Melatonin receptor-deficient mice and uses thereof

Details Melatonin receptor-deficient mice and uses thereof Melatonin receptor-deficient mice and uses thereof

2000-01-07

2001-12-04

Crouch, Deborah (Department: 1632)

Multicellular living organisms and unmodified parts thereof and

Nonhuman animal

Transgenic nonhuman animal

C800S003000, C800S013000, C435S069100, C435S069200, C435S325000

Reexamination Certificate

active

06326526

ABSTRACT:

BACKGROUND OF THE INVENTION
The field of the invention is melatonin and its receptors.
Melatonin, the principal hormone of the pineal gland, influences the timing of mammalian circadian rhythms and regulates the reproductive alterations that occur in response to changes in day length in seasonally breeding mammals (Reppert, S. M. and Weaver, D. T.,
Cell
83:1059-1062, 1995). In humans, melatonin administration has been shown to alleviate the symptoms of jet lag after air travel across several time zones. The hormone also has potent sedative effects in humans and may be a useful hypnotic agent.
Melatonin exerts these effects through specific guanine nucleotide binding protein,(G protein)-coupled receptors. A family of these G protein-coupled melatonin receptors has been cloned from
Xenopus laevis
, chicken and various mammals (U.S. applications Ser. Nos. 08/261,857, filed Jun. 17, 1994; 08/319,887, filed Oct. 7, 1994; and 08/466,103, filed Jun. 6, 1995; Ebisawa, T., et al.
Proc. Natl. Acad. Sci. USA
91:6133-6137, 1994; Reppert, S. M. et al., Neuron 13:1177-1185, 1994; Reppert, S. M. et al.
Proc. Natl. Acad. Sci. USA
92:8734-8738, 1995; Reppert, S. M. et al.,
Neuron
15:1003-1015, 1995). These cloned receptors exhibit affinity and pharmacological characteristics similar to each other and to endogenous receptors, as defined by the melatonin agonist 2- [
125
I]-iodomelatonin (
125
,-Mel). Two mammalian melatonin receptor subtypes have been identified by molecular cloning studies. The mammalian receptor Mel
1a
is expressed in the hypothalamic suprachiasmatic nuclei (SCN) and hypophyseal pars tuberalis, which are presumed sites of the circadian and some of the reproductive actions of melatonin, respectively (Reppert, S. M. et al.,
Neuron
13:1177-1185, 1994). The mammalian Mel
1b
receptor is expressed in retina and brain and may mediate the reported effects of melatonin on retinal physiology in mammals (Reppert, S. M. et al.
Proc. Natl. Acad. Sci. USA
92:8734-8738, 1995). A third receptor subtype, the Mel
1c
melatonin receptor, has been cloned from zebrafish, Xenopus, and chicken, but not from mammals (Reppert, S. M. et al.,
Neuron
15:1003-1015, 1995).
SUMMARY OF THE INVENTION
Mice that are engineered to lack various melatonin receptor subtypes have been generated in accordance with the present invention. Since melatonin can entrain circadian rhythms, the mice of the invention are useful for elucidating the roles of the different receptors in entrainment and control of biological rhythms, including jet lag, disturbed sleep-wake cycle in blind people, sleep disorders in shift workers, establishing a diurnal sleep-wake pattern in neonates, and regulating the initiation and timing of puberty in humans and the mating cycles of seasonally breeding mammals.
In general, the invention features a transgenic non-human animal having a transgene disrupting expression of a melatonin receptor gene, the transgene being chromosomally integrated into the germ cells of the animal, e.g., where the mammal is a mouse and where the melatonin receptor gene encodes the melatonin 1a receptor, or the melatonin 1b receptor. The cells can be homozygous for the transgene, and the disruption can result in a null mutation. The cells can be used to produce a cell line.
In another aspect, the invention features a method of determining if a candidate compound exerts an effect via a melatonin receptor other than the melatonin 1a receptor, the method comprising contacting suprachiasmatic nuclei of the mouse containing the disrupted melatonin 1a receptor gene with the candidate compound, and measuring the phase shift in the suprachiasmatic nuclei, wherein an effect on phase shift in the presence of the candidate compound is an indication that the candidate compound exerts an effect via a melatonin receptor other than the melatonin 1a receptor.
