid clearance and subsequent rapid reversal. Earlier studies have shown that 2 mg/ml Dox produces a rapid switch-off of TG expression (within 1 d) in many tissues and organs [69–71]; however, after 1 mo of treatment, clearance of the antibiotic can take as long as 6 wk [71]. Consequently, we tested lower Dox concentrations in the range of 10 ng/ml to 100 μg/ml and found that 10 μg/ml Dox was optimal. Similar to 2 mg/ml Dox treatment, the Clock^Δ19-HA TG was rapidly turned off at 10 μg/ml (Figure 4); double transgenic mice showed a shortening of their circadian period of about 1 h, while single transgenic or WT mice showed no effect of Dox treatment. No difference in circadian period was observed among the genotypes with 10 μg/ml Dox treatment (F_3,62 = 1.93, p = 0.1339). More important, rapid reversal (switching-on) was achieved when the double transgenic mice were returned to water; their period lengthened by approximately 1 h within one or two cycles (paired t-test, t₂₂ = −17.2177, p < 0.00005). In addition, in situ hybridization revealed that Clock^Δ19-HA transcript in the SCN and throughout the brain is sensitive to the presence of 10 μg/ml Dox. By day 3 of treatment, the transcript is not detectable. Therefore, a dose of 10 μg/ml Dox in the drinking water can easily cross the blood-brain barrier and is sufficient to rapidly switch expression of the TG on and off in the brain. In addition, we found that 100 μg/ml tetracycline in the drinking water also worked effectively (Figure S4). The magnitude and ease by which we can alter circadian wheel running behavior using a low-dose provide flexibility of repeated activation and repression. Importantly, our data argue that the circadian period is regulated through the dynamic and daily expression of Clock and Clock-controlled genes rather than through a static process established during embryonic development.

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Figure 3. Persistent Effects of 2 mg/ml Dox Treatment on Circadian Activity Rhythms of Scg2::tTA/tetO::Clock^Δ19-HA Double Transgenic Mice

Although the effect of Dox treatment was relatively rapid (+Dox, also see Figure 2), the treatment was not immediately reversible upon returning to water (−Dox) from 2 mg/ml Dox. It took at least 3 mo before the double transgenic mice returned to their original longer circadian period.

https://doi.org/10.1371/journal.pgen.0030033.g003

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Figure 4. Rapid Inhibition and Reversal of tTA Mediated tetO::Clock^Δ19-HA Transcription in Scg2::tTA/tetO::Clock^Δ19-HA Double Transgenic Mice to a Lower Concentration of Dox

Representative actograms of (A) a single transgenic tetO::Clock^Δ19-HA mice and (B–D) double transgenic Scg2::tTA/tetO::Clock^Δ19-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 (F_3,78 = 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^Δ19-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^Δ19-HA (*p < 0.0005 for each comparison), indicating that the transgenically induced Clock^Δ19-HA TG expression causes this period difference. With administration of 10 μg/ml Dox, no difference was observed among the genotypes on circadian period (F_3,62 = 1.93, p = 0.1339), thus the transgenically induced Clock^Δ19-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, t₂₂ = −17.2177, p < 0.00005).

(F) In situ hybridization of the Clock^Δ19-HA transcript on coronal brain slices from the single tetO::Clock^Δ19-HA and double transgenic mice (Scg2::tTA/tetO::Clock^Δ19-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^Δ19-HA transcript is “turned off” by day 3 on 10 μg/ml Dox treatment.

https://doi.org/10.1371/journal.pgen.0030033.g004

Regulation of Conditionally Expressed Clock^Δ19 Allele by Doxycycline: Changes in Behavioral Response to Discrete Pulses of Light

