Research reportA short half-life GFP mouse model for analysis of suprachiasmatic nucleus organization
Introduction
Several converging lines of evidence indicate that the Per1 gene is one of the key participants in the molecular feedback loop that drives SCN neuronal clock oscillations. Transcripts of Per1 show a robust circadian rhythm in the SCN [20], [22], [23]. The negative and positive feedback elements which constitute the autofeedback loop of the system have been delineated [15]. Transcription of Per1 is activated by the binding of the CLOCK–BMAL1 complex to the E-boxes in the promoter region of the Per1 gene [6]. This activation is inhibited by PER1 protein and other negative elements [16]
Progress in understanding neural organization has been enhanced by our ability to visualize individual neurons and their activity. The development of jellyfish green fluorescent protein (GFP) as a vital stain has made possible tremendous advances in neuronal imaging [3]. GFP confers a number of advantages over other cellular markers [4]. The pattern of expression is heritable, the cell is labeled in its entirety, the GFP protein can be placed under the control of other promoters and it can be mutated to be unstable, providing a tool for studying dynamic activity. Unlike luciferase, another commonly used reporter, the GFP chromophore is part of the protein and does not require delivery of exogenous substances to express its fluorescence.
GFP, like other transgenic reporters, also presents some disadvantages. While expression is similar among offspring of each transgenic founder, there is substantial variability in pattern of GFP expression among mice generated from the same construct [4]. Transcription factors in different cell types may recognize a transgene differently. Furthermore some chromatin adjacent to the integration site may silence the transgene, a phenomenon termed positive effect variegation. This results in different levels of GFP expression in different cell types [24]. Although the stability of GFP protein has contributed to its wide use as a cellular marker, it also presents some limitations and potential disadvantages. The long half-life of conventional GFP obviates its use in reporting the dynamics of gene promoters and whereas some reports find no toxicity [4], [14], other reports indicate that accumulation of stable GFP induces cell damage or death [9], [12].
We have been exploring the use of a Per1::GFP transgenic mouse, first described in Kuhlman et al. [10], to understand the organization of SCN pacemakers. In this animal, a degradable form of recombinant GFP is driven by the mouse Per1 gene promoter, the half-life of GFP is about 2.1 h, allowing GFP intensity to report promoter dynamics on a circadian time scale and preventing accumulation of GFP. Previous results with this mouse model indicate that in acute slice preparations of the SCN, GFP fluorescence intensity differs at two time points, mid-day and mid-night, suggesting rhythmicity in GFP expression [10].
The first goal of the present study was to assess the usefulness of the Per1::GFP transgenic animal in understanding the organization of the SCN by determining the time course of GFP expression throughout the nucleus. GFP fluorescence was measured for 24 h in an acute SCN slice preparation taken from animals sacrificed at the onset of light in the morning, at zeitgeber time (ZT0). GFP immunoreactivity was measured in animals sacrificed at 4-h intervals in animals housed in light:dark (LD, indexing zeitgeber time, ZT) and in those housed in constant darkness (DD, indexing circadian time, CT), in order to compare GFP expression in vivo and in vitro. Circadian rhythms of GFP, Per1 mRNA, PER1 and PER2 proteins were also determined. A surprising finding was that GFP was absent or not detected in a distinct region of the central SCN. Hence, the second goal was to examine the location of GFP, Per1 mRNA, PER1 and PER2-immunoreactivity within the SCN. Our findings indicate that like GFP, Per1 mRNA, PER1 and PER2 are absent in this region.
Section snippets
Animals and housing
The mPer1::d2EGFP transgenic mouse was made with the B6C3F1 hybrid mouse as described in Kuhlman et al. [10]. The mice used in these studies were hemizygous for the d2EGFP transgene, which is an unstable form of GFP (we use hemizygotes in all our studies. In the few homozygotes we have processed, we do not detect any obvious difference in the amount of GFP expression). At about 3 months of age, animals were housed individually in translucent propylene cages (11.5×7.5×5 inches), in either a
GFP transgenic mouse expresses precise circadian rhythms
As can be seen in Fig. 1 the circadian rhythms of the Per1::GFP mice are precise and have a high amplitude, with an average free-running period of 23.06±0.11 h (n=11). The wheel running activity profile shows most of the running occurring at the beginning of subjective night, and a smaller bout at the end of subjective night. The phase response curve resembles that of normal mice with phase advances and delays to light pulses presented in late or early subjective night, and no phase shifting
Discussion
The Per1::GFP transgenic mouse free-runs in constant conditions with a with very precise onset times (Fig. 1A), a high amplitude rhythm (Fig. 1B) and an activity profile similar to the B6C3F1 strain [21]. Its phase advances and delay in response to light pulses are also similar to that reported for one of its parent strain C57BL/6 mice [17]. This indicates that the Per1::GFP transgene does not adversely affect circadian behavior, and that there are no toxic effects of the transgene.
Acknowledgements
We are grateful to Dr Michael Lehman for his advice on this manuscript. This study was supported by NIH Grants NS-37919 to RS, MH63341 and EY09256 to DGM.
References (25)
- et al.
Suprachiasmatic nucleus in the mouse: retinal innervation, intrinsic organization and efferent projections
Brain Res.
(2001) - et al.
Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP
Neuron
(2000) - et al.
Analysis of clock proteins in mouse SCN demonstrates phylogenetic divergence of the circadian clockwork and resetting mechanisms
Neuron
(2000) - et al.
Is green fluorescent protein toxic to the living cells?
Biochem. Biophys. Res. Commun.
(1999) - et al.
‘Green mice’ as a source of ubiquitous green cells
FEBS Lett.
(1997) - et al.
Two period homologs: circadian expression and photic regulation in the suprachiasmatic nuclei
Neuron
(1997) - et al.
Light-induced resetting of a mammalian circadian clock is associated with rapid induction of the mPer1 transcript
Cell
(1997) - et al.
RIGUI, a putative mammalian ortholog of the Drosophila period gene
Cell
(1997) - et al.
Per1 and Per2 gene expression in the rat suprachiasmatic nucleus: circadian profile and the compartment-specific response to light
Neuroscience
(1999) - et al.
Three period homologs in mammals: differential light responses in the suprachiasmatic circadian clock and oscillating transcripts outside of brain
Neuron
(1998)
Light-induced c-Fos expression in the mouse suprachiasmatic nucleus: immunoelectron microscopy reveals co-localization in multiple cell types
Eur. J. Neurosci.
Green fluorescent protein as a marker for gene expression
Science
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