Unknown,Transcriptomics,Genomics,Proteomics

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Circadian skeletal muscle_wt and Clock mutants

ABSTRACT: One hundred ninety wildtype male C57BL/6J mice age 7-10 weeks were purchased from Jackson Laboratory and entrained to a 12:12 light:dark cycle for 2 weeks. Mice were placed in light-tight boxes on a 12:12 LD cycle for 4 weeks, then released into constant darkness. Starting 30 hours after entry into DD (CT18), the left leg muscle from 5 wildtype mice, and the right leg muscle from the same wildtype mice were collected every 4 hours for 48 hours, for a total of 12 timepoints. Total RNA from each side (left or right) were separately pooled to make a single sample for amplification and hybridization. Therefore, duplicate arrays (from left and right side) were performed at each time point. At timepoints 34 through 58 hours in DD, tissues from age-matched male C57BL/6J Clock/Clock homozygous mutant mice that had been treated with the same light entrainment protocol as the wildtype were collected. Tissues were collected from 5 Clock/Clock mutants at each timepoint except for 34 and 46 hours after the onset of DD, when tissues from 10 Clock/Clock mice were collected. As for the wildtype mice, right and left sides were collected, prepared, amplified, and hybridized in separate pools. Mice were sacrificed by cervical dislocation, and the optic nerves were severed in complete darkness; brain dissection was performed using illumination from an infrared viewer (FJW Industries, Palatine, IL). SCNs were dissected out, pooled at a density of 5 per tube in 100 ?l RNAlater (Ambion, Austin, TX), frozen on dry ice, and stored at â80?C until use. duplicates consisted of pools from independent mice

ORGANISM(S): Mus musculus

SUBMITTER: Brooke Miller

PROVIDER: E-GEOD-3746 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Circadian mutant Overtime reveals F-box protein FBXL3 regulation of cryptochrome and period gene expression.

Siepka Sandra M SM Yoo Seung-Hee SH Park Junghea J Song Weimin W Kumar Vivek V Hu Yinin Y Lee Choogon C Takahashi Joseph S JS

Cell 20070426 5

Using a forward genetics ENU mutagenesis screen for recessive mutations that affect circadian rhythmicity in the mouse, we isolated a long period (approximately 26 hr) circadian mutant named Overtime (Ovtm). Positional cloning and genetic complementation reveal that Ovtm is encoded by the F-box protein FBXL3, a component of the SKP1-CUL1-F-box-protein (SCF) E3 ubiquitin ligase complex. The Ovtm mutation causes an isoleucine to threonine (I364T) substitution leading to a loss of function in FBXL3 ...[more]

PMID: 17462724

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Project description:Recent evidence suggest that the circadian timing system plays an important role in the control of renal function and maintaining blood pressure. Here, we analyzed circadian rhythms of urinary excretion of sodium and potassium in wild-type mice and mice lacking circadian transcriptional activator clock. Analysis of urines collected at hourly intervals over a 24-hour period revealed dramatic changes in rhythms of sodium and potassium excretion in clock(-/-) mice. In parallel, significant differences in circadian pattern of plasma aldosterone levels, but not in the 24-hour mean aldosterone levels, were observed. Microarray-based profiling of renal transcriptomes demonstrated that clock(-/-) mice exhibit dysregulation in multiple mechanisms involved in maintaining sodium and potassium balance by the kidney. The most significant changes were detected in the expression levels of several key enzymes (Cyp4a14, Cyp4a12a and Cyp4a12b) required for the conversion of arachidonic acid to 20-hydroxyeicosatetraenoic acid (20-HETE), a powerful regulator of renal sodium and potassium excretion, renal vascular tone and blood pressure. The 20-HETE levels measured in kidney microsomes of wild-type mice followed a circadian-like temporal pattern. In clock(-/-) mice, the acrophase of this rhythm was shifted by 8 hours and the 24-hour mean levels of 20-HETE were significantly decreased. These results demonstrate that circadian rhythms of urine electrolyte excretion are largely dependent on the circadian clock activity and indicate that circadian oscillations in renal 20-HETE content could be an important mechanism of blood pressure regulation. We examined the temporal profiles of gene expression in mouse whole kidney. Animals were sacrificed for microdissection every 4 hours, i.e. at ZT0, ZT4, ZT8, ZT12, ZT16 and ZT20 (ZT M-bM-^@M-^S Zeitgeber (circadian) time, indicates time of light-on as ZT0 and time of light-off as ZT12). The microarray hybridization was performed in duplicates on pools of RNA composed of equivalent amounts of RNA prepared from teo or three animals at each ZT time-point.

Dataset Information

Circadian skeletal muscle_wt and Clock mutants

Publications

Circadian mutant Overtime reveals F-box protein FBXL3 regulation of cryptochrome and period gene expression.

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