U.S. patent application number 10/432241 was filed with the patent office on 2004-05-13 for transgenic or recombinant non-human mammals and their uses in screening psychoactive medicines.
Invention is credited to Andrieux, Annie, Bosc, Christophe, Denarier, Eric, Job, Didier, Vernet, Muriel.
Application Number | 20040093625 10/432241 |
Document ID | / |
Family ID | 8856873 |
Filed Date | 2004-05-13 |
United States Patent
Application |
20040093625 |
Kind Code |
A1 |
Andrieux, Annie ; et
al. |
May 13, 2004 |
Transgenic or recombinant non-human mammals and their uses in
screening psychoactive medicines
Abstract
The invention concerns transgenic or recombinant non-human
mammals, wherein the expression of the gene coding for a
microtubule associated protein (MAP) is modified (STOP gene)
(inactivation or overexpression) and their uses in screening
medicines useful in schizophrenia and schizo-affective disorders,
with anxious, paranoiac or depressive component.
Inventors: |
Andrieux, Annie; (Grenoble,
FR) ; Job, Didier; (Grenoble, FR) ; Denarier,
Eric; (Grenoble, FR) ; Bosc, Christophe;
(Grenoble, FR) ; Vernet, Muriel; (Grenoble,
FR) |
Correspondence
Address: |
Lowe Hauptman Gostein
Gilman & Berner
Suite 310
1700 Diagonal Road
Alexandria
VA
22314
US
|
Family ID: |
8856873 |
Appl. No.: |
10/432241 |
Filed: |
November 17, 2003 |
PCT Filed: |
November 23, 2001 |
PCT NO: |
PCT/FR01/03701 |
Current U.S.
Class: |
800/14 |
Current CPC
Class: |
C07K 14/4702 20130101;
A61P 25/00 20180101; A01K 67/0276 20130101; A01K 2267/03 20130101;
A01K 2267/0393 20130101; C12N 15/8509 20130101; A01K 2227/105
20130101; A01K 2267/0356 20130101; A01K 2217/075 20130101 |
Class at
Publication: |
800/014 |
International
Class: |
A01K 067/027 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 24, 2000 |
FR |
00/15240 |
Claims
1. A recombinant non-human mammal carrying at least one modified
allele of the gene encoding a STOP protein.
2. The recombinant non-human mammal as claimed in claim 1,
characterized in that said modified allele is obtained from a
construct comprising a nucleic acid sequence derived from the same
mammal or from a mammal which is different from that into which
said construct is inserted, which construct is selected from the
group consisting of constructs containing a sequence encoding a
STOP protein which is antisense, constructs comprising the region
of the STOP promoter in combination with a reporter gene or with
the STOP coding region, constructs comprising at least one portion
of the STOP gene including at least one modification, and
constructs comprising at least one portion of the STOP gene
functionally linked to a promoter and, optionally, to other
regulatory sequences required for expression in the host
animal.
3. The recombinant non-human mammal as claimed in claim 1 or claim
2, characterized in that said constructs are selected from the
following constructs: constructs which include a fragment of the
genomic sequence encoding a STOP protein included between the
initiation codon and the STOP codon, constructs which do not
comprise the region between positions 4118 and 5131 of the genomic
sequence encoding a STOP protein, and constructs comprising 4.1 kb
of the STOP gene, the gene encoding .beta.-galactosidase, placed
under the control of the endogenous STOP promoter, a neomycin
resistance gene under the control of the PGK promoter, 1.57 kb of
sequence of the STOP gene and, finally, the thymidine kinase gene
under the control of PGK promoter.
4. The use of a recombinant non-human mammal as claimed in any one
of claims 1 to 3, for selecting or screening psychoactive
products.
5. A nucleic acid molecule, characterized in that it comprises the
sequence of a modified allele of the gene encoding a STOP protein
as defined in any one of claims 1 to 3, excluding the sequences
corresponding to the GENBANK accession numbers AJ002556 and
Y16032.
6. A probe or a primer for screening recombinant ES clones or
recombinant animals, characterized in that it is selected from the
group consisting of the sequences SEQ ID Nos. 3 to 6.
7. A method for screening and selecting molecules of use in the
treatment of schizophrenia and schizoaffective disorders with a
component of anxiety, paranoia or depression, which method is
characterized in that it comprises at least the following steps:
bringing at least one substance to be screened into contact, in
vitro, with a biological sample consisting of an extract of cells
or organ slices, preferably of neurons or brains, obtained from at
least one recombinant non-human mammal carrying at least one
modified allele of the gene encoding a STOP protein as claimed in
any one of claims 1 to 3, measuring the action of said substance to
be screened on said cells or organ slices, and comparing the values
obtained with those of the cells or of the organ slices of a
biological sample obtained from a non-human mammal of the same
type, carrying two wild-type alleles of the gene encoding a STOP
protein.
8. The method as claimed in claim 7, characterized in that said
measurement is carried out using a protein-protein binding
assay.
9. The method as claimed in claim 7, characterized in that said
measurement is carried out by detection of the variation in
strength of an electrical signal.
10. A vector for homologous recombination of a gene encoding a STOP
protein, characterized in that it comprises a nucleotide sequence
of a modified STOP gene encoding an inactivated STOP protein,
preferably truncated in at least one of the exons, in particular
exon 1.
11. The use of the substances selected using the screening method
as claimed in any one of claims 7 to 9, for preparing a medicinal
product of use in the treatment of schizophrenia and
schizoaffective disorders with a component of anxiety, paranoia or
depression.
12. The use of a primer or of a probe as claimed in claim 6, for
screening recombinant ES clones or recombinant animals.
Description
[0001] The present invention relates to transgenic or recombinant
non-human mammals in which the expression of the gene encoding a
microtubule-associated protein (MAP) is modified (inactivation or
overexpression) and to their uses in screening medicinal products
of use in anxiety, schizophrenia and schizoaffective disorders with
a component of anxiety, paranoia or depression.
[0002] The microtubules of mammalian cells are subjected to
regulation: during interphase, they organize the intracellular
space and are responsible for the intracellular transport of
organelles; during mitosis, they reorganize to form the mitotic
spindle responsible for distributing the chromosomes between the
two daughter cells.
[0003] Microtubules, assembled in vitro using solutions of purified
tubulin, are labile and rapidly depolymerized by exposure to
cold.
[0004] Similar behavior is observed in vivo, but, in this case, it
is regulated by the cellular metabolism. For example, microtubule
depolymerization is promoted by the phosphoprotein stathmin, which
binds and sequesters the tubulin dimers, whereas microtubule
stabilization is mediated by microtubule-associated proteins
(MAPs), which associate with the polymers.
[0005] Neurons contain massive amounts of microtubules and said
microtubules are virtually completely stable in response to cold. A
calmodulin-regulated protein capable of completely stabilizing
microtubules (i.e. able to suppress their dynamic activity and to
make them resistant to cold) has been isolated from preparations of
stable neuronal microtubules; this is the STOP (for Stable Tubulin
Only Polypeptide) protein. The molecular nature of this protein has
remained enigmatic for a long time. A decisive step was taken in
1996 when the cDNA encoding this STOP protein was cloned
(Christophe Bose et al., PNAS., 1996, 93, 2125-2130).
[0006] The STOP protein, which can reversibly block the dynamics of
microtubules by completely abolishing the sensitivity of neuronal
microtubules to cold and to depolymerizing drugs, comprises two
notable repeat domains: a central domain composed of five virtually
complete repeats of a 46 amino acid motif, and a carboxy-terminal
domain made up of twenty-eight incomplete repeats of an 11 amino
acid motif. These two repeat domains are separated by a sequence
containing an abundance of lysine and arginine residues (KR domain)
and by a linker sequence. The N-terminal domain of the STOP protein
contains proline-rich sequences which constitute potential sites
for binding with SH3 domains (src homology domain 3).
