Serotonin 5-HT6 receptor knockout mouse

The present invention features transgenic mice models for gene function, wherein the transgenic mice are characterized by having altered serotonin 5-HT6 receptor gene function. The transgenic mice may be either homozygous or heterozygous for a disruption in the endogenous 5-HT6 gene. Transgenic mice homozygous for a disruption in the endogenous 5-HT6 gene display a phenotype of increased anxiety behavior including diminished investigation of foreign objects and an elevation in stretched attend postures.

INTRODUCTION 
Background 
The biogenic amine serotonin (5-hydroxytryptamine; 5-HT) is a brain 
neurotransmitter that has been strongly implicated in the pathophysiology 
and treatment of a wide variety of neuropsychiatric disorders. It exerts 
its effects through a diverse family of serotonin receptor subtypes. Of 
the 14 different mammalian serotonin receptors to have been cloned, all 
but one are members of the G-protein coupled receptor superfamily. Several 
of these, including the serotonin 5-HT6 receptor, stimulate adenylyl 
cyclase via G coupling. 5-HT6 has a high affinity for several 
therapeutically important antidepressant, antianxiety, hallucinogenic and 
antipsychotic drugs, particularly the atypical antipsychotics such as 
clozapine. The relevance of the 5-HT6 receptor to psychotherapeutics is 
indicated both through its unique anatomical distribution and 
pharmacological properties. 
Messenger RNA encoding the 5-HT6 receptor has been localized by in situ 
hybridization histochemistry to brain regions that regulate emotional 
responses, cognition and motor function. This distribution pattern is 
generally matched by the localization of the 5-HT6 receptor protein 
identified by specific antibodies. This distribution is consistent with 
the binding of psychoactive drugs. Therefore, 5-HT6 receptors are 
implicated in the etiology of major psychiatric disorders and in the 
actions of psychiatric drugs. 
Antipsychotic medications are the treatment of choice for schizoaffective 
disorders. Evidence to date suggests that all of the antipsychotic drugs 
(except clozapine) are similarly effective in treating psychoses, with the 
differences being in milligram potency and side effects. Sometimes 
patients view the side-effects of the antipsychotic drugs as being worse 
than their original psychosis. These include acute dystonic reactions, 
akathisia, Parkinsonism, in which akinesia is a key feature, and tardive 
dyskinesia. Clozapine (Clozaril) has been proven to be more effective than 
all other antipsychotic drugs in treating certain disorders. It has 
reduced movement side-effects, but other serious side-effects limit its 
use. In rare cases coma and death may result from the drug treatment. 
The importance of psychoactive drugs in present treatment of mental 
illness, and the presence of serious and undesirable side-effects with 
their use, makes the development of improved drugs of great interest. 
Animal models useful in screening assays provide a benefit by determining 
candidate agents that have improved specificity of action. 
Relevant Literature 
An overview of 5-hydroxytryptamine receptor subtypes in vertebrates and 
invertebrates may be found in Saudou and Hen (1994) Neurochem Int 
25:503-532. The cloning, characterization, and chromosomal localization of 
a human 5-HT6 serotonin receptor is described in Kohen et al. (1996) J 
Neurochem 66:47-56.Aug. 8, 1997. The human 5-HT6 polypeptide sequence 
diverged significantly from that published for the rat receptor. It was 
determined that the published rat sequence contained a frame shift error. 
The gene for the receptor maps to the human chromosome region 1p35-p36. 
Molecular cloning of the rat receptor is described in Ruat et al. (1993) 
Biochem Biophys Res Commun 193:268-276. 
Boess et al. (1997) Neuropharmacology 36:713-720, stably expressed the rat 
5-HT6 receptor in HEK293 cells and compared the affinity of a range of 
compounds in competition binding experiments. The observed binding was 
LSD&gt;omega-N-methyl-5-HT=bufotenine=5-methoxytryptamine&gt;5-HT&gt;2-methyl-5-HT= 
5-benzyloxytryptamine=tryptamine&gt;5-carboxamidotryptamine&gt;&gt;5-HTQ. Receptor 
antagnosists include methiothepin, clozapine, mianserin and ritanserin. 
The binding of typical and atypical antipsychotic agents to 5-HT6 and 
5-HT7 receptors is discussed in Roth et al. (1994) J Pharmacol Exp Ther 
268:1403-1410. Clozapine and several related atypical antipsychotic agents 
(rilapine, olanzepine, tiospirone, fluperlapine, clorotepine and zotepine) 
had high affinities for 5-HT6 receptor. Several dopamine-selective 
antipsychotic agents (raclopride, rimcazole and penfluridol) had 
essentially no affinity for either the 5-HT6 or 5-HT7 receptors. 