In yet another aspect, the invention features a method of determining if a candidate compound is a melatonin 1b receptor antagonist, the method comprising contacting suprachiasmatic nuclei of the mouse containing the disrupted melatonin 1a receptor gene with melatonin in the presence of the candidate compound, and measuring the phase shift in the suprachiasmatic nuclei, wherein a decrease in phase shift in the presence of both melatonin and the candidate compound, relative to that seen in the presence of melatonin but the absence of the candidate compound, is an indication that the candidate compound is a melatonin antagonist.
The invention also features a method of determining if a candidate compound exerts an effect via the melatonin 1a receptor, the method comprising contacting suprachiasmatic nuclei of the mouse containing the disrupted melatonin 1b receptor gene with the candidate compound, and measuring the suprachiasmatic nuclei neuronal firing, wherein a decrease in suprachiasmatic nuclei neuronal firing in the presence of the candidate compound, relative to that seen in the absence of the candidate compound, is an indication that the candidate compound exerts an effect via the melatonin 1a receptor.
In yet another aspect, the invention features a method of determining if a candidate compound is a melatonin 1a receptor antagonist, the method comprising contacting suprachiasmatic nuclei of the mouse containing the disrupted melatonin 1b receptor gene with melatonin in the presence of the candidate compound, and measuring the suprachiasmatic nuclei neuronal firing, wherein an increase of the suprachiasmatic nuclei neuronal firing in the presence of the candidate compound, relative to that seen in melatonin-treated cells in the absence of the compound, is an indication that the candidate compound is a melatonin la receptor antagonist.
Melatonin's inhibition of neuronal firing is likely due to activation of potassium channels (Wickman, K., and Clapham, D. E.,
Physiol. Rev
. 75:865-885, 1995; Jiang, Z.-G., et al.,
Brain Res
. 687:125-132, 1995), therefore, the invention also features a method of treating a patient having a condition characterized by suprachiasmatic neuronal firing, the method comprising administering to the patient an effective amount of a potassium channel activator. The condition to be treated can be benign prostatic hyperplasia, jet lag, or disturbed sleep-wake cycle, such as that found in blind people, shift workers, or neonates.
In another aspect, the invention features a method of treating a patient having a condition characterized by suprachiasmatic neuronal firing, without affecting the patient's circadian rhythm, the method comprising administering to the patient an effective amount of a potassium channel activator.
The invention also features a method of controlling the estrus cycle of a mammal, the method comprising administering to the mammal an amount of a potassium channel activator effective to induce or maintain the mammal's estrus cycle.
By “high-affinity melatonin receptor polypeptide” is meant all or a functional part of a vertebrate cell surface protein which specifically binds melatonin and signals the appropriate melatonin-mediated cascade of biological events (e.g., a decrease in intracellular cAMP concentration). The polypeptide is characterized as having the ligand binding properties (including the agonist and antagonist binding properties) and tissue distribution described herein.
By “homologous recombination” is meant site-specific insertion or deletion of nucleic acid by a mechanism involving matching up of complementary regions in two different DNA molecules.
By “mutation” is meant a change in a nucleic acid sequence or amino acid sequence, e.g., a deletion, an insertion, a translocation involving one or more exons, introns, or transcription regulatory regions (e.g., a promotor) of a gene. If the mutation reduces the expression or activity level of the protein encoded by the mutated gene (all isoforms included) by more than 80% relative to the unmutated gene, the mutation is called a null mutation, and the mouse harboring the mutation is a knockout mouse.
By “target gene” is meant a gene i

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