[0007] The exon structure of the mouse STOP gene has been
elucidated (Eric Denarier et al., BBRC., 1998, 243, 791-796). This
structure corresponds to the domain structure of the protein: exon
1 encodes the N-terminal domain including the central repeat
domain, exon 2 encodes the linker sequence, exon 3 encodes the KR
domain and exon 4 encodes the carboxy-terminal repeat region.
[0008] The distribution and the role of the STOP protein in neurons
have recently been characterized (Laurent Guillaud et al., Cell
Biol., 1998, 142, 1, 167-179). The distribution of the protein has
been studied at the ultrastructural level in embryonic neuronal
cells, DRG (Dorsal Root Ganglia) cells, which can differentiate in
vitro, and has revealed the existence of isoforms of this protein.
A major isoform (E-STOP protein) has been characterized: this
isoform appears earlier in development than the standard STOP
protein or N-STOP protein, and is the major form in the embryonic
brain. The cDNA of E-STOP has the sequence corresponding to Genbank
accession number AJ002556. The E-STOP protein differs from the
N-STOP protein by the deletion of the carboxy-terminal repeat
sequences, encoded by exon 4; it is therefore a splice variant of
the STOP protein. Another isoform, the F-STOP protein has been
observed in mice fibroblasts (3T3 cells). This protein, which has
an apparent molecular mass of 45 kDa, is much smaller than the
N-STOP protein (115 kDa) or the E-STOP protein (88 kDa). The
cloning and the sequencing of the corresponding cDNA (Genbank
Y16032 and Eric Denarier et al., PNAS., 1998, 95, 6055-6060) have
shown that the sequences encoded by exons 3 and 4 (KR and
carboxy-terminal repeats) are absent in the F-STOP protein. In
addition, the major part of the N-terminal domain of the N-STOP
protein, located upstream of the central repeats and encoded by
exon 1, is absent in the F-STOP protein. The F-STOP protein
therefore comprises the sequences encoded by exon 2 and a part of
those corresponding to exon 1, including the central repeats.
Despite multiple deletions, the F-STOP protein has the same basic
functional properties as the N-STOP protein: the F-STOP protein
binds to calmodulin and has the ability to induce microtubule
stabilization with respect to cold, in vitro and in vivo. Unlike
the N-STOP protein, which appears to be almost permanently
associated with the microtubules, the F-STOP protein remains in the
soluble phase in cells in interphase and only associates with the
microtubules during exposure to cold. Apparently, regulatory
mechanisms prevent the F-STOP protein interacting with the
microtubular cytoskeleton in interphase, thus allowing rapid
microtubular dynamics, and this regulation is inhibited as soon as
the cells are exposed to low temperatures. In mitotic cells, the
F-STOP protein is associated with the microtubule spindles, at
physiological temperature. Thus, a single and same class of
proteins, the STOP proteins, is responsible for microtubule
stabilization in several different cellular types.
[0009] The N, E and F forms of the STOP protein are not the only
isoforms which exist; specifically, the STOP proteins are present
in many tissues, in particular in the lungs, which contain a
specific isoform. Similarly, the F-STOP form appears to be present
in varied tissues. On the other hand, the N-STOP and E-STOP
proteins are, it appears, strictly neuronal (C. Bose et al., Cell
Struct. Function, 1999, 24, 393-399).
[0010] It would seem that microtubule stability is important for
the development and maintenance of the morphology and function of
neurons (Laurent Guillaud et al., mentioned above). Thus, it has
been shown that inhibition of the STOP proteins in vitro by
injecting specific blocking antibodies suppresses microtubule
stability with respect to cold in neuronal or non-neuronal cells
(Eric Denarier, PNAS, 1998, mentioned above). It has also been
shown that inhibition of the STOP proteins in vitro in neurons
impairs neuronal differentiation (Laurent Guillaud et al.,
mentioned above).
[0011] The inventors have found, unexpectedly, that knocking out
the various isoforms of the STOP protein makes it possible to
obtain animals, and in particular mice, which are of particular use
for screening psychoactive medicinal products.
[0012] Consequently, a subject of the present invention is a
recombinant non-human mammal carrying at least one modified allele
of the gene encoding a STOP protein.
[0013] The term "modified STOP gene" is intended to mean both an
altered gene (knock-in animals) and an inhibited or truncated
totally or partially inactivated gene (knock-out animals).
[0014] Advantageously, said recombinant or transgenic animals can
be obtained by homologous recombination in an embryonic stem
cell:
[0015] either by insertion of at least one STOP codon or of an
antisense sequence,
[0016] or by deletion of part or all of the native gene (coding
region or noncoding regions, promoter, 3' regulatory sequences,
activators),
[0017] or by sequence substitution.
[0018] More precisely, a construct in accordance with the invention
is advantageously selected from the group consisting of:
[0019] constructs containing a sequence encoding a STOP protein
which is antisense, which will block expression of the native STOP
sequence,
[0020] constructs comprising the region of the STOP promoter
(positions 1-3400 of FIG. 2) in combination with a reporter gene or
with the STOP coding region. Markers for positive or negative
selection can advantageously be included, such as lacZ, the
regulation and expression of which will lead to the detection of a
change in the phenotype. A preferred reporter gene is the GFP
(green fluorescent protein) gene,
[0021] constructs comprising at least one portion of the STOP gene
(coding region or noncoding regions, promoter, 3' regulatory
sequences, activators) including the desired modification(s)
(deletions, mutations, etc.); advantageously, the DNA constructs
used for a targeted integration should include a region exhibiting
homology with the target sequence (STOP gene), so as to induce a
recombination,
[0022] constructs comprising at least one portion of the STOP gene,
functionally linked to a promoter, which may be constitutive or
inducible, and to other regulatory sequences required for
expression in the host animal. The term "functionally linked" is
intended to mean that a DNA sequence and a regulatory sequence are
combined in such a way that they allow expression of the gene when
the appropriate molecules, for example the transcription-activating
proteins, are bound to the regulatory sequences.
[0023] For the purpose of the present invention, the term "STOP
gene" is intended to mean the STOP genes obtained from any mammal,
such as rat, mouse, bovine or human, or from chicken or from
blowfish, and also the various mutated forms of said STOP gene; it
also includes the various open reading frames, the exons, the
introns, the 3' and 5' noncoding regions involved in regulating the
expression of this gene, up to approximately 4 kb on either side of
the coding region, the promoter and the activators.
[0024] Preferably, the constructs are selected from the following
constructs:
[0025] constructs which include a fragment of the genomic sequence
encoding a STOP protein, included between the initiation codon and
the STOP codon (C. Bose et al., E. Denarier et al., L. Guillaud et
al., mentioned above), including in particular all the introns
normally present in the native chromosome. It may include the 3'
and 5' untranslated regions found in the mature mRNA. It may also
include transcription or translation regulatory sequences
(promoter, activator, etc.), including approximately 4 kb, of the
3' or 5' flanking genomic regions;
[0026] constructs which do not comprise the region between
positions 4118 and 5131 of the genomic sequence encoding a STOP
protein;
[0027] constructs comprising 4.1 kb of the STOP gene (corresponding
to positions 1-4118 of FIG. 2), the gene encoding
.beta.-galactosidase, placed under the control of the endogenous
STOP promoter, a neomycin resistance gene under the control of the
PGK promoter, 1.57 kb of sequence of the STOP gene (corresponding
to positions 5131-6701 of the sequence of FIG. 2) and, finally, the
thymidine kinase gene under the control of the PGK promoter.
[0028] In accordance with the invention, the transgenic animals
obtained constitute two groups, the knock-out animals and the
knock-in animals.