Light remains one of the most well-understood circadian entraining signals and perturbation analyses with light has been exploited to demonstrate functional properties of the circadian clock [72]. The phase-response curve (PRC) can be considered a fundamental pacemaker property [67]. Exposure to light early in the night phase shifts the clock so that subsequent cycles begin at a later time; however, exposure to light late in the night advances the circadian clock. These time-dependent responses to light are important for synchronization to environmental light conditions. Another altered behavioral phenotype in Clock^Δ19/+ mice is high-amplitude phase-resetting responses to a 6-h light pulse (type 0 resetting) compared to WT mice, which exhibit low-amplitude (type 1) phase resetting [73]. In the C57BL/6J inbred strain of mice, a 6-h light pulse near the “break-point” (the transition from phase delays to phase advances at approximately circadian time [CT] 17) produces large phase shifts of about 4 to 6 h; however, Clock heterozygotes display phase shifts of longer than 6 h [73]. In order to assess whether the conditional induction of Clock^Δ19-HA TG, restricted to brain/SCN, recapitulates behavioral properties of Clock^Δ19/+ heterozygous mice, we chose to characterize the phase shifting response. Within the same animal, when Clock^Δ19-HA TG is expressed (or when the mice are on water), their phase shift is significantly larger than when the TG is turned off (when they are on Dox treatment) (Figure 5A). The amplitude of phase shift in Scg2::tTA/tetO::Clock^Δ19-HA double transgenic mice on Dox treatment is comparable to the phase shift/delay described in WT mice after a 6-h light pulse [73]. During subjective night (between CT 12 and CT 17), double transgenic mice showed a significantly larger phase delay on water compared to Dox treatment (Figure 5B). The difference between the two treatments (water versus Dox) is more clearly demonstrated when the data are presented as a PRC (Figure 5C). Therefore, brain/SCN-specific activation of the Clock^Δ19-HA TG affects both the phase resetting, as well as, the period lengthening effects on locomotor activity rhythms, similar to that seen with the original Clock^Δ19/+ heterozygous mutation in the whole animal.

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Figure 5. Light-Induced Phase Shifting Responses in Scg2::tTA/tetO::Clock^Δ19-HA Double Transgenic Mice

(A) Two examples of locomotor activity records of double transgenic mice, where they received a 6-h light pulse, during no treatment (water, expression of Clock^Δ19-HA TG) and during Dox treatment (+Dox, repression of Clock^Δ19-HA TG). All light pulse experiments were carried out after the animals were released into DD. For mouse 230, a 6-h light pulse was given at CT 16 on water treatment and at CT 16.4 on Dox treatment. For mouse 234, a 6-h light pulse was given at the same CT of 16.7 on water and on Dox treatments. The red arrow indicates the time at which the mice were light pulsed.

(B) Comparison of light-induced phase shifts in 20 individual double transgenic mice following a 6-h light pulse either during water or 10 μg/ml Dox treatment. Data shown represent animals that received light pulses between CT 12 to CT 18. The symbol on the left side indicates phase delay of each mouse after a light pulse on water treatment (Clock^Δ19-HA TG expression on). The symbol on the right side indicates phase delay of the same mouse on Dox treatment (Clock^Δ19-HA TG expression off). Animals were light pulsed at a given CT during water treatment and then the animals were later treated with Dox to turn off the TG. Animals were light pulsed again at the closest CT as possible to the original light pulse, within 1-h window (blue lines) or within 1- to 2-h window (purple lines). All double transgenic mice show a larger phase shift after a light pulse with water treatment, when the TG is on, compared to with Dox treatment, when the TG is turned-off (for 0- to 1-h CT interval, paired t-test, t₈ = 6.6026, p = 0.0002; for 1- to 2-h CT interval, paired t-test, t₁₀ = 11.5582, p < 0.00005).

(C) PRC to discrete pulses of light in Scg2::tTA/tetO::Clock^Δ19-HA double transgenic mice. The x-axis indicates the CT at the beginning of the light pulse. The y-axis indicates the phase shift (in hours) produced by the light pulse. When the Clock^Δ19-HA TG is on, the double transgenic mice showed a characteristic increased amplitude PRC, similar to the behavior seen in heterozygous Clock^Δ19 mutant mice after discrete pulses of light; however, when the TG is off, the PRC in double transgenic mice is similar to behavior observed in WT mice.

https://doi.org/10.1371/journal.pgen.0030033.g005

Conditional Regulation of the Clock^wt Allele

In our previous report on the rescue of the Clock mutation using bacterial artificial chromosome (BAC) TGs, we showed that overexpression of WT Clock allele (Clock^wt) shortened free-running period length beyond the WT range [66]. Thus, we explored whether a conditional expression of Clock^wt, restricted to the SCN and brain, could also generate shortened free-running activity rhythms as observed in transgenic mice that overexpress CLOCK^wt ubiquitously. We have crossed one of the tetO::Clock^wt-HA target lines to the Scg2::tTA activator line, and progeny from the mating were examined for their locomotor activity rhythm. We find that the Scg2::tTA/tetO::Clock^wt-HA double transgenic mice exhibit a shortened circadian period by about 1 h compared to the WT circadian period (Figure 6). In addition, similar to transcriptional activation of the Clock^Δ19-HA TG, we demonstrate temporal control of Clock^wt-HA TG expression with rapid switch-off and a rapid reversal. Therefore, conditional expression of the dominant-negative mutant allele, as well as overexpression of the WT allele, restricted to the SCN and brain, is sufficient to alter the period of activity rhythms. Given that the peripheral circadian oscillators in these mice are WT, it will be interesting in future experiments to investigate whether the period of peripheral oscillators in these mice can be regulated as a consequence of period changes induced by the SCN/brain as previously suggested in experiments using transplanted mouse embryonic fibroblasts [20].

