It is known that Clock is involved in the regulation of circadian rhythms in mice. By positional cloning of an N-Ethyl N-Nitrosourea- induced mutant, a dominant negative allele of Clock was identified. The question of the necessity of Clock for rhythmicity has not been answered. In per and tim mutants, null alleles of the Drosophila clock genes period and timeless, respectively, locomotor activity rhythms and molecular oscillations in per and tim mRNAs are abrogated. Thus we predict that the Clock null allele would also result in arrhythmicity in wheel running if it is a bona fide circadian clock component. In addition, the identification of putative period homologues in mice, mPerl and mPer2, allow us to address the question of whether Clock is essential for molecular oscillations in these putative clock components. Gene targeting affords the opportunity to generate a null Clock allele in vivo to determine the effects on behavioral and molecular oscillations in the mouse. Clock is a member of the bHLH-PAS family of transcriptional regulators. We will create a null allele by replacing the exons encoding the start of translation, the bHLH and part of the PAS domains with the neomycin phosphotransferase gene by homologous recombination in embryonic stem cells. Cell lines will then be used to generate mice homozygous for the null allele (knockout) by chimera aggregation and the appropriate breeding. Knockout mice will be examined for alterations in wheel running activity as a behavioral measure of their circadian rhythms. Knockout mice will also be examined for effects of the loss-of-function allele on the molecular oscillations of mPerl and mPer2 by in situ hybridization. Our overall objective is to learn whether Clock is essential for behavioral and molecular rhythmicity in mice.