[0029] In the context of the present invention:
[0030] the knock-out animals have a partial or complete loss of
function in one or both alleles of the gene encoding an endogenous
STOP protein; such a modified gene no longer induces expression of
the corresponding STOP protein. The knock-out animals according to
the invention also include conditional knock-out animals: (i)
modification of the gene encoding a STOP protein, which only
becomes involved after exposure of the animal to a substance which
induces the modification of said gene, (ii) introduction of an
enzyme which induces recombination at a site of the gene encoding a
STOP protein (Cre in the Cre-lox system, for example) or (iii)
another method which induces a modification of the gene encoding a
STOP protein after birth;
[0031] the knock-in animals exhibit a transgene which alters the
endogenous gene encoding a STOP protein. A knock-in animal
corresponds to an alteration in the host's cells which leads to a
modified expression or a modified function of the native STOP gene.
An increased or decreased expression can thus be obtained by
introduction of an additional copy of the STOP gene or by
functional insertion of a regulatory sequence which produces a
significantly increased expression of an endogenous copy of the
STOP gene. These changes can be either constitutive or conditional,
as a function of the presence of an activator or of a repressor.
The exogenous gene is either obtained from a species which is
different from that of the host animal, or is modified in its
coding or noncoding sequence. The gene introduced may be a
wild-type gene or a manipulated sequence, for example exhibiting
deletions, substitutions or insertions in the coding or noncoding
regions.
[0032] The two methods may be combined: first, the gene of origin
is knocked-out, then, secondly, a modified form of said gene is
introduced into said animal.
[0033] The recombinant or transgenic animals thus obtained comprise
an exogenous nucleic acid sequence, either present in the form of
an extrachromosomal element, or stably integrated into all or some
of the cells of said animal, more particularly the germinal
cells.
[0034] Surprisingly, the homozygous mice containing the two alleles
of the inactivated STOP gene (knock-out or STOP KO (-/-) mouse),
obtained by crossing heterozygous animals, are viable and exhibit
no anatomical modification of the brain; on the other hand, they
exhibit deficiencies in synaptic plasticity, associated with
multiple major behavioral disorders comprising a complete lack of
mothering, profound anxiety, an inability to recognize objects, and
abnormal social interactions.
[0035] Advantageously, these multiple behavioral disorders can be
improved by prolonged administration of neuroleptics.
[0036] Consequently, the mice in which the STOP gene has been
inactivated (STOP KO (-/-) mice) constitute a particularly useful
model for studying and treating diseases involving a synaptic
defect which are sensitive to neuroleptics, in particular
schizophrenia and schizoaffective disorders with a component of
anxiety, paranoia or depression.
[0037] A subject of the present invention is also the use of said
recombinant non-human mammal carrying at least one allele of the
gene encoding a modified STOP protein, for selecting or screening
psychoactive products.
[0038] A subject of the present invention is also nucleic acid
molecules comprising the sequence of a modified allele of the gene
encoding a STOP protein as defined above (in particular the
sequences of inactivated STOP genes), excluding the sequences
corresponding to GENBANK accession numbers AJ002556 and Y16032.
[0039] The STOP sequences according to the invention are in
particular obtained by mutation, in various ways known in
themselves, so as to generate the desired targeted modifications:
substitutions, insertions or deletions in a domain or an exon,
which lead to the expression of an inactivated STOP protein or to
the absence of expression of STOP protein. The deletions may
include considerable modifications: deletion of a domain or of an
exon (exon 1 in particular).
[0040] The fragments of said sequences are advantageously obtained
by chemical synthesis of oligonucleotides, by enzyme digestion or
by PCR amplification for example.
[0041] Said fragments comprise at least 15 nucleotides, preferably
approximately 18 nucleotides, and preferably at least 50
nucleotides.
[0042] Such fragments are of use as PCR primers or for screening by
hybridization, of the recombinant ES clones or of the recombinant
animals.
[0043] Said primers or probes for screening recombinant ES clones
or recombinant animals are characterized in that they are selected
from the group consisting of fragments of a STOP gene comprising at
least 15 nucleotides, preferably approximately 18 nucleotides, and
preferably at least 50 nucleotides. Such primers or probes make it
possible to screen cells or animals comprising one of the modified
sequences as defined above.
[0044] Preferably, the following primers are used for the
screening:
[0045] oligonucleotide A4080: positions 4067-4095 of FIG. 2 (SEQ ID
No. 3);
[0046] oligonucleotide 770: positions 4488-4515 of FIG. 2 (SEQ ID
No. 4);
[0047] oligonucleotide AS2: positions 6680-6701 of FIG. 2 (SEQ ID
No. 5).
[0048] Larger fragments (more than 100 nucleotides) are of use for
producing the STOP proteins.
[0049] Sequences homologous to the cloned STOP sequences are
identified by various methods known to those skilled in the
art.
[0050] The nucleic acid sequence similarity is detected by
hybridization under low stringency conditions, for example at
50.degree. C. and 10.times.SSC (0.9 M saline buffer and 0.09 M
sodium citrate).
[0051] Said sequences remain associated when they are subjected to
washing at 55.degree. C. in a 1.times.SSC buffer.
[0052] The identity of the sequences can be determined by
hybridization under stringent conditions, for example at 50.degree.
C. at most and 0.1.times.SSC (9 mM of saline buffer/0.9 mM of
sodium citrate).
[0053] A subject of the present invention is also probes for
detecting and for screening the genomic DNA by hybridization of the
recombinant ES clones or of the recombinant animals, characterized
in that they consist of a fragment of the same STOP gene, located
outside (upstream or downstream) the sequence of the STOP gene
derived from the recombination vector used (region of homologous
recombination).
[0054] Advantageously, said probe corresponds to positions 700-1881
of FIG. 3 (SEQ ID No. 6).
[0055] A subject of the present invention is also a method for
screening and selecting molecules of use in the treatment of
schizophrenia and schizoaffective disorders with a component of
anxiety, paranoia or depression, characterized in that it comprises
at least the following steps:
[0056] bringing at least one substance to be screened into contact,
in vitro, with a biological sample consisting of an extract of
cells or organ slices, preferably of neurons or brains, obtained
from at least one recombinant non-human mammal carrying at least
one modified allele of the gene encoding a STOP protein,
[0057] measuring the action of said substance to be screened on
said cells or organ slices, and
[0058] comparing the values obtained with those of the cells or of
the organ slices of a biological sample obtained from a non-human
mammal of the same type, carrying two wild-type alleles of the gene
encoding a STOP protein.
[0059] The substances tested are in particular obtained from
libraries of substances (natural or synthetic).
[0060] According to an advantageous embodiment of said method, said
measurement is carried out using a protein-protein binding assay;
in such a case, one or more of the molecules used can be labeled
with a label; said label can provide a signal which is detectable
either directly or indirectly.
[0061] Among the labels which can be used, mention may, for
example, be made of radioisotopes, fluorescent or chemiluminescent
molecules, enzymes, specific binding molecules, particles such as
magnetic particles, etc.
[0062] Specific binding molecules include pairs of molecules such
as biotin and streptavidin, digoxin and antidigoxin, etc.
[0063] For the specific binding members, the complementary member
will be labeled with a molecule suitable for detection, in
accordance with known methods.
[0064] Many other reagents can be used in such a screening assay;
it includes, for example, salts, neutral proteins such as albumin,
detergents, etc., which are used to facilitate optimal
protein-protein binding and/or to reduce the nonspecific
interactions or the background noise interactions.
[0065] Reagents which improve the effectiveness of the assay, such
as protease inhibitors, nuclease inhibitors or antimicrobial
agents, can also be used.
[0066] The mixture of components is added in any order, so as to
allow the desired binding.
[0067] The incubations are carried out at a suitable temperature,
usually between 4.degree. C. and 40.degree. C.
[0068] The incubation periods can vary; they are conventionally
between 0.1 and 1 h and are optimized within this time range, in
particular so as to facilitate rapid screening.
[0069] Antibodies specific for STOP protein polymorphisms can be
used in screening immunoassays, more particularly to detect the
binding of the substrate or of STOP protein or to confirm the
absence or presence of a STOP protein in a cell or a sample, such
as a biological sample.