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Figure 6. Locomotor Activity Records of Progeny from Scg2::tTA × tetO::Clock^wt -HA Matings: Representative Actograms

Actograms of (A) WT, (B) single transgenic Scg2-tTA, (C) single transgenic tetO::Clock^wt-HA, and (D and E) double transgenic Scg2::tTA/tetO::Clock^wt-HA mice. All double transgenic mice, expressing the Clock^wt -HA TG, showed a shortening of circadian period of approximately 1 h, beyond the normal WT values. Mice that carry only a single TG (Scg2::tTA or tetO::Clock^Δ19-HA) showed circadian period range similar to their WT littermates. At the first arrow, all animals were given Dox (10 μg/ml) to turn off transgenically induced CLOCK^wt-HA. At the second arrow, Dox was withdrawn to switch back on the Clock^wt-HA TG. A similar effect on circadian period was observed when Dox was administered and withdrawn for the second time in the same animal.

https://doi.org/10.1371/journal.pgen.0030033.g006

TG constructs.

For the generation of tTA-expressing mice (transactivator line), a 1,153-bp fragment upstream of the translation start site of the mouse Secretogranin II gene was amplified by PCR primers 5′-AGTGATTCCTCTTACTAATCCATCTGTGAGAT-3′ (forward) and 5′-GTCTTAAAGATTTCCTGAAAACATAGA-3′ (reverse) using a mouse BAC clone RP23-470F8 as a template (Roswell Park Cancer Institute Mouse BAC Library; Invitrogen, http://www.invitrogen.com). The PCR product was first cloned into the EcoRV restriction site in pBluescript II SK(−) (pBlue) (Stratagene, http://www.stratagene.com). This plasmid was then cut with EcoRI and was used to ligate a 9-kb EcoRI promoter fragment isolated from the BAC clone RP23-470F8, resulting in an intact 9,851-bp promoter fragment upstream of the translation start site. This plasmid was named pBlue-Scg2promoter. A 1,453-bp EcoRI-XmnI fragment coding tTA was released from the PMY20 plasmid (kindly provided by Mark Mayford [35]) and ligated into the SalI restriction site of the pBlue-Scg2promoter plasmid, after T4 DNA polymerase treatment of both fragments. This plasmid was named Scg2::tTA and was linearized with NotI restriction enzyme prior to microinjection. The linearized fragment was isolated using the QIA Quick Gel Extraction Kit (Qiagen, http://www.qiagen.com) and dialyzed for 4 h in 10 mM Tris-HCl (pH 7.5)/0.1 mM EDTA (pH 8.5) buffer before pronuclear injection.

For the generation of tetO target lines, we first generated the 3′-HA–tagged WT Clock and mutant Clock^Δ19 cDNAs from the plasmids pBlue-ClockWT and pBlue-ClockΔ19, respectively, as described previously [57]. Each full-length cDNA also contained 388 bp of 5′ untranslated region of Clock, which was generated from exons 1b, 2, and 3. The following primers were used against the pBlue-ClockWT or pBlue-ClockΔ19 plasmid to generate cDNAs by PCR using Pfu DNA polymerase (Stratagene): 5′-ATAAGAATGCGGCCGCGGGGAGGAGCGCGGCGGTAGCGGTG-3′ (forward) and 5′-CCCAAGCTTCTAAAGAGCGTAATCTGGAACATCGTATGGGTACTGTGGCTGGACCTTGGAAGGGTCA-3′ (reverse). Amplified product was digested with NotI and HindIII, gel purified, and ligated into the NotI-HindIII cloning site of the pTRE2 plasmid (Clontech, http://www.clontech.com). These tetO::Clock^wt-HA and tetO::Clock^Δ19-HA pTRE2 plasmids were linearized using DrdI/XmnI restriction enzymes to exclude most of the vector backbone sequence prior to pronuclear injection. All plasmids were sequence verified using the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit and analyzed on an ABI377 automated sequencer (Applied Biosystems, http://www.appliedbiosystems.com).

Transgenic animals.