Ward and Dorsa (1996) J Comp Neurol 370:405-414 describe the striatal 
distribution of the mRNAs of the serotonin2A (5-HT2A), serotonin2C 
(5-HT2C), and serotonin6 (5-HT6) receptors in relation to enkephalin, 
substance P, and dynorphin expressing output neurons. Pierce et al. (1996) 
Neuroscience 70:553-559 assayed for the presence of messenger RNA for rat 
serotonin receptor subtypes in peripheral sensory and sympathetic ganglia 
was detected using the method of polymerase chain reaction. The presence 
of messenger RNA for 5-HT6 receptor was found within superior cervical 
ganglia, but not lumbar sympathetic ganglia. 
The effects of administering 5-HT6 antisense oligonucleotides to the rat 
brain are described in Sleight et al. (1996) Behav Brain Res 73:245-248; 
and Bourson et al. (1995) J Pharmacol Exp Ther 274:173-180. A behavioral 
syndrome of yawning, stretching and chewing was observed in treated rats. 
SUMMARY OF THE INVENTION 
Non-human transgenic animal models are provided for serotonin 5-HT6 
receptor (5-HT6) function, where the transgenic animal is characterized by 
having an altered 5-HT6 gene. Alterations to the gene include deletions or 
other loss of function mutations, introduction of an exogenous gene having 
a nucleotide sequence with targeted or random mutations, introduction of 
an exogenous gene from another species, or a combination thereof. The 
transgenic animals may be either homozygous or heterozygous for the 
alteration. The animals and cells derived therefrom are useful for 
screening biologically active agents that may modulate 5-HT6 receptor 
function. The screening methods are of particular use for determining the 
specificity and action of antianxiety, antidepressant or antipsychotic 
drugs, particularly serotoninergic drugs, or drugs that may interact with 
serotonin receptors. The animals are useful as a model to investigate the 
role of serotonin 5-HT6 receptors in normal brain function.

DATABASE REFERENCES FOR GENETIC SEQUENCES 
The human serotonin 5-HT6 receptor gene has the Genbank accession number 
L41146. The rat serotonin 5-HT6 receptor gene has the Genbank accession 
number L41147. 
DESCRIPTION OF THE SPECIFIC EMBODIMENTS 
Non-human transgenic animal models useful for screening psychoactive drugs 
are provided. The animals have genetically altered serotonin 5-HT6 
receptors. Alterations to the gene include deletion or other loss of 
function mutations, introduction of an exogenous gene having a nucleotide 
sequence with targeted or random mutations, introduction of an exogenous 
gene from another species, or a combination thereof. The transgenic 
animals may be either homozygous or heterozygous for the genetic 
alteration. 
The subject animals are useful for testing the specificity of drugs 
developed as 5-HT6 receptor-selective agonists and antagonists. Completely 
selective compounds will not interact with other receptors, and thus will 
be inert in 5-HT6 knockout mice. In addition, these animals provide a 
useful model for the behavioral testing of psychoactive compounds. 
Antipsychotic, antidepressant and antianxiety drugs can be assayed by 
behavioral testing appropriate to each class of drugs. The animals are 
also used to determine the extent to which 5-HT6 receptors contribute to 
the efficacy of drugs in current use. 
TRANSGENIC ANIMALS 
The term "transgene" is used herein to describe genetic material that has 
been or is about to be artificially inserted into the genome of a 
mammalian cell, particularly a mammalian cell of a living animal. The 
transgene is used to transform a cell, meaning that a permanent or 
transient genetic change, preferably a permanent genetic change, is 
induced in a cell following incorporation of exogenous DNA. A permanent 
genetic change is generally achieved by introduction of the DNA into the 
genome of the cell. Vectors for stable integration include plasmids, 
retroviruses and other animal viruses, YACs, and the like. Of interest are 
transgenic mammals, e.g. cows, pigs, goats, horses, etc., and particularly 
rodents, e.g. rats, mice, etc. 
Transgenic animals comprise an exogenous nucleic acid sequence present as 
an extrachromosomal element or stably integrated in all or a portion of 
its cells, especially in germ cells. Unless otherwise indicated, it will 
be assumed that a transgenic animal comprises stable changes to the 
germline sequence. During the initial construction of the animal, 
"chimeras" or "chimeric animals" are generated, in which only a subset of 
cells have the altered genome. Chimeras are primarily used for breeding 
purposes in order to generate the desired transgenic animal. Animals 
having a heterozygous alteration are generated by breeding of chimeras. 
Male and female heterozygotes are typically bred to generate homozygous 
animals. 
Transgenic animals fall into two groups, colloquially termed "knockouts" 
and "knockins". In the present invention, knockouts have a partial or 
complete loss of function in one or both alleles of the endogenous 5-HT6 
gene. Knockins have an introduced transgene with altered genetic sequence 
and function from the endogenous gene. The two may be combined, such that 
the naturally occurring gene is disabled, and an altered form introduced. 
