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Title:  5-HT3 receptor assay using transgenic mammal

United States Patent:  6,630,612

Issued:  October 7, 2003

Inventors:  Allan; Andrea M. (Albuquerque, NM); Engel; Sharon R. (Damascus, MD); Lyons; Clifford R. (Albuquerque, NM)

Assignee:  University of New Mexico (Albuquerque, NM)

Appl. No.:  892311

Filed:  June 26, 2001

Abstract

The invention relates to transgenic mammals characterized by 5-HT3 receptor over-expression in the central nervous system (CNS). The mammals have particular utility as models for studying the role of 5-HT3 receptors in the CNS, especially for the study of reward pathways for alcohol and other substances of abuse.

SUMMARY OF THE DISCLOSURE

The inventions comprise transgenic mammals carrying an exogenous 5-HT3 transgene in their genome for over-expression in the CNS, particularly the forebrain. The inventions further comprise mammalian embryos carrying the 5-HT3 transgene capable of developing into viable transgenic animals whose progeny carry the transgene after breeding forward by sexual reproduction. The inventions additionally include DNA constructs comprising selected promoter+intron+5-HT3 cDNA or DNA segments cloned into plasmids for ultimate insertion into the genome of a mammal.

The transgenic mammals of the invention are characterized by a high density of functional 5-HT3 receptors in the CNS of the mammals. They also exhibit a decreased desire for alcohol as compared to genetically similar non-transgenic mammals and decreased aggressiveness.

DETAILED DESCRIPTION OF THE INVENTION

Transgenic mammals according to the invention comprise mammals containing within their genomes endogenous 5-HT3 gene which naturally encode and express 5-HT3 receptors and further contain within their genomes at least one cDNA exogenous copy of this gene for over-expression of 5-HT3 receptors in the CNS of the transgenic mammals.

1. 5-HT3 Receptor Gene

The primary structure and functional expressions of certain 5-HT3 serotonin cell surface receptors are known and described, for example, in Maricq et al., Science 254: 432-437, 1991, incorporated herein by reference in its entirety. The nucleotide sequence encoding 5-HT3 receptor (5-HT3 R-A) according to this publication is deposited in GenBank under Accession Number M74425. The publication describes cDNA encoding mouse 5-HT3 receptor from cloned NCB-20 cells (a hybrid cell line derived from mouse neuroblastoma cells and Chinese hamster embryonic brain cells), which is useful in the practice of the present invention.

5-HT3 nucleotide sequences from other mammalian sources including humans, comprising known and 5-HT3 family members as yet to be discovered, are also useful as transgenes in the practice of this invention, provided the product transgenic mammal exhibits the characteristics described and claimed herein. The transgene(s) may be either homologous or heterologous with the starter mammals. Further, 5-HT3 nucleotide sequences which have been truncated, for example to reduce potential immunogenicity of their encoded proteins expressed in heterologous species, may be employed with the proviso that the truncated sequence employed encodes and expresses functional 5-HT3 receptor binding sites.

2. Engineering Constructs

DNA constructs comprising 5-HT3 transgene(s) for production of the transgenic mammal of the invention typically also comprise selected nucleic acid regions associated with the transgene (as by fusion therewith) for mediation of, for example, its introduction into the target genome, its expression loci in the transgenic mammal, on/off external regulation of transgene expression, and other desired features, as generally known in the art. Inducible and tissue-specific promotors are exemplary.

The construct of the present invention is engineered to produce 5-HT3 over-expression substantially restricted to the CNS, especially the forebrain of the product transgenic mammal. One criticism of over-expressing transgenic mammals is that expression of the transgene not only occurs at sites where the native gene is expressed to obtain over-expression at these sites, but also is expressed in locations where the endogenous receptor is not expressed. The present invention obviates this predicted problem.

To obtain animal models according to the present invention which facilitate the study of the role of endogenous 5-HT3 receptors in vivo, it is essential to limit over-expression of these receptors to endogenous binding site areas for 5-HT3 receptor ligands and/or functional receptor expression sites, in particular, the central nervous system (CNS). 5-HT3 receptors in the CNS have been identified in the enteric system, on sympathetic, para-sympathetic, and sensory nerve fibers in the CNS and on several mouse neuroblastoma lines. According to Maricq et al. (Science, ibid.), 5-HT3 receptors are known to modulate intestinal contraction and transmission of pain sensations, but the function of 5-HT3 receptors in the CNS remained obscure at the time of this publication.

