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Functional identification of the mouse circadian Clock gene by transgenic BAC rescue.

ABSTRACT: As a complementary approach to positional cloning, we used in vivo complementation with bacterial artificial chromosome (BAC) clones expressed in transgenic mice to identify the circadian Clock gene. A 140 kb BAC transgene completely rescued both the long period and the loss-of-rhythm phenotypes in Clock mutant mice. Analysis with overlapping BAC transgenes demonstrates that a large transcription unit spanning approximately 100,000 base pairs is the Clock gene and encodes a novel basic-helix-loop-helix-PAS domain protein. Overexpression of the Clock transgene can shorten period length beyond the wild-type range, which provides additional evidence that Clock is an integral component of the circadian pacemaking system. Taken together, these results provide a proof of principle that "cloning by rescue" is an efficient and definitive method in mice.

SUBMITTER: Antoch MP

PROVIDER: S-EPMC3764491 | biostudies-literature | 1997 May

REPOSITORIES: biostudies-literature

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Functional identification of the mouse circadian Clock gene by transgenic BAC rescue.

Antoch M P MP Song E J EJ Chang A M AM Vitaterna M H MH Zhao Y Y Wilsbacher L D LD Sangoram A M AM King D P DP Pinto L H LH Takahashi J S JS

Cell 19970501 4

As a complementary approach to positional cloning, we used in vivo complementation with bacterial artificial chromosome (BAC) clones expressed in transgenic mice to identify the circadian Clock gene. A 140 kb BAC transgene completely rescued both the long period and the loss-of-rhythm phenotypes in Clock mutant mice. Analysis with overlapping BAC transgenes demonstrates that a large transcription unit spanning approximately 100,000 base pairs is the Clock gene and encodes a novel basic-helix-loo ...[more]

PMID: 9160756

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Project description:PurposeMice lacking rods and cones retain pupillary light reflexes that are mediated by intrinsically photosensitive retinal ganglion cells (ipRGCs). Melanopsin is necessary and sufficient for this nonvisual photoreception. The mammalian inner retina also expresses the potential blue light photopigments cryptochromes 1 and 2. Previous studies have shown that outer retinal degenerate mice lacking cryptochromes have lower nonvisual photic sensitivity than retinal degenerate mice, suggesting a role for cryptochrome in inner retinal photoreception.MethodsNonvisual photoreception (pupillary light responses, circadian entrainment, and in vitro sensitivity of intrinsically photosensitive retinal ganglion cells) were studied in wild-type, rd/rd, and circadian clock-mutant mice with and without rd/rd mutation.ResultsLoss of cryptochrome in retinal degenerate mice reduces the sensitivity of the pupillary light response at all wavelengths but does not alter the form of the action spectrum, suggesting that cryptochrome does not function as a photopigment in the inner retina. The authors compounded the rd/rd retinal degeneration mutation with mutations in other essential circadian clock genes, mPeriod and Bmal1. Both mPeriod1?/?; mPeriod2?/?;rd/rd and Bmal1?/?;rd/rd mice showed significantly lower pupillary light sensitivity than rd/rd mice alone. A moderate amplitude (0.5 log) circadian rhythm of pupillary light responsiveness was observed in rd/rd mice. Multielectrode array recordings of ipRGC responses of mCryptochrome1?/?;mCryptochrome2?/? and mPeriod1?/?;mPeriod2?/? mice showed minimal sensitivity decrement compared with wild-type animals. mCryptochrome1?/?;mCryptochrome2?/?;rd/rd, mPeriod1?/?;mPeriod2?/?;rd/rd and Bmal1?/?;rd/rd mice all showed comparable weak behavioral synchronization to a 12-hour light/12-hour dark cycle.ConclusionsThe effect of cryptochrome loss on nonvisual photoreception is due to loss of the circadian clock nonspecifically. The circadian clock modulates the sensitivity of nonvisual photoreception.

Dataset Information

Functional identification of the mouse circadian Clock gene by transgenic BAC rescue.

Publications

Functional identification of the mouse circadian Clock gene by transgenic BAC rescue.

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