5/26/2023 0 Comments Mper1 snapgenePlots represent analysis of 10 days of data from representative wild-type (A and B), mPER1-deficient (C and D), and mPer2ldc mice (E and F) in LD (A, A′, C, C′, E, and E′) and DD (B, B′, D, D′, F, and F′). Another mPER1-deficient mice maintained robust rhythmicity (period = 24.0 hr) for the duration of study and was not readily distinguishable from wild-type mice (see supplemental material). ![]() One mPER1-deficient mouse had markedly reduced amplitude of circadian rhythmicity but with a significant residual component in the circadian range (period = 21.75 hr) for 3 weeks in DD ( Figure 4, center row, left panel). The transition from rhythmicity to arrhythmicity was gradual and was accompanied by an increase in the amplitude of ultradian components. In some cases, very faint rhythmicity was apparent by eye but did not reach the criterion for rhythmicity described in Experimental Procedures (see Figure 4 and supplemental material ). Most of these mPER1-deficient mice maintained rhythmicity for the first 10–14 days in DD, followed by a severe to total loss of circadian amplitude in the second and third 10-day intervals in DD (see Experimental Procedures Figure 5). Nine of 11 mPER1-deficient mice became arrhythmic following transfer to DD ( Figure 4). All three mPer2ldc mutant mice shown were arrhythmic by the end of the record Of the mPER1-deficient mice, the animal on the left maintained weak rhythmicity in DD while the other two animals became arrhythmic by the third 10-day segment in DD. All three wild-type mice shown maintained robust rhythmicity in DD, as did all of the wild-type mice studied. The timing of the light-dark cycle is indicated by the bar above the record. Animals were initially housed in a 12L:12D light-dark cycle (LD) and then were transferred to constant darkness (DD). Vertical bars represent periods of wheel running. Each horizontal line represents a 48 hr period the second 24 hr period is plotted to the right and below the first. Similar results were obtained in two additional experimentsĪctivity records (actograms) of representative wild-type mice (+/+ top row), mPER1-deficient (center row), and homozygous mPer2ldc mutant (bottom row) mice are shown in double-plotted format. Note that the 3′ probe hybridized more effectively with target mRNA than the 5′ probe both images were generated from a single blot that was stripped and reprobed. Minor, long transcripts were also detectable in the homozygous mutant mice with both the 5′ and 3′ probes, likely corresponding to transcripts in which exon 4 is directly spliced to exon 7 (C) as well as transcripts containing a portion of the neomycin cassette between exons 4 and 7. In mice homozygous for the targeted allele (−/−), the major transcript identified with the 5′ probe (B) was ∼0.8 kb: this transcript also hybridized with a probe based on sequence from intron 4 (data not shown). In wild-type mice (+/+), a major transcript band at ∼7 kb was detected with probes directed to both the 5′ and 3′ cDNA sequence relative to the site of insertion of the neomycin resistance cassette (band A). ![]() ![]() We conclude that this mPer2 allele represents a null allele at the level of the SCN. The immunohistochemistry data in Figure 2 show that this possible deletion mutant mPER2 protein is not present at detectable levels in the SCN. The antibody used for assessment of mPER2 immunoreactivity in the SCN is directed to the carboxyl terminus of mPER2, a region that would be present in the deletion mutant protein. This transcript could, if translated, result in a mutant mPER2 protein lacking only the 108 amino acids of the PAS domain encoded by exons 5 and 6 (residues 148–255). This splicing event maintains the reading frame as in native mPER2. A relatively rare transcript form was also detected in which exon 4 was spliced directly to exon 7. These exons encode the first 147 residues of mPER2 the potential products of these transcripts would lack the entire PAS domain and the remainder of the coding region. Several transcript forms, including the major product of the mutant mPer2 allele, could generate truncated proteins from the first four exons of mPer2 ( Figure 3 and Experimental Procedures). However, RT-PCR and RACE analysis of brain RNA revealed the presence of several mPer2 transcripts in mice homozygous for the mPer2 targeted allele ( Figure 3 and Experimental Procedures). Immunohistochemical analysis of the SCN confirmed that mice homozygous for the targeted mPer2 allele were devoid of detectable mPER2 immunoreactivity ( Figure 2).
0 Comments
Leave a Reply. |