Resetting central and peripheral circadian oscillators in transgenic rats

Science

Volumn 288, Issue 5466, 2000, Pages 682-685

  Yamazaki, Shin ^a   Numano, Rika ^b   Abe, Michikazu ^a   Hida, Akiko ^b   Takahashi, Ri Ichi ^c   Ueda, Masatsugu ^c   Block, Gene D ^a   Sakaki, Yoshiyuki ^b   Menaker, Michael ^a   Tei, Hajime ^b  

^a UNIVERSITY OF VIRGINIA   (United States)

^b UNIVERSITY OF TOKYO   (Japan)

^c YS New Technology Institute Inc   (Japan)

Author keywords

[No Author keywords available]

Indexed keywords

ANIMAL EXPERIMENT; ARTICLE; CIRCADIAN RHYTHM; INFRARED RADIATION; LOCOMOTION; NONHUMAN; OSCILLATION; PHOTOSTIMULATION; PRIORITY JOURNAL; RAT; TRANSGENIC MOUSE;

ANIMALS; ANIMALS, GENETICALLY MODIFIED; BIOLOGICAL CLOCKS; CELL CYCLE PROTEINS; CIRCADIAN RHYTHM; CULTURE TECHNIQUES; DARKNESS; GENES, REPORTER; LIGHT; LIVER; LUCIFERASES; LUNG; MALE; MICE; MOTOR ACTIVITY; MUSCLE, SKELETAL; NUCLEAR PROTEINS; PROMOTER REGIONS (GENETICS); RATS; SUPRACHIASMATIC NUCLEUS;

ANIMALIA; MAMMALIA; MUS MUSCULUS;

EID: 0034724728     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.288.5466.682     Document Type: Article

References (27)

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7. A mouse Per1 genomic fragment of 6.7 kb was ligated directly to the second codon of the firefly luciferase cDNA flanked by the SV40 late polyadenylation signal. The mPer1 fragment includes five functional E box regions, a transcription initiation site, the first and second exons, which are split by the first intron, and a translational start codon in the second exon (Fig. 1A). As expected, the reporter gene was induced by the concerted action of Clock and Bmal1, and repressed by either Cry1 or Cry2 in a transient cotransfection assay (A. Hida et al., Genomics, in press). The linearized reporter fragment was microinjected into 302 fertilized eggs of Wistar rats (Charles River Japan Inc.); [S. Hochi, T. Ninomiya, M. Homma, A. Yuki, Anim. Biotechnol. 1, 175 (1990)]. Transgenic rats were identified by polymerase chain reaction, and the copy number of the transgene was determined by Southern analysis. Six transgenic rats were obtained by the screening of 60 weaned pups. All of the six founder rats developed normally, although one was sterile and one was mosaic. Luciferase activity in brain extracts of the four transgenic lines was roughly proportional to the copy number of the reporter gene. A transgenic line W(perl)1], which showed circadian oscillation of luciferase activity in the SCN, was selected for further study. There are approximately 12 copies per genome of the transgene integrated in W(perl)1, and the luciferase activity in the brain is 1431 relative luminescence units per mg protein. We measured the period of the wheel-running activity rhythm in constant darkness for both male Per1-luc and male wild-type controls. The period of the transgenic rats (at 8 to 10 weeks of age) was 24.43 ± 0.02 hours (SEM, n = 20), which is very close to the period of the wild-type animals (24.33 ± 0.01 hours, n = 26) at 6 to 8 weeks of age, indicating that the transgene does not disrupt molecular circadian time keeping.
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27. We thank M. E. Geusz and T. Kondo for technical information for bioluminescence measurements, M. Katsuki for helpful discussion, and E. D. Herzog for comments on the manuscript. Supported in part by the NSF Center for Biological Timing, NIH grant (MH56647 to M.M.) and by research grants from the Japanese Ministry of Education, Science, Sports, and Culture, and the Japanese Ministry of Health and Welfare (to H.T.).