Photic and circadian expression of luciferase in mPeriod1-luc transgenic mice invivo - PubMed (original) (raw)

Photic and circadian expression of luciferase in mPeriod1-luc transgenic mice invivo

Lisa D Wilsbacher et al. Proc Natl Acad Sci U S A. 2002.

Abstract

A conserved transcription-translation negative feedback loop forms the molecular basis of the circadian oscillator in animals. Molecular interactions within this loop have been relatively well characterized in vitro and in cell culture; however, in vivo approaches are required to assess the functional significance of these interactions. Here, regulation of circadian gene expression was studied in vivo by using transgenic reporter mouse lines in which 6.75 kb of the mouse Period1 (mPer1) promoter drives luciferase (luc) expression. Six mPer1-luc transgenic lines were created, and all lines express a daily rhythm of luc mRNA in the suprachiasmatic nuclei (SCN). Each mPer1-luc line also sustains a long-term circadian rhythm of luminescence in SCN slice culture. A 6-h light pulse administered during the early subjective night rapidly induces luc mRNA expression in the SCN; however, high luc mRNA levels are sustained, whereas endogenous mPer1 mRNA levels return to baseline, suggesting that posttranscriptional events mediate the down-regulation of mPer1 after exposure to light. This approach demonstrates that the 6.75-kb mPer1 promoter fragment is sufficient to confer both circadian and photic regulation in vivo and reveals a potential posttranscriptional regulatory mechanism within the mammalian circadian oscillator.

PubMed Disclaimer

Figures

Figure 1

Figure 1

mPer1_-luc construct and activity in transfection-based reporter assay. (A) The 6.75-kb_mPer1 promoter contains five E-box sites, five putative cAMP response element-binding protein (CREB) sites, eight putative Elk1 sites, and three putative serum response factor (SRF) sites as well as exon 1 and the 5′ UTR of exon 2. (B) Transfection-based_luc_ assays in NIH 3T3 fibroblasts using the_mPer1_-luc reporter. +, indicates the presence of an expression plasmid; *, indicates a significant difference from the condition of_mPer1_-luc reporter alone; †, indicates a significant difference from the condition of_mPer1_-luc reporter in the presence of only mCLOCK and mBMAL [generalized linear model ANOVA,F(5,35) = 147.91; P < 1.0 × 10−6; Scheffé's post hoc comparison,P ≤ 0.05]. Results represent two independent experiments with n = 3 of each condition.

Figure 2

Figure 2

Diurnal variation of luc mRNA expression in the SCN of P1L mice. P1L mice were killed at ZTs 6 and 18, and coronal brain sections were hybridized in situ with a_luc_ riboprobe. In all lines, higher expression occurs in the SCN (arrows) at ZT 6, whereas low-to-undetectable expression occurs at ZT 18. Diurnal variation was not observed in other brain regions. TG copies, indicates the transgene copy number for each line.

Figure 3

Figure 3

Circadian rhythm of luc expression in P1L mice. Mice were maintained in constant darkness for at least 7 days before being killed, and the endogenous circadian rhythm of each animal was measured. Three to five animals were collected at each CT point beginning at 18. (A) Expression of luc. (B) Expression of endogenous_mPer1_.

Figure 4

Figure 4

Persistent circadian rhythms of luminescence in SCN culture. Animals were entrained to a 12-h light/12-h dark cycle. Mice were killed 1 h before lights-off, and SCN explants were cultured for 5 days in the presence of luciferin.

Figure 5

Figure 5

Light-responsiveness of mPer1_-luc expression in transgenic mice. Mice were maintained in constant darkness for at least 7 days before the experiment, and the endogenous circadian rhythm of each animal was measured. At CT 17, mice were transferred to a light box. Time-course graphs indicate the fold induction from baseline expression at CT 17. The line through each data set is the nonlinear least-squares fit to the mean for each time point assuming a biexponential function (see_Results). (A) Time course of_luc_ mRNA levels. *, denotes significant differences in signal intensity between time = 0 (dark control) and the indicated time [generalized linear model ANOVA,F(6,22) = 5.32; P = 0.004039]. Biexponential function parameters: λ = 0.46439 and β = 0.46547. (B) Time course of endogenous_mPer1_ mRNA levels. *, denotes significant differences in signal intensity between time = 0 (dark control) and the indicated time [generalized linear model ANOVA,F(6,22) = 9.32; P = 0.000232]. Biexponential function parameters: λ = 0.74934 and β = 0.93453.

Similar articles

Cited by

References

    1. Dunlap J C. Cell. 1999;96:271–290. - PubMed
    1. Wilsbacher L D, Takahashi J S. Curr Opin Genet Dev. 1998;8:595–602. - PubMed
    1. King D P, Takahashi J S. Annu Rev Neurosci. 2000;23:713–742. - PubMed
    1. Wager-Smith K, Kay S A. Nat Genet. 2000;26:23–27. - PubMed
    1. Miyamoto Y, Sancar A. Proc Natl Acad Sci USA. 1998;95:6097–6102. - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources