Multicellular living organisms and unmodified parts thereof and – Method of using a transgenic nonhuman animal in an in vivo...
Reexamination Certificate
2001-09-07
2004-06-22
Falk, Anne-Marie (Department: 1636)
Multicellular living organisms and unmodified parts thereof and
Method of using a transgenic nonhuman animal in an in vivo...
C800S018000, C800S025000
Reexamination Certificate
active
06753456
ABSTRACT:
FIELD OF INVENTION
The invention relates generally to animal model systems useful for examining and manipulating neurobehaviors mediated by nicotine. More specifically, the invention relates to knock-in mice having a leucine-to-serine mutation of the &agr;4 nicotinic receptor subunit gene resulting in nicotine hypersensitivity, and to methods of using the knock-in mice to identify agents that modulate nicotine addiction and other neurobehaviors.
BACKGROUND OF THE INVENTION
The mechanism leading from nicotine intake to addiction begins with the activation of neuronal nicotinic acetylcholine receptors (nAChR). Nicotine elicits dopamine release in several regions of the brain, leading to reward, motor learning, and addictive effects. The highest-affinity and most abundant nicotine binding in the brain corresponds to a nAChR formed by &agr;4 and &mgr;2 subunits. The &agr;4 subunit is the principal partner for the &bgr;2 subunit in brain; &bgr;2-containing receptors play an important role in nicotine self-administration, in nicotine-stimulated electrophysiological responses in midbrain neurons, and in nicotine-stimulated dopamine release in the ventral striatum. The &agr;4 subunit is localized in dopaminergic neurons with tyrosine hydroxylase. The &agr;4 and &bgr;2 subunits are also the site of at least five point mutations that cause the human disease, autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE).
Nicotine is known to reduce anxiety, or to produce a bimodal effect on anxiety. Nicotinic receptors modulate the release of neurotransmitters, for example &ggr;-aminobutyric acid, dopamine, and serotonin, that have critical roles in the regulation of anxiety. The mechanism by which nicotine reduces anxiety is not well understood, but results from several studies suggest that nicotine-mediated effects on neurons in which nicotine is a co-transmitter may play important roles. Accordingly, a model system for studying nicotinic neurotransmission and the role of the nicotinic acetylcholine receptor is of key importance.
BRIEF DESCRIPTION OF INVENTION
In one embodiment of the invention there is provided a transgenic non-human animal having a transgene comprising a leucine-to-serine mutation of the &agr;4 nicotinic receptor subunit chromosomally integrated into germ cells of the animal. The leucine to serine mutation is located in the M2 transmembrane region of the acetylcholine receptor.
In another embodiment of the invention there is provided a transgenic mouse comprising a transgene having a leucine-to-serine mutation at 9′ of the &agr;4 nicotinic receptor subunit. Expression of the receptor subunit gene results in a mouse that displays modified behavior compared to a normal mouse. The transgenic mouse displays nicotinic hypersensitivity, increased anxiety, increased sensitivity to seizures, poor motor learning, excessive ambulation, a reduction in dopaminergic neuron function upon aging, or any combination thereof.
In another embodiment of the invention there is provided a transgenic mouse comprising a transgene having a single codon change in the &agr;4 nicotinic receptor subunit. Expression of the receptor subunit gene results in a mouse that displays modified behavior compared to a normal mouse. The transgenic mouse displays nicotinic hypersensitivity, increased anxiety, increased sensitivity to seizures, poor motor learning, excessive ambulation, a reduction in dopaminergic neuron function upon aging, susceptibility to seizure, spontaneous seizures, or any combination thereof.
In yet another embodiment of the invention there is provided a method for screening a candidate agent for the ability to modulate nicotine-mediated behavior in a transgenic animal. The method includes administering to a first transgenic animal a candidate agent and comparing nicotine-mediated behavior in the animal to the nicotine-mediated behavior of a second transgenic animal not administered the candidate agent. A difference in nicotine-mediated behavior in the animal administered the candidate agent compared to the animal not administered the agent is indicative of an agent that modifies nicotine-mediated behavior.
In still another embodiment of the invention there is provided a method for screening for candidate agents that modulate nicotine hypersensitivity. The method includes administering a candidate agent to a transgenic animal and determining the effect of the agent upon a cellular or molecular process associated with nicotinic hypersensitivity compared to an effect of the agent administered to a non-transgenic animal.
In another embodiment, there is provided a method for screening for candidate agents that modulate seizures associated with epilepsy. The method includes administering a candidate agent to a transgenic animal and determining the effect of the agent upon seizure activity associated with epilepsy compared to an effect of the agent administered to a non-transgenic animal.
BRIEF DESCRIPTION OF FIGURES
FIG. 1
shows the physiological design, recombinant construction, and genomic characterization of the &agr;4 knock-in mouse strains.
FIGS. 1A-C
show the agonist concentration-response relations of WT and mutated (&agr;4 Leu9′Ser) rat &agr;4&bgr;2 receptors expressed in oocytes (five oocytes for each curve).
In
FIG. 1A
, the agonist is acetylcholine; in
FIG. 1B
, the agonist is nicotine and in
FIG. 1C
, the agonist is choline. The choline responses of the WT receptor were not studied systematically, because there is no response at choline concentrations up to 1 mM, and higher concentrations of choline block the channel.
FIG. 1C
insert shows the time course of the response to 30 &mgr;M choline, showing partial desensitization.
FIG. 1D
shows the targeting construct containing exon 5 with the Leu9′Ser mutation, the neomycin resistance gene (neo) flanked by loxP sites, the diphtheria toxin A chain gene (DT), and the pKO V907 vector (pKO).
FIG. 1E
shows that deletion of the neo cassette by transfecting the neo-intact ES cells with a cytomegalovirus-Cre plasmid generates neo-deleted ES cell lines.
FIG. 1F
shows sequence analysis of DNA extracted from WT (SEQ ID NO:2) heterozygous (het: SEQ ID NO:3), and homozygous (hom: SEQ ID NO:5) neo-intact mice. The WT sequence at nucleotide position 142, corresponding to the codon at position 9′ in the M2 region, is CTT, encoding leucine; the mutant sequence is TCT, encoding serine.
FIG. 2
shows the pathophysiological basis of dopaminergic neuron deficits in mutant mice.
FIG. 2A
shows cell counts of tyrosine hydroxylase (TH)-positive neurons in substantia nigra of ED 16 to ED 18 embryos from WT, neo-intact, and neo-deleted mice. The heterozygote (het) cell counts do not differ significantly from WT, but both the homozygous (homo) neo-intact (P, 0.01, f test) and the neo-deleted cell counts (P, 0.05, t test) differ significantly from WT.
FIG. 2B
shows whole-cell voltage-clamp recording of responses to two consecutive puffs of choline (100 &mgr;M, 20 ms) in neuron-like cells differentiated from ED 16 midbrain neuronal progenitor cells. Upper trace, cell from a WT embryo; lower trace, cell derived from a heterozygous neo-intact ED 16 embryo.
FIG. 2C
shows the mean ±SEM of responses in neuron-like cells derived from heterozygous animals (n=5 cells) but little or no response in cells from WT animals (n=7 cells; significant difference, P, 0.05, t test).
FIG. 3
shows spontaneous and drug-modulated locomotion of WT and heterozygous (het) neo-intact mutants.
FIG. 3A
shows the effect of no treatment. Heterozygotes showed significantly higher locomotion than WT mice at the beginning of the experiment (P, 0.001).
FIG. 3B
shows locomotion after nicotine, 0.02 mg/kg, was injected 30 min after the start of behavioral monitoring. The plot shows data averaged over the time periods, 10 min before baseline (BL) and 5-15 min after injection. Heterozygous mice showed a significant reduction of locomotor activity after nicotine injection (P, 0.05). There was no significant difference in non-injected animals (rig
Fonck Carlos
Labarca Cesar
Lester Henry A.
Schwarz Johannes
California Institute of Technology
Falk Anne-Marie
Gray Cary Ware & Freidenrich LLP
Qian Celine
LandOfFree
Point mutant mice with hypersensitive alpha 4 nicotinic... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Point mutant mice with hypersensitive alpha 4 nicotinic..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Point mutant mice with hypersensitive alpha 4 nicotinic... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3342389