[0070] According to another advantageous embodiment of said method,
said measurement is carried out by detection of the variation in
intensity of an electrical signal; specifically, it is possible to
observe, as regards the nerve cells of the recombinant animals
according to the invention, an alteration in the organization of
the synapse (positioning and transport of neuroreceptors) in the
recombinant animals according to the invention.
[0071] The present invention also relates to a method for screening
and selecting molecules of use in the treatment of anxiety,
schizophrenia and schizoaffective disorders with a component of
anxiety, paranoia or depression, which method is characterized in
that it comprises:
[0072] administering at least one substance to be screened to at
least one recombinant non-human mammal carrying at least one
modified allele of the gene encoding a STOP protein; and
[0073] studying the behavior of said mammal compared to a series of
control animals and/or determining the location of the medicinal
products after their administration.
[0074] Such animals can advantageously be used as models for
screening psychoactive molecules exhibiting low toxicity in
humans.
[0075] A subject of the present invention is also a vector for
homologous recombination of a gene encoding a STOP protein,
characterized in that it comprises a nucleotide sequence of a
modified STOP gene encoding an inactivated STOP protein, preferably
truncated in at least one of the exons, in particular exon 1.
[0076] A subject of the present invention is also a method for
producing recombinant non-human mammals carrying at least one
allele of the gene encoding an inactivated STOP protein,
characterized in that:
[0077] an allele of the gene encoding a STOP protein is
truncated;
[0078] said modified sequence is introduced into a segment of the
genomic DNA of a non-human mammal of the same type, associated with
a suitable label, so as to obtain a labeled sequence M containing
said modified allele;
[0079] said sequence M is integrated, in vitro, into the stem cells
of germinal lines of embryos of a non-human mammal by transfection
and the cells which have said allele through homologous
recombination events are selected; then
[0080] said selected stem cells are reinjected into an embryo which
is reimplanted into a non-human mammal of the same type, in order
to obtain chimeric animals; and
[0081] in the F1 generation, recombined heterozygous non-human
mammals are obtained and, in the F2 generation, recombined STOP -/-
homozygous non-human mammals, recognizable by the presence of the
label, and "wild-type" (+/+) mice are obtained.
[0082] A subject of the present invention is also the use of the
substances selected using the screening method as defined above,
for preparing a medicinal product of use in the treatment of
schizophrenia and schizoaffective disorders with a component of
anxiety, paranoia or depression.
[0083] Besides the preceding arrangements, the invention also
comprises other arrangements, which will emerge from the following
description, which refers to examples of implementation of the
method which is the subject of the present invention, with
reference to the attached drawings in which:
[0084] FIG. 1 illustrates the genomic organization of the STOP gene
at exon 1 and the establishment of knock-out mice for the STOP gene
[STOP KO (-/-) mice] by alteration of exon 1. A: restriction map of
a fragment of the STOP gene (wild-type allele) used for producing a
genomic homology fragment, structure of the homologous
recombination vector or screening vector ptSTOP, and predicted
structure of mutant allele. EV: EcoRV; EI: EcoRI; TK: thymidine
kinase; pgk: phosphoglycerate kinase; neo: neomycin; NTR:
Nucleotide Translation Region. B: Southern blotting profiles for
the STOP gene in wild-type mice (+/+; 8 kb) and heterozygous mice
(+/-; 5.3 kb);
[0085] FIG. 2 represents the sequence of the STOP gene at exon 1
(positions 3333-5150) (7.2 kb genomic clone); the fragments used to
establish the STOP KO (-/-) mice are as follows: 5' homologous
sequence: 4.118 kb, positions 1-4118; 3' homologous sequence: 1.57
kb, positions 5131 to 6701;
[0086] FIG. 3: FIG. 3A represents the genomic sequence of the STOP
gene, located in the 5' position relative to the sequence of FIG.
2, and FIG. 3B represents the probe used for the screening, which
is an EcoR V-EcoR I fragment (positions 701-1881 on FIG. 3A);
[0087] FIG. 4 illustrates the Western blotting analysis, using the
polyclonal antibody 23C (Laurent Guillaud et al., mentioned above),
of the expression of the STOP (E-STOP and N-STOP) proteins in the
brain of the STOP KO (-/-) or wild-type mice. This figure shows the
absence of STOP proteins in the brain of the STOP KO (-/-) mice
compared with the wild-type mice; the presence of an equivalent
amount of proteins in the two types of sample loaded onto the gel
is demonstrated by the signal obtained with an anti-.beta.-tubulin
(.beta.-tub) antibody;
[0088] FIGS. 5 to 8 illustrate the alteration in the long-term
depression (LTD) and in the long-term potentiation (LTP) in the
STOP KO (-/-) mice;
[0089] FIG. 5 illustrates the basal synaptic response of the
Schaffer collaterals: the "in/out"-type curves represent the slope
of the curve of the excitatory post-synaptic potential (EPSP) as a
function of the excitability of the fibers of the Schaffer
collaterals, based on a section from wild-type mice (a) or from
STOP KO (-/-) mice (b). Summary of the results obtained on six
wild-type mice and six STOP KO (-/-) mice (c). The slopes of the
curves are not significantly different, indicating normal basal
synaptic transmission in the STOP KO (-/-) mice;
[0090] FIG. 6 illustrates the results of the experiments for
long-term potentiation (LTP) at the synapses of the Schaffer
collaterals and of the pyramidal cells of the CA1 region of the
hippocampus:
[0091] FIG. 6(a) shows that a high frequency stimulation (tetanic
stimulation induced by 4 stimuli of 100 Hz for 1 s, applied at
intervals of 10 to 20 s) leads to a long-term increase in the slope
of the EPSP curve, in a section from a wild-type mouse,
[0092] FIG. 6(b) shows that, on the other hand, an identical
stimulation leads to only a small increase in the slope of the EPSP
curve in a section from a STOP KO (-/-) mouse, and
[0093] FIG. 6(c) represents a summary of the results obtained in
the wild-type mice and the STOP KO (-/-) mice. The initial values
of the slopes of the EPSP curves were standardized in each
experiment, using the mean value of the curve obtained during the
control period (-10 to 0 min). The results, expressed as
mean.+-.s.e.m., correspond to the values obtained on 13 and 9
sections derived, respectively, from seven wild-type mice and six
STOP -/- mice. These results show a significant deficiency in
long-term potentiation in the STOP KO (-/-) mice (p=0.0007,
measurement recorded after 30 to 40 minutes);
[0094] FIG. 7 illustrates the results of the experiments of
long-term depression (LTD) at the synapses of the Schaffer
collaterals and of the CA1 pyramidal cells:
[0095] FIG. 7(a) shows that low frequency stimulation (LFS, 1 Hz
for 15 min) induces a long-term decrease in the slope of the EPSP
curve in the sections from wild-type mice,
[0096] FIG. 7(b) shows that, on the other hand, the low frequency
stimulation does not induce a long-term decrease in the slope of
the EPSP curve in the sections from KO -/- mice, and
[0097] FIG. 7(c) represents a summary of the LTD experiments in the
STOP KO (-/-) mice and in the wild-type mice. The results,
expressed as mean.+-.s.e.m., correspond to the values obtained on
15 and 9 sections of, respectively, nine wild-type mice and six
STOP KO (-/-) mice. These results show a significant alteration in
long-term depression (LTD) in the STOP KO (-/-) mice (p=0.01,
results recorded after 40-45 minutes);
[0098] FIG. 8A illustrates the NMDA/AMPA ratio at the synapses of
the Schaffer collaterals and of the CA1 pyramidal cells
corresponding to the ratio of the values of the EPSP curves for the
NMDA (N-methyl-D-aspartate) receptor and for the AMPA
(alpha-amino-3hydroxy-5-- methyl-4-isoxazole propionic acid)
receptor on 14 and 9 sections of, respectively, six wild-type mice
and six STOP KO (-/-) mice. The slopes for the NMDA receptors and
the AMPA receptors were measured for a stimulus strength
corresponding to twice the threshold value. No significant
difference was observed between the wild-type mice and the STOP KO
(-/-) mice;
[0099] FIG. 8B illustrates the depolarization during a tetanic
stimulation of the Schaffer collaterals: the graph represents the
summary of the results of quantifying the depolarization during a
tetanic stimulation. The depolarization is calculated 300 ms after
the start of the first stimulus of 100 Hz. The experiments were
carried out on 11 sections from wild-type mice and 8 sections from
STOP KO (-/-) mice originating, respectively, from seven wild-type
mice and six STOP KO (-/-) mice. The results are not significantly
different in the wild-type mice and the STOP KO (-/-) mice;
[0100] FIG. 9 illustrates the alteration in synaptic plasticity in
the short term in the STOP KO (-/-) mice:
[0101] FIG. 9A illustrates the results of the experiments of
post-tetanic potentiation of the synaptic transmission of the
Schaffer collaterals. A high frequency stimulation in the presence
of the NMDA receptor antagonist D-APV (50-100 .mu.M) induces a
transient increase in the EPSP slope. The results were obtained
using 6 and 10 sections originating, respectively, from four
wild-type mice and five STOP KO (-/-) mice. The results show an
alteration in the post-tetanic potentiation in the STOP KO (-/-)
mice (p=0.04, measurements carried out from 0 to 30 s after tetanic
stimulation),
[0102] FIG. 9B illustrates the results of the experiments of paired
pulse facilitation (PPF) of the synaptic transmission of the
Schaffer collaterals. The results obtained correspond to 7 and 12
sections originating, respectively, from four wild-type mice and
five STOP KO (-/-) mice. The paired pulse facilitation is not
significantly modified in the STOP -/- mice, and
[0103] FIG. 9C illustrates the experiments of hippocampal mossy
fiber frequency facilitation. The results were obtained using 10
and 12 sections obtained from 7 and 8 sections originating,
respectively, from six wild-type mice and seven STOP KO (-/-) mice.