In a knockout, preferably the target gene expression is undetectable or 
insignificant. A knock-out of a 5-HT6 gene means that function of the 
5-HT6 receptor has been substantially decreased so that expression is not 
detectable or only present at insignificant levels. This may be achieved 
by a variety of mechanisms, including introduction of a disruption of the 
coding sequence, e.g. insertion of one or more stop codons, insertion of a 
DNA fragment, etc., deletion of coding sequence, substitution of stop 
codons for coding sequence, etc. In some cases the exogenous transgene 
sequences are ultimately deleted from the genome, leaving a net change to 
the native sequence. Different approaches may be used to achieve the 
"knock-out". A chromosomal deletion of all or part of the native gene may 
be induced, including deletions of the non-coding regions, particularly 
the promoter region, 3' regulatory sequences, enhancers, or deletions of 
gene that activate expression of 5-HT6 genes. A functional knock-out may 
also be achieved by the introduction of an anti-sense construct that 
blocks expression of the native genes (for example, see Li and Cohen 
(1996) Cell 85:319-329). "Knock-outs" also include conditional knock-outs, 
for example where alteration of the target gene occurs upon exposure of 
the animal to a substance that promotes target gene alteration, 
introduction of an enzyme that promotes recombination at the target gene 
site (e.g. Cre in the Cre-lox system), or other method for directing the 
target gene alteration postnatally. 
A "knock-in" of a target gene means an alteration in a host cell genome 
that results in altered expression or function of the native 5-HT6 gene. 
Increased (including ectopic) or decreased expression may be achieved by 
introduction of an additional copy of the target gene, or by operatively 
inserting a regulatory sequence that provides for enhanced expression of 
an endogenous copy of the target gene. These changes may be constitutive 
or conditional, i.e. dependent on the presence of an activator or 
represser. 
The exogenous gene is usually either from a different species than the 
animal host, or is otherwise altered in its coding or non-coding sequence. 
The introduced gene may be a wild-type gene, naturally occurring 
polymorphism, or a genetically manipulated sequence, for example having 
deletions, substitutions or insertions in the coding or non-coding 
regions. The introduced sequence may encode a 5-HT6 polypeptide, or may 
utilize the 5-HT6 promoter operably linked to a reporter gene. Where the 
introduced gene is a coding sequence, it is usually operably linked to a 
promoter, which may be constitutive or inducible, and other regulatory 
sequences required for expression in the host animal. By "operably linked" 
is meant that a DNA sequence and a regulatory sequence(s) are connected in 
such a way as to permit gene expression when the appropriate molecules, 
e.g. transcriptional activator proteins, are bound to the regulatory 
sequence(s). 
Specific constructs of interest, but are not limited to, include anti-sense 
5-HT6, which will block native 5-HT6 expression, expression of dominant 
negative 5-HT6 mutations, and over-expression of a 5-HT6 gene. A 
detectable marker, such as lac Z may be introduced into the locus, where 
upregulation of expression will result in an easily detected change in 
phenotype. Constructs utilizing the 5-HT6 promoter region, in combination 
with a reporter gene or with the coding region are also of interest. 
A series of small deletions and/or substitutions may be made in the 5-HT6 
gene to determine the role of different exons in DNA binding, 
transcriptional regulation, etc. By providing expression of 5-HT6 protein 
in cells in which it is otherwise not normally produced, one can induce 
changes in cell behavior. 
DNA constructs for homologous recombination will comprise at least a 
portion of the 5-HT6 gene with the desired genetic modification, and will 
include regions of homology to the target locus. DNA constructs for random 
integration need not include regions of homology to mediate recombination. 
Conveniently, markers for positive and negative selection are included. 
Methods for generating cells having targeted gene modifications through 
homologous recombination are known in the art. For various techniques for 
transfecting mammalian cells, see Keown et al. (1990) Methods in 
Enzymology 185:527-537. 
For embryonic stem (ES) cells, an ES cell line may be employed, or 
embryonic cells may be obtained freshly from a host, e.g. mouse, rat, 
guinea pig, etc. Such cells are grown on an appropriate fibroblast-feeder 
layer or grown in the presence of appropriate growth factors, such as 
leukemia inhibiting factor (LIF). When ES cells have been transformed, 
they may be used to produce transgenic animals. After transformation, the 
cells are plated onto a feeder layer in an appropriate medium. Cells 
containing the construct may be detected by employing a selective medium. 
After sufficient time for colonies to grow, they are picked and analyzed 
for the occurrence of homologous recombination or integration of the 
construct. Those colonies that are positive may then be used for embryo 
manipulation and blastocyst injection. Blastocysts are obtained from 4 to 
6 week old superovulated females. The ES cells are trypsinized, and the 
modified cells are injected into the blastocoel of the blastocyst. After 
injection, the blastocysts are returned to each uterine horn of 
pseudopregnant females. Females are then allowed to go to term and the 
resulting litters screened for mutant cells having the construct. By 
providing for a different phenotype of the blastocyst and the ES cells, 
chimeric progeny can be readily detected. 
The chimeric animals are screened for the presence of the modified gene and 
males and females having the modification are mated to produce homozygous 
progeny. If the gene alterations cause lethality at some point in 
development, tissues or organs can be maintained as allogeneic or congenic 
grafts or transplants, or in in vitro culture. 