Transgenic mouse lines were generated by pronuclear injection using standard techniques [91] and essentially as described in Antoch et al. [66]. Briefly, the linearized DNA fragment was injected into fertilized mouse oocytes isolated from crosses of WT CD1 matings at a concentration of 1 ng/μl. Transgenic mice were identified by PCR analysis of genomic DNA prepared from tail biopsy samples. PCR amplification of the transactivator TG (Scg2::tTA) was carried out using primers (forward primer 5′-AGACAAGCTTGATGCAAATGAG-3′; reverse primer 5′-CAAGTGTATGGCCAGATCTCAA-3′) that generate a 482-bp fragment. Two Scg2::tTA founder lines were obtained and characterized, and one line was chosen for the experiments presented here. For the tetO::Clock^wt-HA and tetO::Clock^Δ19-HA TGs, genotyping was performed using 5′-ATATGCAAGGCCAGGTTGTC-3′ (forward primer) and 5′-TCTGTGGCATACTGGATGGA-3′ (reverse primer), which generates a 258-bp fragment. We produced eight tetO::Clock^Δ19-HA lines and 13 tetO::Clock^wt-HA target lines. Each founder animal was maintained as a congenic line by backcrossing to the C57BL/6J inbred strain for at least four generations. All animals were raised in a 12/12-h light–dark cycle from birth. All animal studies were conducted in accordance with the regulation of the Committee on Animal Care and Use at Northwestern University.

Dox treatment.

Doxycycline hydrochloride (Sigma-Aldrich, http://www.sigmaaldrich.com) was supplied in the drinking water at a concentration of 2 mg/ml or 10 μg/ml. The Dox-containing water was renewed every 2 to 3 d. Mice were supplied with regular food and water with or without Dox ad libitum.

Circadian behavioral analysis.

Wheel-running activity of singly housed animals was recorded and analyzed essentially as described [65]. Mice were entrained to a 12/12-h light–dark cycle for a minimum of 7 d before they were released into constant darkness. Activity data were collected as number of events per minute and recorded continuously by a PC system (Chronobiology Kit; Stanford Software Systems, http://www.query.com); data were analyzed using ClockLab software (Actimetrics, Wilmette, Illinois, United States). The free-running period was calculated (days 1 to 20 in DD) by using a χ² periodogram [92]. For the light pulse experiments, a 6-h light pulse (approximately 100 lux) was provided at a given CT, where CT 0 denotes the beginning of the subjective day and CT 12 denotes the beginning of the subjective night. The magnitude of phase shift was determined by measuring the phase difference, based on the activity onset as a phase reference point, between regression lines fit immediately before the light pulse and at least seven consecutive activity-onset times after the light pulse (excluding the four cycles immediately after the pulse) as described previously [73]. The magnitude of phase shift was corrected for circadian period as estimated before the light pulse for each individual. All other statistical analyses were performed using the Stata Statistical/Data Analysis software (version Stata/SE 9.0; StataCorp, http://www.statacorp.com).

Western blotting.

Tissues were homogenized in a buffer containing 150 mM NaCl, 50 mM Tris (pH 7.4), 1% Triton X-100, 0.1% SDS, and protease inhibitor cocktail (Complete; Roche Applied Science, http://www.roche.com). Homogenates were cleared by centrifugation at 10,000g for 10 min at 4 °C, and supernatants were collected and protein concentration was estimated using Bio-Rad DC Protein Assay (Bio-Rad, http://www.bio-rad.com) according to the manufacturer's instructions. Total protein (40 μg) was mixed with sample buffer according to the protocol of [93] and resolved on an 8% SDS–polyacrylamide gel by electrophoresis. Thereafter, proteins were electrotransferred onto a Poly Screen PVDF transfer membrane (Perkin Elmer Life Sciences, http://www.perkinelmer.com). The membranes were blocked with PBST (PBS + 0.1% Tween-20) containing 5% powder milk for 1 h and then incubated with the 3F10 mouse monoclonal anti-HA–peroxidase antibody (1:500; Roche Applied Science) or anti–actin-peroxidase antibody (1:1,000; Santa Cruz Biotechnology, http://scbt.com) according to the manufacturer's protocol. Anti-CLOCK guinea pig antibody (1:1,000) was followed by anti–guinea pig IgG secondary antisera horseradish peroxidase (1:1,000; Jackson ImmunoResearch Laboratories, http://www.jacksonimmuno.com). CLOCK guinea pig antibody was generously provided by Choogon Lee (University of Florida, Tallahassee, Florida, United States). Proteins were visualized with a chemiluminescence detection system (ECL Western blotting detection analysis system; Amersham Pharmacia, http://www.amersham.com) and with subsequent exposure to autoradiographic film.

In situ hybridization.