In the wild-type mice, repeated stimulation of the synapses of the
mossy fiber using stimulation frequencies of between 0.033 and 1 Hz
caused a reversible 3-fold increase in the amplitude of the
response of the mossy fiber. The facilitation is significantly
altered in the STOP KO (-/-) mice (p=0.03, values recorded at a
stimulation frequency of 1 Hz);
[0104] FIG. 10 illustrates the disorders of maternal behavior of
the STOP KO (-/-) mice:
[0105] FIG. 10a: the survival of the newborns, derived from
primiparous mothers, carrying the wild-type or mutated (STOP -/-)
STOP allele is analyzed on the second day after birth;
[0106] FIGS. 10b and 10c: the manifestation of a maternal behavior
is analyzed in the STOP KO (-/-) young primiparous females and
young males, compared with the wild-type mice. The results are
expressed in the form of mean.+-.s.e.m.; n=9 for the wild-type and
STOP KO (-/-) female mice and n=10 for the wild-type and STOP KO
(-/-) male mice;
[0107] FIG. 11 illustrates the behavioral disorders in the STOP KO
(-/-) mice; the activities of the mice (sleeping, eating, grooming,
walking and remaining immobile while awake) were recorded on video
for a period of 3 hours, n=11 for the wild-type mice (wt for
wild-type) and the STOP KO (-/-) mice:
[0108] FIG. 11a: time given to each activity. Each box corresponds
to a different activity, as indicated in the left-hand panel. The
STOP KO (-/-) mice spend more time walking and remaining mobile
than the wild-type mice, to the detriment of the time spent
sleeping or eating;
[0109] FIG. 11b: number of changes in activity. Compared to the
wild-type mice, the STOP KO (-/-) mice show a higher number of
activity changes, with a higher number of walking and resting
phases;
[0110] FIG. 11c: percentage of phases of grooming followed by a
sleeping phase (GS) out of total number of sleeping phases (S)
expressed as mean.+-.s.e.m. The percentages are calculated for each
mouse before calculating the mean. The G-S sequence which is
typical in the wild-type mice is frequently interrupted in the STOP
KO (-/-) mice;
[0111] FIG. 12 illustrates the state of anxiety of the STOP KO
(-/-) mice, evaluated by the light/dark test. FIG. 12a: time spent
in the lit box, and FIG. 12b: the number of passages between the
two boxes are respectively recorded over a period of 5 minutes,
from the first time the animals enter the box in darkness. The
wild-type mice (+/+) are used as controls. The values correspond to
mean value.+-.standard error of the mean (s.e.m). The differences
between the KO mice and the control mice are indicated with a risk
of p<0.01 (**):
[0112] FIG. 13 illustrates the short-term memory disorders of the
STOP KO (-/-) mice, evaluated by the object recognition test. The
wild-type mice (+/+) are used as controls. The results are
expressed by the recognition index (RI) : RI values significantly
greater than 50% correspond to a positive recognition test. The
values correspond to the mean value.+-.standard error of the mean
(s.e.m);
[0113] FIG. 14 illustrates the social behavior of the STOP KO (-/-)
mice; the wild-type mice (+/+) are used as controls: FIG. 14a:
evaluation of the time spent by a male in social investigation with
respect to an intruder [n=11 for the wild-type mice and n=13 for
the STOP KO (-/-) mice]; FIGS. 14b and 14c: inter-male aggression;
the aggression tests are performed for two consecutive days [n=11
for the wild-type mice and n=10 for the STOP KO (-/-) mice]; the
number of attacks and the time spent fighting (mean.+-.standard
error of the mean (s.e.m)) are recorded on the second day; *:
p<0.05, **: p<0.01, Mann-Whitney U test;
[0114] FIG. 15 illustrates the effect of neuroleptics on the
maternal behavior of the STOP KO (-/-) mice:
[0115] FIG. 15A: reinstallation in the nest of the newborns derived
from wild-type (wt) and STOP KO (-/-) post-partum females. The
reinstallation of the young mice to the nest was tested during the
first day post-partum, in the mice treated with neuroleptics
(mixture of haloperidol and chlorpromazine) or an anxiolytic
(diazepam) or in untreated mice. The mice received a dose of 0.5
mg/kg/day from 6-8 days before birth until the day of birth. The
females were placed in the presence of 3 newborns and the
reinstallation in the nest was recorded for each female. The mean
of the values obtained is given for each genotype (mean.+-.s.e.m.,
n=6 for each group of wild-type and STOP KO (-/-) mice. *
p<0.05, ** p<0.02, *** p<0.01, nonparametric Mann and
Whitney U test;
[0116] FIG. 15B: survival of the newborns among the wild-type mice
and the STOP KO (-/-) mice. The survival of the newborns is
analyzed among the STOP KO (-/-) mice subjected to various
treatments. The newborns are considered to be survivors when they
are raised until weaning. No survival of the newborns was observed
among the untreated STOP KO (-/-) mice (n=20) or the STOP KO (-/-)
mice treated in the short term (FIG. 15A). On the other hand,
survival of the newborns is observed in four of the seven STOP KO
(-/-) mice treated in the long term (4 months) with neuroleptics.
Survival of the newborns is observed in all the wild-type mice
(n=7) given the same long-term treatment with neuroleptics. *
p<0.05, ** p<0.02, *** p<0.01, Fisher exact test.
EXAMPLE 1
Establishment of Knock-out (KO) Mice in which the STOP Gene is
Inactivated: STOP KO (-/-) Mice
[0117] 1 - Materials and Methods
[0118] 1-1 Construction of the Genomic Homology Fragment and of the
Homologous Recombination Vector (Screening Vector)
[0119] The genomic DNA fragments used to construct the homologous
recombination vector are derived from a genomic DNA library from
mice of the strain 129, cloned into the P1 phage, and screened by
hybridization with a cDNA of the STOP gene or a cDNA probe for said
gene (Eric Denarier et al., BBRC, 1998, mentioned above).
