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 in the reverse orientation. The 5′ and 3′ arms of the targeting construct were ca. 6 and 9 kb, respectively. Restriction sites introduced by the PGK-NEO cassette and used for Southern blot analysis were EcoRV (R5) and SpeI (S). Intron and exon structure is shown only for the first five exons (E1 to E5). Boxes represent exons, and the lines between the boxes indicate introns. Open boxes represent untranslated regions. Filled boxes represent putative coding regions based on translation from the first ATG with a Kozak consensus sequence at nt 358 of AF050182. (A second Kozak consensus sequence present in exon 2 could begin translation at nt 523). Horizontal filled boxes indicate the locations of probes used for Southern blotting. (B) Southern blot identification of the targeting event in ES cells. Representative autoradiograms are shown. The upper band in each case represents the wild-type allele, and the lower band represents the targeted allele (see panel A). (C) Southern blot analysis of DNA extracted from mouse tails. Genotypes are shown above lanes. (D) PCR genotyping of DNA extracted from mouse tails. Genotypes are shown above lanes. M, size marker (pUC19 DdeI with bands at 910, 540, 426, 409, 266, and 166 bp).

Northern blot analysis done to identify transcripts arising from the targeted mPer3 allele. Skeletal muscle RNA was hybridized with probes directed to the mPer3 cDNA sequence 5′ or 3′ of the PGK-NEO cassette, to the NEO cassette, and to the portion of mPer3 cDNA deleted by the targeting construct. RNA size standards (in kilobases) are shown on the left.

Rhythmic gene expression in skeletal muscle. Autoradiograms from Northern blots illustrate rhythmic expression of mPer1, mPer2, and mPer3 in −/− and +/+ mice on the first day in DD. Hybridization of the actin control probe verified equal loading of the lanes (bottom autoradiogram). Graphs illustrate temporal profiles of mPer gene expression in skeletal muscle. Values are expressed relative to those for actin, and each value represents the mean of two blots. See the legend to Fig. 4 for plotting conventions.

mPER3 protein is absent in mice with targeted disruption of the mPer3 gene. (A) Western blot incubated with antisera to mPER3 showing immunoreactive proteins from wild-type (+/+) and mPER3-deficient (−/−) mice. Tissues examined were whole brain minus cerebellum and hypothalamus (Brain), whole hypothalamus (Hypo), and anterior hypothalamus containing SCN (AH/SCN). The specific mPER3 band corresponds in mobility to in vitro-translated mPER3. Results shown are representative of three experiments. (B) Immunoprecipitation. Brain proteins extracted from +/+ or −/− mice were precipitated with antiserum to mCRY1, mCRY2, or mPER3 and separated by SDS-polyacrylamide gel electrophoresis, and the blot was incubated with antiserum to mPER3. The mPER3 band indicated by the arrow corresponds in mobility to in vitro-translated mPER3. Similar results were found in a replicate experiment.

Rhythmic gene expression in the SCN, as assessed by in situ hybridization. Significant rhythms of expression of mPer1, mPer2, mCry1, and Bmal1 were detected in both wild-type and mPER3-deficient mice, and there were no differences between the genotypes. mPer3 RNA levels were significantly reduced in the SCN of mPer3-deficient mice. Each value represents the mean ± standard error of the mean for six to eight mice. Adjacent sections from the same group of mice were used for all probes. Tissues were collected on the first day in DD. The horizontal bar at the bottom of each panel represents the lighting cycle that the animals experienced prior to entry into DD: subjective day is gray, and subjective night is black. (Circadian times 0 and 12 represent the times at which the lights would have been turned on and off had the animals remained in LD.) Data from circadian times 3, 21, and 24 are double plotted.

Representative actograms illustrating rhythmic wheel-running behavior in a wild-type mouse (left) and an mPER3-deficient mouse (right). Each horizontal line represents a 48-h period; the second 24-h period is double plotted, appearing both to the right of and below the first 24-h period. Vertical deflections represent periods of wheel running. Animals were maintained in 12L:12D for the first 8 days of the record and then entered DD. The timing of the lighting cycle is indicated by the horizontal bar at the top. Records shown are from isogenic male mice from study 5 (Table 1).