5-HT6 NUCLEIC ACID COMPOSITIONS 
The terms "serotonin 5-HT6 receptor gene" is used generically to designate 
serotonin 5-HT6 receptor genes, e.g. homologs from rat, human, mouse, 
guinea pig, etc., and their alternate forms. The gene is also intended to 
mean the open reading frame encoding specific polypeptides, introns, and 
adjacent 5' and 3' non-coding nucleotide sequences involved in the 
regulation of expression, up to about 1 kb beyond the coding region, but 
possibly further in either direction. The DNA sequences encoding 5-HT6 
receptor may be CDNA or genomic DNA or a fragment thereof. The gene may be 
introduced into an appropriate vector for extrachromosomal maintenance or 
for integration into the host. 
A genomic sequence of interest comprises the nucleic acid present between 
the initiation codon and the stop codon, as defined in the listed 
sequences, including all of the introns that are normally present in a 
native chromosome. It may further include the 3' and 5' untranslated 
regions found in the mature mRNA. It may further include specific 
transcriptional and translational regulatory sequences, such as promoters, 
enhancers, etc., including about 1 kb, but possibly more, of flanking 
genomic DNA at either the 5' or 3' end of the transcribed region. The 
genomic DNA may be isolated as a fragment of 100 kbp or smaller; and 
substantially free of flanking chromosomal sequence. 
The sequence of this 5' region, and further 5' upstream sequences and 3' 
downstream sequences, may be utilized for promoter elements, including 
enhancer binding sites, that provide for expression in tissues where 
serotonin 5-HT6 receptor is expressed. The tissue specific expression is 
useful for determining the pattern of expression, and for providing 
promoters that mimic the native pattern of expression. Naturally occurring 
polymorphisms in the promoter region are useful for determining natural 
variations in expression, particularly those that may be associated with 
disease. Alternatively, mutations may be introduced into the promoter 
region to determine the effect of altering expression in experimentally 
defined systems. Methods for the identification of specific DNA motifs 
involved in the binding of transcriptional factors are known in the art, 
e.g. sequence similarity to known binding motifs, gel retardation studies, 
etc. For examples, see Blackwell et al. (1995) Mol Med 1:194-205; Mortlock 
et al. (1996) Genome Res. 6:327-33; and Joulin and Richard-Foy (1995) Eur 
J Biochem 232:620-626. 
The regulatory sequences may be used to identify cis acting sequences 
required for transcriptional or translational regulation of 5-HT6 receptor 
expression, especially in different tissues or stages of development, and 
to identify cis acting sequences and trans acting factors that regulate or 
mediate expression. Such transcription or translational control regions 
may be operably linked to a 5-HT6 receptor gene in order to promote 
expression of wild type or altered 5-HT6 or other proteins of interest in 
cultured cells, or in embryonic, fetal or adult tissues, and for gene 
therapy. 
The nucleic acid compositions used in the subject invention may encode all 
or a part of the 5-HT6 receptor polypeptides as appropriate. Fragments may 
be obtained of the DNA sequence by chemically synthesizing 
oligonucleotides in accordance with conventional methods, by restriction 
enzyme digestion, by PCR amplification, etc. For the most part, DNA 
fragments will be of at least 15 nt, usually at least 18 nt, more usually 
at least about 50 nt. Such small DNA fragments are useful as primers for 
PCR, hybridization screening, etc. Larger DNA fragments, i.e. greater than 
100 nt are useful for production of the encoded polypeptide. For use in 
amplification reactions, such as PCR, a pair of primers will be used. 
Homologs of cloned serotonin 5-HT6 receptor are identified by various 
methods known in the art. Nucleic acids having sequence similarity are 
detected by hybridization under low stringency conditions, for example, at 
50.degree. C. and 10.times. SSC (0.9 M saline/0.09 M sodium citrate) and 
remain bound when subjected to washing at 55.degree. C. in 1.times. SSC. 
Sequence identity may be determined by hybridization under stringent 
conditions, for example, at 50.degree. C. or higher and 0.1.times. SSC (9 
mM saline/0.9 mM sodium citrate). By using probes, particularly labeled 
probes of DNA sequences, one can isolate homologous or related genes. The 
source of homologous genes may be any species, e.g. primate, rodents, 
canines, felines, bovines, ovines, equines, etc. 
The 5-HT6 receptor sequence, including flanking promoter regions and coding 
regions, may be mutated in various ways known in the art to generate 
targeted changes in promoter strength, sequence of the encoded protein, 
etc. The sequence changes may be substitutions, insertions or deletions. 
Deletions may include large changes, such as deletions of a domain or 
exon. Other modifications of interest include epitope tagging, e.g. with 
the FLAG system, HA, etc. For studies of subcellular localization, fusion 
proteins with green fluorescent proteins (GFP) may be used. Such mutated 
genes may be used to study structure-function relationships of 5-HT6 
receptor polypeptides, or to alter properties of the proteins that affect 
their function or regulation. 