[0120] The genomic homology fragment of the STOP gene is
constructed from the 7.2 kb clone, the sequence of which is given
in FIG. 2, according to the following steps: a 1012 pb fragment,
containing the repeat sequences of the coding region of the STOP
gene, which extends from positions 4118 to 5131 of the sequence
given in FIG. 2, was deleted and replaced with an expression
cassette containing the neomycin (neo) resistance gene under the
control of the PGK promoter and the .beta.-galactosidase (lacZ)
gene under the transcriptional control of the endogenous STOP
promoter. In addition, an EcoRV site was introduced in a 5'
position of the lacZ gene.
[0121] The homologous recombination vector (ptSTOP) is obtained by
cloning the homology fragment of the STOP gene described above into
the vector PGK-TK. The vector pGK-TK derives from the vector pPNT
constructed by Tybulewicz et al. (Cell, 1991, 65, 1153-1163) by
insertion of the herpes simplex virus (HSV) thymidine kinase gene
under the control of the phosphoglycerate kinase (PGK)
promoter.
[0122] 1-2 Homologous Recombination in ES Cells and Genotyping
[0123] The vector ptSTOP is linearized with the Notl enzyme and
electroporated into ES cells (ES-R1, A. Naguy et al., PNAS, 1993,
90, 8428-8428) or into ES-AT1 cells isolated from 3.5-day
blastocysts derived from F1 mice (129 Sv Pas.times.129 Sv Pas).
Next, the electroporated ES cells are seeded onto a layer of
neomycin-resistant fibroblasts pretreated with mitomycin, and
cultured in DMEM medium rich in glucose (INVITROGEN) containing 15%
of fetal calf serum and 1000 IU/ml of leukemia inhibiting factor
(Esgron, CHEMICON). Two days after transfection, geneticin (G418,
INVITROGEN) is added to the culture medium, at the final
concentration of 250 .mu.g/ml. Gancyclovir (SYNTEX) is added from
the fourth to the eighth day after transfection. The recombinant ES
cell clones are removed 10 days after transfection and amplified
before being frozen or analyzed. The genotype of the clones
resistant to G418 and to gancyclovir is verified by Southern
blotting analysis of the genomic DNA digested with EcoRV and
hybridized with a probe specific for the STOP gene, located in the
5' region flanking the homologous recombination region (see FIG. 3)
and corresponding to positions 698-1875, after EcoRV-EcoRI
digestion, of FIG. 3. The size of the restriction fragments is 8 kb
for the wild-type allele and 5.3 kb for the mutated allele (FIG.
1B).
[0124] 1-3 Microinjection of the Recombinant ES Cells and
Production of Transgenic Mice Homozygous for the Mutated Allele of
the STOP Gene [STOP KO (-/-) mice]
[0125] The recombinant ES cells carrying the mutated allele are
microinjected into OF1 mouse embryos at the morula stage, and the
injected embryos are then reimplanted into the uterus of the
surrogate mother, so as to produce chimeric mice (Gene targeting: A
practical approach, A. L. Joyner Ed., New York, Oxford University
Press, 1993, pages 174-179). Crossing these chimeras with BalB/c or
129/sv mice (Laboratoires CHARLES RIVER) produces heterozygous F1
descendants in which the transmission of the STOP gene mutation is
verified by Southern blotting analysis of the genomic DNA
originating from a tail sample. The F1 descendants are crossed with
one another to give homozygous F2 descendants.
[0126] 1-4 Western Blotting Analysis of the Expression of the STOP
Gene in the Brain of the STOP KO (-/-) Mice
[0127] Extracts of brains from STOP KO (-/-) mice and from
wild-type mice are prepared and analyzed by Western blotting using
the polyclonal antibody 23C, according to the protocols described
in Guillaud et al., mentioned above.
[0128] 1-5 Histological Analysis, Immunolabeling of the STOP
Proteins and Detection of the .beta.-galactosidase Activity of the
Brain of the STOP KO (-/-) Mice
[0129] a) Histological Analysis
[0130] 10- to 12-week-old wild-type and STOP KO (-/-) male mice are
perfused with a solution of paraformaldehyde (4% PFA). The brains
are fixed in the same solution for 2 h at 4.degree. C. A cytochrome
oxidase detection assay (Y. Liu et al., J. Neurosci. Methods, 1993,
49, 181-184) and staining with crystal violet are carried out on
100 .mu.m sections of the brains.
[0131] b) Immunolabeling of the STOP Proteins
[0132] The brains are prepared as described in paragraph a) and are
then frozen in sucrose (20% in PBS). 20 .mu.m brain sections are
incubated successively in the following solutions: 1%
H.sub.2O.sub.2 (15 min), 3% of BSA (30 min), and a mixture of the
polyclonal antibody 23C (100 .mu.g/ml) and of the
peroxidase-coupled anti-rabbit antibody conjugate (overnight), and
then the STOP proteins are revealed with ethylcarbazole (AEC,
DAKO).
[0133] c) Detection of the .beta.-galactosidase Activity
[0134] Brain sections (100 .mu.m) are fixed in 0.2% glutaraldehyde
and 2% formaldehyde. The .beta.-galactosidase activity is detected
by staining the sections in a solution of PBS containing 5 mM of
potassium ferricyanide, 5 mM of potassium ferrocyanide, 2 mM of
magnesium chloride and 1 mg/ml of X-Gal, at 30.degree. C. for 3 to
5 hours.
[0135] 1-6 Analysis of the Microtubule Stability in the Neurons and
the Glial Cells of the STOP KO (-/-) Mice
[0136] Neurons and glial cells from embryos of wild-type and of
STOP KO (-/-) mice are kept at ambient temperature or subjected to
a temperature of 0.degree. C. for 45 minutes. After extraction of
the free tubulin, according to the protocol described in Laurent
Guillaud et al., mentioned above, the microtubules are stained with
an anti-tubulin antibody and the nuclei are stained with Hoechst
solution.
[0137] 2 - Results
[0138] 2-1 Establishment of STOP KO (-/-) Mice
[0139] The genotypic profile of the STOP KO (-/-) homozygous mutant
shows the presence of a 5.3 kb fragment (FIG. 1B) which indicates
the deletion of the 1012 bp fragment containing the repeat
sequences of the coding region of the STOP gene.
[0140] The analysis of the various heterozygous crosses shows that
the mutated STOP allele is transmitted in a mendelian manner.
[0141] The STOP KO (-/-) homozygous mice are viable, appear to be
in good health, and exhibit no visible macroscopic lesions.
[0142] A null phenotype is obtained; the mice carrying the mutated
allele in the homozygous state do not express any STOP protein:
[0143] the analysis of the brain extracts from the STOP KO (-/-)
mice, by Western blotting using the polyclonal antibody 23C
(Guillaud et al., mentioned above), shows an absence of STOP
proteins (E-STOP and N-STOP) in these mice, whereas these two
isoforms are detected in the wild-type adult mice (FIG. 4);
[0144] the immunohistological analysis of the brain sections from
the STOP KO (-/-) mice shows an absence of specific labeling of the
STOP proteins (E-STOP and N-STOP) in these mice, whereas specific
labeling of these STOP proteins is observed in all the nervous
tissues of the wild-type adult mice.
[0145] 2-2 Lack of Stability with Respect to Cold of the
Microtubules of Cells Derived from STOP KO (-/-) Mice
[0146] Due to the absence of STOP protein in the STOP KO (-/-)
mice, depolymerization of the microtubules is observed
simultaneously after exposure to cold, in the neurons, glial cells
and the fibroblasts.
[0147] 2-3 Absence of Anatomical Lesions in the Brain of the STOP
KO (-/-) Mice
[0148] Analysis by optical microscopy of the anatomy of the brain
of the STOP KO (-/-) mice, using parasagital sections stained with
crystal violet to visualize the nuclei, shows no differences
between the KO -/- mice and the wild-type mice.