In a particular embodiment of the invention, as exemplified below, a transgenic mammal is engineered to obtain 5-HT3 over-expression restricted to the forebrain by controlling transgene expression with the tissue-specific Ca2+ calmodulin (CAM) kinase II.alpha. promoter. In this embodiment of the invention, no expression of 5-HT3 receptors could be detected in the cerebellum or other body sites of the transgenic animals where 5-HT3 receptor activity is not normally found in corresponding non-transgenic animals.

3. Utility of Products of the Invention.

The transgenic animal of this embodiment is thus an excellent model for studying the role of 5-HT3 receptors in regulating dopamine release in the forebrain, particularly the nucleus accumbens, in various psychotrophic diseases and conditions in addition to addictive or substance abuse disorders in which the presence or absence of dopamine is a significant factor, and in characterizing neuronal processes involved in these phenomena.

The product transgenic animal is particularly useful for the study of reward pathways for substances of abuse, as the nucleus accumbens and, more generally, the mesolimbic dopamine system are recognized as playing a major role in the rewarding properties of substances of abuse (Pharmacol. Biochem. Behavior 51: 835-842, 1995, incorporated herein by reference in its entirety).

The usefulness of this model is illustrated herein by a demonstration of the effect of 5-HT3 over-expression in the forebrain on voluntary alcohol consumption. According to Campbell, et al. (Pharmacol. Biochem. Behavior, ibid.) 5-HT3 receptors have a specific role in mediating dopamine release in the nucleus accumbens, and their data support the involvement of 5-HT3 receptors in enhanced dopamine release in this region, suggesting prima facie that the normal high voluntary alcohol consumption in the founder strain (B6SJL) of the present transgenic mice before genetic alteration according to the invention is at least in part due to a natural increase in ethanol-stimulated, 5-HT3 modulated, dopamine release in pleasure pathways of the forebrain (see also Belknap et al. Psychopharmacol. 112: 503-510, 1993). Most unexpectedly, however, over-expression of 5-HT3 receptors in these mice resulted in a marked decrease in ethanol consumption (see Examples).

The transgenic animals of the invention are accordingly useful inter alia for the development of pharmacotherapies for the treatment of substances of abuse which are positive modulators at 5-HT3 receptors; for the development of 5-HT3 receptor agonists or antagonists for clinical use or basic research; and as models for evaluating psychiatric conditions such as anxiety, aggression, or depression.

The exemplified transgenic mammal strains of the invention are very docile, as compared to the mammals prior to genetic altering (starter mammals), and are particularly suitable for the pet trade.

The invention is thus useful for imparting traits accompanying over-expression of 5-HT3 receptors into a line or breed of mammals by genetically engineering one or more mammals of the line according to the invention to obtain transgenic mammals carrying at least one 5-HT3 transgene in each of their genomes, which overexpress 5-HT3 receptors in the CNS, preferably in the forebrain. Transgenic product mammals are then selected for desired traits and inbred into the line by assortative mating on the basis of phenotypic similarities as known in the art to promote homozygosity at the 5-HT3 transgene locus.

Particularly contemplated for genetic intervention are lines or breeds of mammals that exhibit undesirable behavior such as aggressiveness or over-excitability, but that otherwise have highly desirable qualities, for example, exceptional show conformation, racing speed, herding or retrieving abilities, service work, or companionship. In such cases, the invention has particular veterinary or breeder use for imparting docility to the breed, while otherwise retaining the breed genotype.

4. Production of Transgenic Animals of the Invention

Specific methods of preparing the transgenic animals of the invention are described herein. However, numerous methods for preparing transgenic animals are now known in the art, and other methods will undoubtedly be developed. Any such methods which provide the 5-HT3 over-expressing transgenic animals described herein can be used in the practice of the invention. The microinjection technique described herein is particularly suitable for addition of transgenes to the genome for over-expression without accompanying removal of other genes. An overview of techniques useful for engineering a transgenic mammal according to the present invention is set forth in U.S. Pat. No. 5,766,879 to Gerald et al. (incorporated herein by reference), who describe in some detail various general techniques and principles for engineering transgenic mammal expressing DNA encoding mammalian 5-HT4 transgenic receptors.

The transgenic engineering processes described herein are generally believed to provide randomly altered genomes, and the transgenic products have not yet been genotyped. The transgenic mammals of the present invention are accordingly defined herein by phenotype, especially a characeristic high density expression of 5-HT3 receptors, typically at least 100 fold greater density, in the forebrain and undetectable amounts thereof in the cerebellum, according to the binding experiments described in the Examples. Densities up to 2200 fmol receptor protein per mg of frontal cortex protein have been obtained; densities from about 1500 fmol and higher, particularly 2000 fmol and higher, are characteristic. The transgenic products are further characterized by their capacity to pass genetic over-expression of 5-HT3 receptors to their progeny by normal breeding within selected strains of these animals. Strains described in the Examples (B6SJL, C57, DBA, and 129) have been identified for frozen embryo deposit with the American Type Culture Collection, Manassas, Va., USA, in conformance with the relevant provisions of the Budapest Treaty on the Deposits of Microorganisms for the Purpose of Patent Procedure, during the pendency of this or related applications.