Techniques for in vitro mutagenesis of cloned genes are known. Examples of 
protocols for scanning mutations may be found in Gustin et al., 1993 
Biotechniques 14:22; Barany, 1985 Gene 37:111-23; Colicelli et al., 1985 
Mol Gen Genet 199:537-9; and Prentki et al., 1984 Gene 29:303-13. Methods 
for site specific mutagenesis can be found in Sambrook et al., 1989 
Molecular Cloning: A Laboratory Manual, CSH Press, pp. 15.3-15.108; Weiner 
et al., 1993 Gene 126:35-41; Sayers et al., 1992 Biotechniques 13:592-6; 
Jones and Winistorfer, 1992 Biotechniques 12:528-30; Barton et al., 1990 
Nucleic Acids Res 18:7349-55; Marotti and Tomich, 1989 Gene Anal Tech 
6:67-70; and Zhu 1989 Anal Biochem 177:120-4. 
Drug Screening Assays 
Through use of the subject transgenic animals or cells derived therefrom, 
one can identify ligands or substrates that bind to, modulate, antagonize 
or agonize 5-HT6 receptors. Screening to determine drugs that lack effect 
on these receptors is also of interest. Areas of investigation are the 
development of psychoactive therapies, e.g. antipsychotic, antianxiety, 
antidepressant, etc. treatments. Of particular interest are screening 
assays for agents that have a low toxicity for human cells. 
A wide variety of assays may be used for this purpose, including in vivo 
behavioral studies, determination of the localization of drugs after 
administration, labeled in vitro protein-protein binding assays, 
protein-DNA binding assays, electrophoretic mobility shift assays, 
immunoassays for protein binding, and the like. Depending on the 
particular assay, whole animals may be used, or cell derived therefrom. 
Cells may be freshly isolated from an animal, or may be immortalized in 
culture. Cell of particular interest include neural and brain tissue. 
The term "agent" as used herein describes any molecule, e.g. protein or 
pharmaceutical, with the capability of affecting the biological action of 
serotonin 5-HT6 receptor. Generally a plurality of assay mixtures are run 
in parallel with different agent concentrations to obtain a differential 
response to the various concentrations. Typically, one of these 
concentrations serves as a negative control, i.e. at zero concentration or 
below the level of detection. 
Candidate agents encompass numerous chemical classes, though typically they 
are organic molecules, preferably small organic compounds having a 
molecular weight of more than 50 and less than about 2,500 daltons. 
Candidate agents comprise functional groups necessary for structural 
interaction with proteins, particularly hydrogen bonding, and typically 
include at least an amine, carbonyl, hydroxyl or carboxyl group, 
preferably at least two of the functional chemical groups. The candidate 
agents often comprise cyclical carbon or heterocyclic structures and/or 
aromatic or polyaromatic structures substituted with one or more of the 
above functional groups. Candidate agents are also found among 
biomolecules including, but not limited to: peptides, saccharides, fatty 
acids, steroids, purines, pyrimidines, derivatives, structural analogs or 
combinations thereof. 
Candidate agents are obtained from a wide variety of sources including 
libraries of synthetic or natural compounds. For example, numerous means 
are available for random and directed synthesis of a wide variety of 
organic compounds and biomolecules, including expression of randomized 
oligonucleotides and oligopeptides. Alternatively, libraries of natural 
compounds in the form of bacterial, fungal, plant and animal extracts are 
available or readily produced. Additionally, natural or synthetically 
produced libraries and compounds are readily modified through conventional 
chemical, physical and biochemical means, and may be used to produce 
combinatorial libraries. Known pharmacological agents may be subjected to 
directed or random chemical modifications, such as acylation, alkylation, 
esterification, amidification, etc. to produce structural analogs. 
Screening may be directed to known pharmacologically active compounds and 
chemical analogs thereof. Known antipsychotic drugs include the following. 
Drugs with a significant serotoninergic effect are of particular interest. 
______________________________________ 
Name Class Serotonergic Effect 
______________________________________ 
Chlorpromazine 
Phenothiazine, Aliphatic 
++++ 
Fluphenazine Phenothiazine, Piperazine + 
Perphenazine Phenothiazine, Piperazine ++++ 
Trifluoperazine Phenothiazine, Piperazine +++ 
Thioridazine Phenothiazine, Piperidine ++++ 
Mesoridazine Phenothiazine, Piperidine ++++ 
Haloperidol Butyrophenone + 
Clozapine Dibenzodiazepine Atypical ++++ 
Agents 
Loxapine Dibenzodiazepine ++++ 
Molindone Dihydroindolone + 
Thiothixene Thioxanthene + 
Risperidone Benzisoxazole 0 
______________________________________ 
Known antidepressant drugs include the following. Of particular interest 
are serotonin-norepinephrine reuptake inhibitors, serotonin-receptor 
modulators and serotonin selective reuptake inhibitors. 
