Generation of an mPer3 targeting construct.
Genomic DNA clones were isolated from a 129/sv mouse library (Stratagene) using a probe generated from a portion of the mPer3 cDNA corresponding to nucleotides (nt) 187 to 1139 of the sequence assigned GenBank accession number AF050182. The targeting construct was generated by inserting a 1.8-kb neomycin resistance cassette (PGK-NEO) in reverse orientation in place of a 1.6-kb EcoRI fragment of the genomic clone (Fig. A). The excised EcoRI fragment of the genomic clone began in intron 2, approximately 150 bp 5′ of exon 3, and contained exon 3, intron 3, and the 5′ portion of exon 4. The excised regions encode amino acid residues 92 to 154 of mPER3. The PGK-NEO cassette contains the phosphoglycerate kinase (PGK) promoter driving the expression of the neomycin (NEO) resistance gene and was provided by En Li (Massachusetts General Hospital, Boston).
FIG. 1 mPer3 targeting construct and genotyping strategies. (A) Schematic representation of the mPer3 gene, the targeting construct, and the targeted allele. A 1.6-kb portion of the mPer3 gene was excised with EcoRI (R1), and a PGK-NEO cassette was inserted (more ...) Generation of mPER3-deficient mice.
The targeting construct was linearized and then introduced into J1 embryonic stem (ES) cells by electroporation. Genomic DNA extracted from neomycin-resistant ES cell lines was digested with EcoRV, subjected to agarose gel electrophoresis, and hybridized with a 0.5-kb probe from the 5′ region flanking the targeting cassette. Four positive clones were identified out of 196 screened. Homologous recombination in these four positive clones was confirmed by digestion with SpeI and hybridization with a probe from the 3′-flanking region (Fig. ). Two of the four positive ES cell lines (no. 12 and 135) were used for microinjection to generate chimeric founders.
Chimeric males were bred to females of the C57BL/6, C3H/HeJ, and isogenic (129/sv) strains. C57BL/6 and C3H/HeJ mice were purchased from Charles River Laboratories. Isogenic females were provided by En Li. For studies of gene expression, F1 heterozygotes generated from crosses to the C57BL/6 strain were crossed to produce F2 mice homozygous for the targeted allele (mPER3 deficient, or mPer3−/−) or homozygous for the wild-type allele (+/+). Behavioral studies were conducted with male mice from line 12 in a C57BL/6 background, from line 135 in a C3H background, and from both line 12 and line 135 in an isogenic (129/sv) background.
Genotypes were determined by PCR and/or Southern blot analysis of tail biopsy DNA (Fig. C and D). Probes for Southern blots were the 5′- and 3′-flanking probes used to identify the ES cell lines (see above) radiolabeled with [32P]dCTP (New England Nuclear [NEN]) by use of random hexamer primers (Oligolabeling kit; Boehringer Mannheim Biochemicals).
The PCR method was done with a cocktail of three primers, a forward primer in intron 3 (3-43; 5′ TCTGTGAGTTCTTCCGTGTCTGTT) (present only in the wild-type [WT] allele), a primer located in the NEO cassette (Neo6-2; 5′ TGCCCCAAAGGCCTACCCGCTTCC), and a common reverse primer in exon 4 (3-41; 5′ GTCTTGAGGGGCAAGCAGGTCGAC). The presence of the WT allele led to the amplification of a ca. 200-bp band from primers 3-43 and 3-41, while the presence of the targeted allele was detected by amplification of a ca. 400-bp band with primers Neo6-2 and 3-41 (Fig. D). PCR was performed with Taq DNA polymerase (Fisher) in buffer H (Epicentre Technologies). The PCR protocol consisted of 3 min at 95°C, 30 cycles of amplification (each consisting of 30 s at 94°C, 30 s at 60°C, and 90 s at 72°C), and a final extension phase (10 min at 72°C). Products were separated on 1.5% agarose gels and viewed by UV transillumination with ethidium bromide.
Mice used for analysis of proteins were housed in a light-dark cycle consisting of 12 h of light and 12 h of dark (12L:12D). Mice were euthanized by inhalation of carbon dioxide 30 min before lights were turned off. Tissues were removed, dissected, and frozen on dry ice. Tissue pools consisted of anterior hypothalamus containing SCN, whole hypothalamus, and brain (whole brain minus hypothalamus and cerebellum).
Tissue extracts were prepared as described previously (14
). Briefly, tissues were homogenized at 4°C in 3 volumes of buffer 1 (0.4 M NaCl, 20 mM HEPES [pH 7.5], 1 mM EDTA, 5 mM NaF, 1 mM dithiothreitol, 0.3% Triton X-100, 5% glycerol, 0.25 mM phenylmethylsulfonyl fluoride [PMSF], 10 mg of aprotinin per ml, 5 mg of leupeptin per ml, 1 mg of pepstatin A per ml). Homogenates were cleared by centrifugation (twice, 12 min each, 12,000 × g
). Supernatants were mixed with 2× sample buffer (14
) and boiled. Proteins were separated by electrophoresis through sodium dodecyl sulfate (SDS)–6% polyacrylamide gels and then transferred to nitrocellulose membranes. Membranes were blocked with 5% nonfat dry milk in Tris-buffered saline containing 0.05% Tween 20 and then incubated with affinity-purified antisera to mPER3, mCRY1, or mCRY2 (Alpha Diagnostics International). Immunoreactive bands were visualized using anti-rabbit immunoglobulin G secondary antisera and enhanced chemiluminescence detection (NEN). Full-length mPer3
ligated into the pcDNA3.1-HA vector (12
) was translated in vitro using a TnT T7 Quick kit (Promega), and approximately 0.03 fmol was loaded as a positive control.
Frozen brain tissue was collected and homogenized as described above. After the second centrifugation step, supernatants were diluted with 2 volumes of buffer 2 (identical in composition to buffer 1 except that Triton X-100 was reduced to 0.05% and buffer 2 contains no NaCl). Extracts (500 mg of total protein) were incubated with 20 μl of protein G-Sepharose 4 Fastflow beads (Pharmacia) for 30 min at 4°C and then centrifuged. A slurry of antibody (1 μl of anti-PER3, 3 μl of anti-CRY1, or 3 μl of anti-CRY2) plus protein G-Sepharose 4 Fastflow beads was added to the clarified supernatants. After being mixed by gentle rotation for 2 h at 4°C, the beads were collected by centrifugation. Immune complexes were washed three times, mixed with sample buffer, boiled, and centrifuged. The final immune complexes in the supernatants were analyzed by Western blotting to detect mPER3 as described above. Blots were also analyzed for mCRY1 and mCRY2 to rule out the possibility that failure to immunoprecipitate mPER3 in mice homozygous for the targeted allele was due to an altered abundance of mCRY proteins; comparable amounts of mCRY1 and mCRY2 were immunoprecipitated in +/+ and mPer3−/− mice (data not shown).
Analysis of gene expression.
Mice used for analysis of gene expression were housed in 12L:12D for at least 10 days prior to analysis. The lighting timer was disabled on the day of study so that the animals remained in constant darkness (DD) on the day of tissue collection. Mice were euthanatized by carbon dioxide inhalation with the aid of dim-red illumination; tissues then were rapidly dissected and frozen (−80°C).
Gene expression in the SCN was examined by in situ hybridization using methods described previously (33
). Coronal, 15-μm sections through the SCN were hybridized overnight with 35
S-labeled cRNA probes generated by in vitro transcription (Promega). The templates for probe generation were PCR-generated fragments of cDNAs subcloned into the TA vector (Invitrogen). Probes were mPer1
(nt 340 to 761 of the sequence assigned GenBank accession number AF022992
(nt 9 to 489, accession number AF035830
(nt 108 to 622, accession number AF050182
(nt 1081 to 1793, accession number AB000777
), and Bmal1
(nt 864 to 1362, accession number AF015203
Northern blot analysis was performed as previously described (38
). RNA was extracted from skeletal muscle samples using Ultraspec reagent (Biotecx). Poly(A)+
RNA was isolated using oligo(dT) (Qiagen). RNA samples [1 μg of poly(A)+
RNA per lane] were separated by electrophoresis through agarose-formaldehyde gels and blotted to GeneScreen (NEN). Probes for Northern blots were labeled by the method of random priming (Oligolabeling kit) with [32
P]dCTP (NEN). Templates were mPer3
5′ (nt 108 to 622, AF050182
deletion-specific probe (nt 629 to 824, AF050182
3′ (nt 1637 to 2223, AF050182
), NEO (690-bp Pst
I fragment), mPer1
(nt 340 to 761, AF022992
), and mPer2
(nt 1572 to 2338, AF035830
). To verify equal loading across lanes, blots were stripped and reprobed with a probe generated from human β-actin cDNA (Clontech).
Reverse transcription (RT)-PCR was performed to define the mPer3 transcripts present in mPER3-deficient mice. RNA was extracted from whole brain of WT mice and mice homozygous for the targeting construct (Ultraspec RNA isolation; Biotecx). RT with random hexamer primers was performed using an RT-PCR kit with a slight modification of the manufacturer's instructions (Perkin-Elmer Cetus). PCR was performed using Taq DNA polymerase and a PCR protocol consisting of 3 min at 95°C, 30 cycles of amplification (each consisting of 30 s at 94°C, 30 s at 60°C, and 90 s at 72°C), and a final extension phase (10 min at 72°C).
Primers for RT-PCR were as follows: exon 1 forward, 5′-GCAAACTGAGAGAAGCAGGCTGAG-3′; exon 2 forward, 5′-ACTAATCCCAGTACCCTAGATGCTCT-3′; Neo3, 5′-CGAGCTCATTCCTCCACTCATGATCTA-3′; Neo32147, 5′-CTGTCATCTCACCTTGCTCCTGCC-3′; Neo6-2, 5′-TGCCCCAAAGGCCTACCCGCTTCC-3′; exon 4 reverse, 5′-TTGGGTCCAGTTGTTCCAGAAAGG-3′; exon 4 reverse, 5′-GTCTTGAGGGGCAAGCAGGTCGAC-3′; and exon 5 reverse, 5′-TTCGCTGGTGCACATTCATACTGCG-3′.
PCR products were subcloned (TA vector), and nucleotide sequences were determined manually using the Sanger method (Sequenase; U.S. Biochemicals) or an ABI automated sequencer (Department of Molecular Biology, Massachusetts General Hospital).
Assessment of behavioral rhythms.
Mice used for behavioral analysis were housed individually within light-tight, ventilated environmental compartments in a temperature- and humidity-controlled facility. After 2 to 14 days of being monitored in 12L:12D, mice entered DD starting at the time when lights were normally turned off (2100 Eastern Standard Time). In most studies, dim-red light from fluorescent bulbs was present within each compartment at all times, including those designated as representing darkness. In study 5, the dim-red bulbs also were disabled.
For monitoring locomotor activity (spontaneous wheel running), male mice were housed individually in cages equipped with running wheels. Magnetic reed switches mounted near each running wheel detected the movement of a magnet mounted on the wheel. Switch closures were detected by a computer-based system (DataCol3; MiniMitter Corp.) and saved to a disk at 5-min intervals. Data were plotted in actogram format using the software within the DataCol package and were double plotted by photocopying. The free-running period was estimated without knowledge of the genotype of the animals. The period was determined from the slope of a hand-drawn line through activity onsets of each animal housed in DD. The first 5 days after discontinuation of the lighting cycle were excluded from the analysis to allow stabilization to constant conditions.