[0149] More precisely:
[0150] analysis of the layers of cells of the cerebellum, of the
neocortex, of the hippocampus and of the olfactory bulb, which
correspond to those showing considerable expression of the STOP
proteins in the wild-type mice, shows a completely normal
organization in the KO -/- mice,
[0151] examination of the somatosensory cortex, using tangential
sections stained to reveal the cytochrome oxidase activity, showed
a normal organization of the barrel fields in the KO -/- mice,
[0152] the .beta.-galactosidase expression profile in the brain of
the STOP KO (-/-) mice is identical to that observed in the
heterozygous mice, which demonstrates that the cells which, in the
normal state, express considerable amounts of STOP proteins are
still present in the STOP -/- mice.
[0153] The STOP (-/-) homozygous mice which do not exhibit
anatomical brain lesions detectable by microscopy exhibit, however,
behavioral disorders.
EXAMPLE 2
Electrophysiological Analysis of the Synaptic Transmission of the
KO STOP -/- Mice
[0154] 2 - Materials and Methods
[0155] To prepare the sections of hippocampus, 1- to 3-month-old
mice were deeply anesthetized with nembutal. Brain sections
(300-400 .mu.m) were prepared in an artificial cerebrospinal fluid
(124 mM NaCl, 26 mM NaHCO.sub.3, 2.5 mM KCl, 1.25 mM
NaH.sub.2PO.sub.4, 2.5 mM CaCl.sub.2 and 1.3 mM MgCl.sub.2), at a
temperature of between 4.degree. C. and 8.degree. C. More
precisely, the sections were maintained at ambient temperature for
at least 1 h and were then submerged in a chamber containing
artificial cerebrospinal fluid equilibrated with 95% O.sub.2 and 5%
CO.sub.2, and transferred into a superfusion chamber.
[0156] The excitatory post-synaptic potential (EPSP) of the
extracellular fields was recorded using microelectrodes (1 to 3
M.OMEGA.) filled with artificial cerebrospinal fluid. The
measurements were carried out at a temperature of approximately 22
to 25.degree. C. Bipolar steel electrodes were used to stimulate
the Schaffer collateral and the mossy fiber (stimulation of 10 to
100 mA, for 0.1 ms with intervals of 10 to 30 s between each
stimulation).
[0157] For all the analyses carried out in the CA1 region, the
stimulating electrodes and the extracellular measuring electrodes
were placed in the stratum radiatum, and picrotoxin (final
concentration of 100 .mu.M, SIGMA) was added to the artificial
cerebrospinal fluid. In these series of analyses, the CA1 region
was separated from the CA3 region by sectioning the brain section
with a knife before measurement.
[0158] For the in/out-curves, the excitability of the fibers was
analyzed after blocking the activation of the glutamate receptor
with the AMPA (alpha-amino-3hydroxy-5-methyl-4-isoxazole propionic
acid) receptor antagonist NBQX (5 to 10 .mu.M, TOCRIS). The
responses of the AMPA receptor were measured and then the responses
of the NMDA (N-methyl-D-aspartate) receptor were revealed after
suppression of the extracellular magnesium and isolated by adding
NBQX (10 .mu.M), in order to perform a quantitative analysis.
[0159] For the post-tetanic potentiation (PTP)-type analyses, a
high dose of D-APV (50 to 100 .mu.M; provided by TOCRIS) was added
to the solution of the bath, at least 10 minutes before the tetanic
shock.
[0160] For the paired pulse experiments, the Schaffer collaterals
were stimulated repeatedly with two stimuli of the same strength
separated by short intervals of varying duration. The result is
expressed by the ratio between the amplitude of the response to the
second stimulus and to the first stimulus, determined from an
average of 15 to 20 responses, for each value of the interval.
[0161] The responses of the mossy fibers were analyzed by applying
a bath of the selective glutamate receptor agonist DCG IV (type 2
metabotropic group). The inhibitory effects of the DCG IV (10 mM,
provided by TOCRIS) on the entries into the mossy fibers are
similar in the wild-type mice and the STOP KO (-/-) mice. NBQX (5
to 10 mM) was applied at the end of each analysis of the mossy
fiber, in order to determine the excitability of these fibers.
[0162] The data acquisition and the analysis of the long-term
potentiation (LTP) and long-term depression (LTD) experiments were
carried out blind, relative to the genotype of the mice. All the
results are expressed in the form of mean.+-.standard error of the
mean (s.e.m).
[0163] 2 - Results
[0164] The functioning of the synapses of the STOP KO (-/-) mice
was analyzed in the hippocampus, where there is considerable
expression of the STOP proteins.
[0165] First of all, in order to selectively analyze glutamatergic
transmission, the synaptic transmission in the CA1 region of the
hippocampus was analyzed in the presence of picrotoxin, a GABA type
A receptor antagonist.
[0166] The basal synaptic transmission was evaluated by analyzing
the relationship between the excitability of the fibers of the
Schaffer collaterals and the amplitude of the excitatory
post-synaptic potentials in the CA1 region of the hippocampus. The
analysis was carried out for various stimulation strengths. The
in/out-type curves are qualitatively similar in the STOP KO (-/-)
mice and the wild-type mice (FIGS. 5a and 5b). The quantitative
analysis carried out on the slopes of the in/out curves of six
wild-type mice and six STOP KO (-/-) mice shows no difference
between the 2 groups of mice (FIG. 5c), indicating normal basal
synaptic transmission in the STOP (-/-) mice.
[0167] For the analysis of the synaptic plasticity, the synaptic
response to a standard stimulus was evaluated by the slope of the
EPSP curve. The basal values of the slopes are determined by repeat
low-frequency stimulations (0.03-0.1 Hz). At time zero, a
conditioning stimulation protocol is applied. The synaptic
adaptation is demonstrated by a stable deviation of the values of
the EPSP slopes, compared to the basal values.
[0168] A high-frequency (100 Hz) conditioning protocol, applied to
the Schaffer collateral-pyramidal cell of the CA1 region synapse,
produces a stable increase in the slopes of the curves, in the
sections from the wild-type mice (FIG. 6a), indicating synaptic
potentiation in these mice. This potentiation persisted for more
than 30 minutes, and such a persistence represents a long-term
potentiation (LTP).
[0169] In the STOP KO (-/-) mice, a weaker potentiation of the
synaptic transmission is observed (FIG. 6b).
[0170] This difference is confirmed by the quantitative analysis of
all the results obtained in the 2 groups of mice (FIG. 6c).
[0171] The long-term depression (LTD) was analyzed at the same
synapses of the Schaffer collaterals and of the CA1 pyramidal
cells. The conventional low-frequency stimulation protocol was used
(LFS, 900 stimulations of 1 Hz). Sections from STOP KO (-/-) mice
showed a significant decrease in the LTD amplitude (FIG. 7b).
[0172] These results show that the LTP and the LTD are altered in
the STOP KO (-/-) mice.
[0173] The LTP and the LTD depend crucially on the activity of the
NMDA receptor. However, the basal activity of the NMDA receptor,
measured by the ratio of the NMDA/AMPA response to stimuli (FIG.
8A), and the activation of the NMDA receptor during the tetanic
stimulation (FIG. 8B) are comparable in the STOP KO (-/-) mice and
the wild-type mice.
[0174] These results demonstrate a deficiency in the 2 major forms
of synaptic plasticity (LTP and LTD) in the STOP KO (-/-) mice and
indicate that this deficiency is not linked to a deficiency in
expression of the NMDA receptor in the STOP KO (-/-) mice.