______________________________________ 
Generic Name 
Trade Names Class 
______________________________________ 
amitriptyline 
Elavil, Endep TCA 
amoxapine Asendin Heterocyclic 
bupropion Wellbutrin 
Other 
clomipramine Anafranil TCA 
desipramine* Norpramin, Pertofrane TCA 
doxepin Adapin, Sinequan TCA 
fluoxetine Prozac SSRI 
fluvoxamine Luvox SSRI 
imipramine SK-Pramine, Tofranil, Janimine TCA 
isocarboxazid Marplan MAOI 
maprotiline Ludiomil Heterocyclic 
nefazodone Serzone 
SRM 
nortriptyline* Aventyl, Pamelor TCA 
paroxetine* Paxil SSRI 
phenelzine Nardil MAOI 
protriptyline Vivactil TCA 
sertraline* Zoloft SSRI 
tranylcypromine Parnate MAOI 
trazodone* Desyrel SRM 
trimipramine Surmontil 
TCA 
venlafaxine Effexor 
______________________________________ 
SNRI 
MAOI = monoamine oxidase inhibitor 
SNRI = Serotoninnorepinephrine reuptake inhibitor 
SRM = serotoninreceptor modulator 
SSRI = serotonin selective reuptake inhibitor 
TCA = tricyclic antidepressant 
Known antianxiety drugs include the following. 
______________________________________ 
Generic Name Brand Name 
______________________________________ 
alprazolam Xanax 
chlordiazepoxide Librium 
clorazepate Tranxene 
diazepam Valium 
lorazepam Ativan 
oxazepam Serax 
prazepam Centrax 
triazolam Halcion 
buspirone Buspar 
chloral hydrate Noctec, others 
______________________________________ 
Where the screening assay is a binding assay, one or more of the molecules 
may be joined to a label, where the label can directly or indirectly 
provide a detectable signal. Various labels include radioisotopes, 
fluorescers, chemiluminescers, enzymes, specific binding molecules, 
particles, e.g. magnetic particles, and the like. Specific binding 
molecules include pairs, such as biotin and streptavidin, digoxin and 
antidigoxin etc. For the specific binding members, the complementary 
member would normally be labeled with a molecule that provides for 
detection, in accordance with known procedures. 
A variety of other reagents may be included in the screening assay. These 
include reagents like salts, neutral proteins, e.g. albumin, detergents, 
etc that are used to facilitate optimal protein-protein binding and/or 
reduce non-specific or background interactions. Reagents that improve the 
efficiency of the assay, such as protease inhibitors, nuclease inhibitors, 
anti-microbial agents, etc. may be used. The mixture of components are 
added in any order that provides for the requisite binding. Incubations 
are performed at any suitable temperature, typically between 4 and 
40.degree. C. Incubation periods are selected for optimum activity, but 
may also be optimized to facilitate rapid high-throughput screening. 
Typically between 0.1 and 1 hours will be sufficient. 
Antibodies specific for 5-HT6 receptor polymorphisms may be used in 
screening immunoassays, particularly to detect the binding of substrates 
to 5-HT6 receptors, or to confirm the absence or presence of a 5-HT6 
receptor in a cell or sample. Samples, as used herein, include biological 
fluids such as tracheal lavage, blood, cerebrospinal fluid, tears, saliva, 
lymph, dialysis fluid and the like; organ or tissue culture derived 
fluids; and fluids extracted from physiological tissues. Also included in 
the term are derivatives and fractions of such fluids. The number of cells 
in a sample will generally be at least about 10.sup.3, usually at least 
10.sup.4 more usually at least about 10.sup.5. The cells may be 
dissociated, in the case of solid tissues, or tissue sections may be 
analyzed. Alternatively a lysate of the cells may be prepared. 
For example, detection may utilize staining of cells or histological 
sections, performed in accordance with conventional methods. The 
antibodies of interest are added to the cell sample, and incubated for a 
period of time sufficient to allow binding to the epitope, usually at 
least about 10 minutes. The antibody may be labeled with radioisotopes, 
enzymes, fluorescers, chemiluminescers, or other labels for direct 
detection. Alternatively, a second stage antibody or reagent is used to 
amplify the signal. Such reagents are well known in the art. For example, 
the primary antibody may be conjugated to biotin, with horseradish 
peroxidase-conjugated avidin added as a second stage reagent. Final 
detection uses a substrate that undergoes a color change in the presence 
of the peroxidase. The absence or presence of antibody binding may be 
determined by various methods, including flow cytometry of dissociated 
cells, microscopy, radiography, scintillation counting, etc. 
An alternative method for diagnosis depends on the in vitro detection of 
binding between antibodies and 5-HT6 receptor in a lysate. Measuring the 
concentration of binding in a sample or fraction thereof may be 
accomplished by a variety of specific assays. A conventional sandwich type 
assay may be used. For example, a sandwich assay may first attach specific 
antibodies to an insoluble surface or support. The particular manner of 
binding is not crucial so long as it is compatible with the reagents and 
overall methods of the invention. They may be bound to the plates 
covalently or non-covalently, preferably non-covalently. 