5. Methods and Products Described in the Examples

The Examples describe the production of transgenic mice over-expressing 5-HT3 receptors. The mice develop normally through adulthood and can pass the transgene to offspring by normal sexual reproduction. The 5-HT3 receptors are over-expressed exclusively in the forebrain, with no increase in receptor or RNA being detected in cerebellum, liver, lung, heart, spleen, kidney, gut, testis, or skeletal muscle.

The KD values reported here (1.7 nM for transgenic mouse membranes and 2.4 nM for control mouse membranes) are comparable to those reported by other researchers for this ligand. In contrast, Bmax values for transgenic mice membranes (2.5 pmol/mg protein) are much higher than the values for control mice membranes (21 fmol/mg protein). The control membrane values are comparable to those reported by other researchers. Thus, cortical membranes from the transgenic mouse show approximately 100-fold increase in 5-HT3 receptor binding sites in homogenates of whole cortical membrane, with no appreciable change in affinity of the receptor.

5-HT3 receptor mRNA can be detected as early as embryonic day 10 in the rat and this receptor may play a role in secondary induction in fetal development, and early over-expression of 5-HT3 receptors could compromise mouse development. Because the CAM kinase II.alpha. protein is not highly expressed until after birth (Burgin et al., J. Neuroscience 10: 1788-1798, 1991), this promoter region ws used to limit the transgene expression until after birth, thereby limiting the interference of receptor over-expression during fetal development. Also, the transgenic 5-HT3 receptor distribution in the forebrain closely resembles that reported for CAM kinase II.alpha. protein. The CAM kinase II.alpha. promotor region thus appears to have limited expression of the transgene to the forebrain where CAM kinase II.alpha. is normally produced. Therefore, this promoter region is successful in limiting appreciable expression until after birth. Thus, it appears that this genetic manipulation has not disrupted normal mouse development.

The transgenic 5-HT3 receptor appears to function the same as the endogenous receptor. Data to support this came from three types of experiments. First, ligand binding experiments in which an increased Bmax with no change in KD showed that more receptors were produced and that the receptor binding site binds ligand as does the endogenous receptor. The binding experiments were accomplished in washed membrane preparations which suggest that the receptor was incorporated into the plasma membrane as was the endogenous receptor. Second, electrical recordings of transgenic mice cells versus B6SJL/F1 controls showed an enhanced inward cationic current when stimulated with a 5-HT3 agonist, demonstrating that the transgenic receptors formed functional ion channels in the membrane. Third, 5-HT3 over-expression resulted in a behavioral change in the mice. The decrease in ethanol consumption by the transgenic mice reflects an alteration in the rewarding effects of ethanol for these mice due to 5-HT3 receptor over-expression.

Gene insertion to produce the exemplified transgenic animals resulted in eight lines of mice being initially produced. Alcohol (ethanol) drinking behavior was tested in the four lines that showed over-expression of 5-HT3 receptors by ligand binding (see below). The amount of ethanol consumption was related to the degree of 5-HT3 over-expression (data not shown). The line that showed the least increase in receptor expression showed no statistical difference in ethanol consumption over control mice. However, the other three lines showed significant decrease in ethanol consumption over controls with the amount of receptor over-expression being inversely related to the amount of ethanol consumed. Thus, the transgene insertion locus alone is not responsible for the effect on ethanol consumption. The ethanol free choice test was used as one of the criteria for deciding which line would represent the best model in which to study the models and to breed forward onto the different inbred backgrounds (e.g., C57, DBA, 129 mice).

Claim 1 of 17 Claims

What is claimed is:

1. A method for determining the effect of a substance on 5-HT3 receptors comprising:

administering the substance to a first transgenic mouse whose genome comprises its endogenous 5-HT3 receptor and a 5-HT3 transgene encoding the 5-HT3 receptor, wherein said transgene comprises a murine 5-HT3 transgene operably linked to a rat CAM kinase II.alpha. promoter wherein the mouse is characterized by at least one characteristic selected from the group consisting of decreased craving for alcohol and decreased aggressiveness relative to non-transgenic mice of the parental strain; and

determining a parameter associated with 5-HT3 receptor expression of the first transgenic mouse administered the substance.




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