In situ hybridization procedures were performed as described [94]. Briefly, animals were sacrificed by cervical dislocation; the brains were removed immediately, frozen on dry ice, and stored at −80 °C. Sectioning, fixation, hybridization, and washing were performed as described. Sections were hybridized using an antisense-HA oligo probe (5′- AAGAGCGTAATCTGGAACATCGTATGGGTACTGTGGCTGG-3′), a WT Clock oligo probe (5′-GCTCTAGCTGGTCTTTTAGATGCTGCATGGCTCCTAACTGAGCTG-3′), or an Scg2 oligo probe (5′-TTCAGCAGCTCCAGGGCGGAGTTGATCACCTTGGACTTGTCCAGGCGGGACAT-3′). Primers were 5′-end-labeled with [γ-³³P]ATP by using recombinant terminal deoxynucleotidyl transferase (Invitrogen).

Immunocytochemistry.

Animals were anesthetized with ketamine/xylazine/saline cocktail (10 mg/ml ketamine, 2 mg/ml xylazine; Phoenix Scientific, http://www.psiqv.com) at 0.01 ml/g body weight and then perfused intracardially with 50 ml of 0.9% saline solution followed by 50 ml of 4% paraformaldehyde (Sigma Aldrich) in 0.1 M phosphate buffer (pH 7.2). The brains were removed and postfixed for 24 to 48 h at 4 °C in 4% paraformaldehyde in 0.1 M phosphate buffer. For immunocytochemistry, 50-μm coronal sections were collected through the entire SCN using a VibroSlice microtome (World Precision Instruments, http://www.wpiinc.com) and processed free floating. Sections were incubated with mouse anti-HA.11 biotin-labeled monoclonal antibody (BIOT-101L-100, 1:200; Covance Research Products, http://www.crpinc.com) followed by anti-biotin, mouse IgG1, monoclonal 2F5 conjugated with Alexa Fluor 488 secondary antibody (1:1,000; Invitrogen). A primary antibody to either VIP (1:5,000; ImmunoStar Inc, http://www.immunostar.com) or AVP (1:10,000 ImmunoStar Inc) was followed by Alexa Fluor 568 goat anti-rabbit IgG (H+L) secondary antibody (1:1,000; Invitrogen). The Alexa Fluor 488 signal was assigned a green pseudo-color, while the Alexa Fluor 568 signal was assigned a red pseudo-color. Sections were viewed with a Leica DMRXE7 confocal microscope with Ar (488 nm) and green HeNe (543 nm) lasers in the Biological Imaging Facility at Northwestern University (Evanston, Illinois, United States). Images were captured by sequential excitation with each laser to avoid crosstalk between the two wavelengths using the Leica Confocal Software (version 2.61 build 1537).

β-Galactosidase staining.

Mice carrying a tetO promoter-lacZ reporter construct (lines lac1 and lac2) were generously provided by Mark Mayford. All procedures were performed as described by Low-Zeddies and Takahashi [80]. Briefly, mice were anesthetized with ketamine/xylazine/saline cocktail and then transcardially perfused with chilled 0.1% heparin PBS. Brains were removed and postfixed for 30 min in 4% paraformaldehyde PBS on ice and then stored overnight in 20% sucrose PBS at 4 °C. Brains were frozen on dry ice, embedded in Shandon Lipshaw M1 embedding matrix (Pittsburgh, Pennsylvania, United States), and sectioned coronally at 50-μm thickness. Free-floating sections were collected in a wash buffer (PBS with 2 mM MgCl₂, 0.02% NP-40 [Sigma] [pH 7.3]) and then incubated for 24 h at 37 °C in an X-gal staining solution (1 mg/ml 5-bromo-4-chloro-3-indolyl-β-d-galactoside; Gold Biochemical, Long Beach, New York, United States) dissolved in dimethyl sulfoxide buffer containing 5 mM K₃Fe(CN)₆ and 5 mM K₄Fe(CN)₆). Finally, sections were rinsed three times in the wash buffer and then mounted in aqueous mounting medium (3:1 glycerol/PBS) on gelatin-coated glass slides. Stained sections were viewed and photographed under bright-field illumination.

Accession numbers

Accession numbers for genes used in this paper are mouse BAC clone RP23-470F8 (GenBank [http://www.ncbi.nlm.nih.gov/Genbank] AC084213), Clock (GenBank AF000998 [mRNA], AF146793 [genomic], Entrez Gene ID 12753, UniProt O08785), Bmal1 (Arntl) (Entrez Gene ID 11865, UniProt Q9WTL8), Scg2 (Entrez Gene ID 20254), Avp (Entrez Gene ID 11998, UniProt P35455), and Vip (Entrez Gene ID 22353, UniProt P32648).