The insoluble supports may be any compositions to which polypeptides can be 
bound, which is readily separated from soluble material, and which is 
otherwise compatible with the overall method. The surface of such supports 
may be solid or porous and of any convenient shape. Examples of suitable 
insoluble supports to which the receptor is bound include beads, e.g. 
magnetic beads, membranes and microtiter plates. These are typically made 
of glass, plastic (e.g. polystyrene), polysaccharides, nylon or 
nitrocellulose. Microtiter plates are especially convenient because a 
large number of assays can be carried out simultaneously, using small 
amounts of reagents and samples. 
A number of assays are known in the art for determining the effect of a 
drug on animal behavior. Behavioral abnormalities in animal models are 
useful for testing the effect, interactions, and specificity of a 
candidate biologically active agent. Some examples are provided, although 
it will be understood by one of skill in the art that many other assays 
may also be used. The subject animals may be used by themselves, or in 
combination with control animals. Control animals may have the native 
5-HT6 receptor intact, or may be a combination transgenic, where the 
normal 5-HT6 gene is disrupted, and has been replaced with an exogenous 
gene, e.g. the human gene, a mutated mouse gene, etc. 
Prepulse inhibition (PPI) of an acoustic startle response is impaired in 
schizophrenics. PPI can be studied in rodents, where a PPI deficit is 
introduced by drug treatment or conditions of social isolation. (See Varty 
and Higgins (1995) Psychopharmacology 122:15-26). The ability of 
neuroleptic drugs to restore a PPI disruption may be studied by inducing a 
deficit, and administering a candidate drug to determine if function is 
restored. 
The ability of a candidate serotonin receptor antagonist to inhibit 
psychostimulant, e.g. amphetamine, cocaine, etc.--induced increases in 
extracellular levels of 5-HT or dopamine may be determined by in vivo 
microdialysis (see Kuroki et al. (1996) Brain Res. 743:357-361). 
Memory and learning deficits are studied by various means, e.g. 3 runway 
panel for working memory impairment (attempts to pass through two 
incorrect panels of the three panel-gates at four choice points) (Ohno et 
al. (1997) Pharmacol Biochem Behav 57:257-261). Candidate drugs are 
assessed for their ability to restore or inhibit memory function. Anxiety 
testing in combination with drug therapy may be assessed with a variety of 
known tests, including elevated plus maze, light/dark box, fear 
potentiated startle responses, and the like. 
Ethanol, cocaine, etc. can produce a significant increase in locomotor 
activity (LMA) in mice (Le, et al. (1997) Pharmacol Biochem Behav 
57:325-332). Candidate drugs may be assessed for their ability to suppress 
this increase in locomoter activity. Candidate drugs may be assessed for 
the effect on self-administration by lever-pressing, etc. of drugs, e.g. 
cocaine, in rodent models. 
The therapeutic agents may be administered in a variety of ways, orally, 
topically, parenterally e.g. subcutaneously, intraperitoneally, by viral 
infection, intravascularly, etc. Inhaled treatments are of particular 
interest. Depending upon the manner of introduction, the compounds may be 
formulated in a variety of ways. The concentration of therapeutically 
active compound in the formulation may vary from about 0.1-100 wt. %. 
The pharmaceutical compositions can be prepared in various forms, such as 
granules, tablets, pills, suppositories, capsules, suspensions, salves, 
lotions and the like. Pharmaceutical grade organic or inorganic carriers 
and/or diluents suitable for oral and topical use can be used to make up 
compositions containing the therapeutically-active compounds. Diluents 
known to the art include aqueous media, vegetable and animal oils and 
fats. Stabilizing agents, wetting and emulsifying agents, salts for 
varying the osmotic pressure or buffers for securing an adequate pH value, 
and skin penetration enhancers can be used as auxiliary agents. 
It is to be understood that this invention is not limited to the particular 
methodology, protocols, cell lines, animal species or genera, constructs, 
and reagents described, as such may, of course, vary. It is also to be 
understood that the terminology used herein is for the purpose of 
describing particular embodiments only, and is not intended to limit the 
scope of the present invention which will be limited only by the appended 
claims. 
It must be noted that as used herein and in the appended claims, the 
singular forms "a", "and", and "the" include plural referents unless the 
context clearly dictates otherwise. Thus, for example, reference to "a 
construct" includes a plurality of such constructs and reference to "the 
serotonin 5-HT6 receptor-encoding nucleic acid" includes reference to one 
or more serotonin 5-HT6 receptor-encoding nucleic acids and equivalents 
thereof known to those skilled in the art, and so forth. 
Unless defined otherwise, all technical and scientific terms used herein 
have the same meaning as commonly understood to one of ordinary skill in 
the art to which this invention belongs. Although any methods, devices and 
materials similar or equivalent to those described herein can be used in 
the practice or testing of the invention, the preferred methods, devices 
and materials are now described. 
All publications mentioned herein are incorporated herein by reference for 
the purpose of describing and disclosing, for example, the cell lines, 
constructs, and methodologies that are described in the publications which 
might be used in connection with the presently described invention. The 
publications discussed above and throughout the text are provided solely 
for their disclosure prior to the filing date of the present application. 
