hCRY2-A260T BAC transgenic mice express the full-length CRY2 human gene incorporating an A260T missense mutation that is correlated with the Familial Advanced Sleep Phase (FASP) phenotype.
Louis Ptacek, University of California, San Francisco
Familial Advanced Sleep Phase (FASP) is a heritable human sleep phenotype characterized by very early sleep and wake times. A correlated A260T missense mutation in the human CRY2 (cryptochrome 2 (photolyase-like)) gene, a principal component of mammalian circadian clocks, has been identified in one affected family.
This hCRY2-A260T BAC transgenic line expresses the full-length CRY2 human gene carrying the A260T missense mutation. Line 24 carries an insertion of 15 copies of the BAC. Under conditions of 12 hours of light and 12 hours of dark (LD 12:12), these mice show significant advances in their peak time of resting behavior and activity onset/offset times. Mutant mice demonstrate a shortened circadian period and reduced phase-shift to early night light pulse associated with phase-advanced behavioral rhythms in the light-dark cycle.
The A260T mutation is located in the phosphate loop of the flavin adenine dinucleotide (FAD) binding domain of CRY2. The mutation alters the conformation of CRY2 protein, increasing its accessibility and affinity for FBXL3 (F-box and leucine-rich repeat protein 3; an E3 ubiquitin ligase), thus promoting its degradation. These results demonstrate that CRY2 stability controlled by FBXL3 plays a key role in the regulation of human sleep wake behavior.
BAC mice carrying the wildtype CRY2 gene (see Stock No. 031672) entrain stably to the light/dark cycle.
Crosses of the hCRY2 BAC transgenic strains with mPer2Luc animals (see Stock No. 006852) enable analysis of circadian rhythms via bioluminescence in tissue culture.
Mouse embryonic fibroblasts (MEFs) derived from crosses of hCRY2 BAC transgenic mice with mouse Cry2 knock-out animals (see Stock No. 016185) helped define circadian period shortening.
Also of potential interest, hCRY2-A260T BAC transgenic line 25 (carrying 10 copies of the transgene) are also available - see Stock No. 031794. Shortened circadian period has been detected in both lines 24 and 25. There may be slight differences in the phenotypes, but they have not been studied in depth.
Human BAC RP11-1084E2, encoding full-length CRY2 and incorporating an A260T (amino acid)/GAACGCCA to GAACACCA (nucleotide) mutation was microinjected into C57BL/6 x SJL F1 embryos. Resultant mice were subsequently backcrossed to C57BL/6J for 12-13 generations by the donating laboratory. Founder line 24 carries 15 copies of the transgene.
|Expressed Gene||CRY2, cryptochrome circadian regulator 2, human|
|Site of Expression|
|Allele Name||transgene insertion 24, Louis Ptacek|
|Allele Type||Transgenic (Humanized sequence, Inserted expressed sequence)|
|Gene Symbol and Name||Tg(CRY2*A260T)24Ljp, transgene insertion 24, Louis Ptacek|
|Promoter||CRY2, cryptochrome circadian regulator 2, human|
|Expressed Gene||CRY2, cryptochrome circadian regulator 2, human|
|Strain of Origin||(C57BL/6 x SJL)F1|
|Molecular Note||The transgenic construct contains human BAC RP11-1084E2, encoding full-length CRY2 and incorporating an A260T (amino acid)/GAACGCCA to GAACACCA (nucleotide) mutation. Founder line 24 carries 15 copies of the transgene. The A260T missense mutation was identified in one family affected with Familial Advanced Sleep Phase (FASP). The mutation is located in the phosphate loop of the flavin adenine dinucleotide (FAD) binding domain.|
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