Nothing herein is to be construed as an admission that the inventors are 
not entitled to antedate such disclosure by virtue of prior invention. 
The following examples are put forth so as to provide those of ordinary 
skill in the art with a complete disclosure and description of how to make 
and use the subject invention, and are not intended to limit the scope of 
what is regarded as the invention. Efforts have been made to ensure 
accuracy with respect to the numbers used (e.g. amounts, temperature, 
concentrations, etc.) but some experimental errors and deviations should 
be allowed for. Unless otherwise indicated, parts are parts by weight, 
molecular weight is average molecular weight, temperature is in degrees 
centigrade; and pressure is at or near atmospheric. 
Experimental 
Generation of 5-HT6 Receptor Mutant Mice 
FIG. 1 depicts restriction maps of a portion of the mouse 5-HT6 receptor 
gene, the pHT6-TKN targeting construct, and the genetic alteration 
expected in a gene targeting event. The gene fragment was obtained from a 
strain 129 mouse P1 genomic library that was screened using PCR primers 
corresponding to the coding region of the rat 5-HT6 receptor. 
Alternatively, such a fragment is obtained by probing a strain 129 genomic 
phage library with a rat 5-HT6 receptor cDNA probe using publically 
available sequences. 
The pHT6-TKN construct was made by deleting a 300 bp Pst I (P) restriction 
fragment from the coding region of the 5-HT6 receptor gene. The deleted 
region contained sequences encoding a region of the 5-HT6 receptor protein 
from the third transmembrane domain to the fifth intracellular loop. In 
its place, a neomycin resistance cassette (Neo) was inserted. The mutated 
fragment was subcloned into the pGK-TK plasmid, which contains the HSV 
thymidine kinase (TK) gene driven by the PGK promoter in a Bluescript 
vector. 
Embryonic stem (ES) cells were electroporated with linearized pHT6-TKN 
vector, and then plated on fibroblast feeder layers. ES cells were treated 
with geneticin (neomycin analog) to select for cells that had incorporated 
the targeting construct. ES cell clones surviving drug selection were 
screened for homologous recombination events by Southern blot analysis. In 
the process of constructing the pHT6-TKN vector, an exogenous Xba I 
restriction site (X) was introduced at the 3' end of the Neo cassette. 
Therefore, ES cell genomic DNA was digested with Xba I and DNA blots were 
hybridized with a probe corresponding to a 5-HT6 receptor gene region 
located 5' to the integration site of the construct. With this strategy, 
the native allele is indicated by an 11 Kb band and a mutant allele 
produced by homologous recombination is indicated by a 7.5 Kb band (see 
FIG. 1). 
ES cell clones with targeted 5-HT6 receptor gene mutations were then 
isolated, expanded, and used to generate chimeric mice. Chimeras were 
generated by microinjecting targeted ES cells into the cavities of 
blastocysts derived from the C57BL/6 mouse strain. Injected embryos were 
transferred into uteri of surrogate mothers, and chimeric mice were born. 
The chimeras were then bred with C57BL/6 mice, and germ line transmission 
of the targeted mutation was verified by Southern blots of DNA from the 
tails of the offspring. Crosses were then performed between male and 
female mice that were heterozygous for the targeted mutation. These 
crosses resulted in the production of offspring with the expected 
Mendelian ratios of sexes and genotypes. The absence of intact 5-HT6 
receptor mRNA in homozygous mutant mice was verified by Northern blot 
analysis of brain RNA, using a probe corresponding to the deleted protein 
coding region. Homozygous mutant mice appear healthy. 
Behavioral studies: 5HT6 receptor mutant and wild type littermate mice were 
acclimated to an open field enclosure (50.times.50.times.38 cm) in 3 daily 
10 minute sessions. On the 4th day, a novel object (a small wooden block 
(approx 18.times.18.times.36 mm)) was placed in the center of the field 
and the following behaviors observed: activity in the vicinity of the 
object, stretched attend postures (SAPs), and sniffs of the object. 
A trend was present, with the knock-out animals displaying reduced sniffing 
of the object and elevated SAPs. The diminished investigation of the 
object, and elevation in SAPs, a "risk assessment" behavior, are both 
consistent with an elevation in trait anxiety in the mutant mice. For 
methodology, see Blanchard & Blanchard (1969) J. Comp. Physiol. Psychol. 
67:370-375, "Crouching as an index of fear"; and Blanchard et al. (1990) 
Psychopharmacol. 101:511-518, "Diazepam changes risk assessment in an 
anxiety/defense test battery". 
In another model of rodent anxiety, the elevated zero maze, knock-out 
animals displayed trends toward decreased exploration of the open arms and 
a significant elevation of stretched attend postures. These measures are 
also consistent with elevated anxiety (Shepherd et al. (1994) Psychopharm. 
116:56-64, "Behavioural and pharmacological characterization of the 
elevated "zero-maze" as an animal model of anxiety"). These results, 
together with the known distribution of 5HT6 receptor expression in brain 
regions associated with anxiety, implicate 5HT6 receptors in the 
regulation of anxiety state.