[0175] FIG. 9B shows that, in the STOP KO (-/-) mice, the synaptic
potentiation is altered during the first minutes after tetanic
stimulation, and also at later times. Consequently, the existence
of a possible deficiency in short-term plasticity, at the synapses
of the Schaffer collaterals and of the CA1 pyramidal cells was
analyzed by measuring post-synaptic potentiation (PTP) and paired
pulse facilitation (PPF). Like LTP, PTP is a form of potentiation
subsequent to a tetanic stimulation (stimulus of 1 Hz for 1 s), but
it is induced in the presence of the NMDA receptor antagonist
(D-APV) in order to block post-synaptic events involved in LTP, and
persists only for a few minutes, subsequent to the tetanic
stimulation. PPF is another form of synaptic plasticity observed
when the synapses are stimulated by paired pulses. PPF is defined
by an increase in the synaptic response in response to the second
stimulus. The PTP is reduced in the STOP KO (-/-) mice (FIG. 9A).
On the other hand, the paired pulse facilitation (PPF) is similar
in the wild-type mice and the STOP KO (-/-) mice (FIG. 9B), for an
extended range of decreasing values of extracellular calcium
concentration.
[0176] The synaptic plasticity at the synapses of the mossy fibers
and of the pyramidal cells of the CA3 region was then analyzed. No
difference in the long-term potentiation (LTP) nor in the paired
pulse facilitation (PPF) was observed between the STOP KO (-/-)
mice and the wild-type mice. In order to study the short-term
plasticity, the mossy fibers were stimulated with increasing
frequencies ranging from 0.033 to 1 Hz. This protocol normally
induces a considerable and transient increase in the amplitude of
the response of the mossy fibers, a phenomenon known as frequency
facilitation. The amplitude of the frequency facilitation was
significantly decreased in the STOP KO (-/-) mice, in comparison
with the wild-type mice (FIG. 9C).
[0177] All of these results show that several distinct forms of
long-term and short-term plasticity are altered in various regions
of the hippocampus, in the STOP KO (-/-) mice.
EXAMPLE 3
Analysis of the Behavioral Disorders of the STOP KO (-/-) Mice
[0178] 1 - Materials and Methods
[0179] All the behavioral tests were carried out on litters of STOP
KO (-/-) mice and of control wild-type mice derived from the same
colony (genetic background BALBc/129 Sv).
[0180] 1-1 Maternal Behavioral Tests (Mothering)
[0181] The maternal behavior is assessed by the performing of the
following acts:
[0182] 1. preparing a nest
[0183] 2. reinstalling the newborns in the nest.
[0184] Tests Carried Out in Nulliparous Females and in Males
[0185] Young nulliparous females, 28 to 49 days old, were reared
individually for at least one day before the beginning of the
experiment, and they were then provided with cotton to construct a
nest.
[0186] On D1, each female is placed together with 3 1- to 3-day-old
newborns in the following way: the newborns are each placed in one
of the corners of the cage, at a distance from the nest, and, after
30 minutes, the newborns are returned to their natural mother.
[0187] On D2, each female is again placed together with the
newborns and, for each female, the number of young mice reinstalled
in the nest is evaluated for a period of 30 minutes.
[0188] Young males, 30 to 45 days old, were used under the same
conditions as the nulliparous females, with the only difference
that they were placed together with the young mice for 2
consecutive days before being tested on D3, instead of D2 for the
nulliparous females.
[0189] Tests Carried out in Primiparous or Multiparous Mothers
[0190] Post-partum females (second gestation) were reared
individually from the beginning of their gestation. On the day of
giving birth, the young mice were removed and kept in the warm for
one hour. The mother was then removed from her usual cage and three
newborns were each placed in a corner of this same cage, at a
distance from the nest. Next, the mother was returned to her nest
and the number of young mice reinstalled in the nest, over a period
of 20 minutes, was evaluated.
[0191] 1-2 "Light/dark test"
[0192] The test of the choice between light and dark, known as
"light/dark test", is used to reveal a state of anxiety caused by
an anxiety-generating stimulus. This method, validated by Misslin
et al. (1990, Neuroreport, I, 267-270), is based on the natural
tendency of rodents to prefer a dark environment, and makes it
possible to evaluate the emotional response of animals subjected to
a stress consisting of light.
[0193] The animals are maintained in individual cages placed in an
incubator having a temperature of between 21.degree. C. and
22.degree. C. and an inverted light/dark cycle of 12 h/12 h, with
as much water and food as desired. All the experiments are carried
out in accordance with the institutional directives relating to
animal experimentation.
[0194] The device consists of two polyvinylcarbonate boxes (20
cm.times.20 cm.times.14 cm) covered with perspex. One of the boxes
is made dark and the other is lit using a 100 W desk lamp placed at
a distance of 15 cm (4400 1.times.). An opaque plastic tunnel (5
cm.times.7 cm.times.10 cm) separates the darkened box from the lit
box.
[0195] The animals are individually placed in the lit box with the
head directed toward the tunnel. The time spent in the lit box
(TLB) and the number of passages between the two boxes are recorded
over a period of 5 minutes, from the first time the animals enter
the darkened box.
[0196] The overall analysis of the results is carried out using the
Mann and Whitney U test. The risk (p) is fixed at p<0.05. The
results are expressed by the mean value.+-.standard error of the
mean (s.e.m).
[0197] 1-3 Object Recognition Test
[0198] The short-term memory is evaluated by the object recognition
test previously described (Ennaceur et al., 1988, Behav. Brain
Res., 31, 47-59; Dodart et al., 1997, Neuroreport, 8, 1173-1178),
which is based on the natural tendency of rodents to explore a new
object, in preference to a familiar object.
[0199] The animals are maintained under the conditions as described
in example 3, section 1-2.
[0200] The object recognition test is carried out in an open space
made of perspex (52 cm.times.52 cm.times.40 cm). The floor is
divided into 9 squares of equal size. The objects to be
distinguished are a bead and a dice. The animals are given 30 min
to become familiar with the open area.
[0201] The following day, they are subjected to a learning test of
10 min (first test) during which they are individually placed in
the open space, in the presence of an object A (dice or bead).
During this period, the following are recorded:
[0202] the locomotor activity, evaluated by the number of squares
crossed, and
[0203] the time spent by the animal in exploring object A, i.e. the
time during which the animal's nose is directed at a distance from
the object of less than 1 cm.
[0204] Three hours later, they are subjected to a recognition test
of 10 min (second test). For this test, object A and the other
object (B) are placed in the open space and the locomotor activity,
and also the amount of time spent exploring object A (t.sub.A) and
object B (t.sub.B); are recorded. Next, the recognition index (RI,
on the Y-axis in FIG. 13) is calculated from the following formula
RI=t.sub.A/(t.sub.A+t.sub.B).times.- 100. A recognition test is
considered to be positive if the value of the recognition index is
significantly greater than 50%.
[0205] The overall analysis of the results is carried out as
described in example 3, section 1-2.
[0206] 1-4 Social Behavior Test, Intruder Test
[0207] 1-4-1 Social Investigation
[0208] The social behavior is evaluated on young males, 4 weeks
old, isolated for one week in a cage (resident young males); a male
intruder reared in a group is introduced into the cage and the
social investigation time (approach, sniffing, sexual posturing) of
the resident young males is evaluated for 6 minutes. The results
are analyzed using the Mann and Whitney U test.
[0209] The results are expressed by the mean value.+-.standard
error of the mean.
[0210] 1-4-2 Inter-male Aggression
[0211] Resident males are isolated for one month and an intruder
(male reared in a group) is placed in the cage. The number of
attacks and the time spent fighting by the residents is measured
over a period of 5 minutes. The results are analyzed using the Mann
and Whitney U test. The results are expressed by the mean
value.+-.standard error of the mean.
[0212] 2 - Results
[0213] 2-1 Maternal Behavior of the STOP KO (-/-) Mice
[0214] The STOP KO (-/-) mice exhibit major deficiencies in
maternal behavior which result in a complete lack of interest for
their progeny, as shown by the results given in FIG. 10, obtained
from 161 young mice derived from 20 female STOP KO (-/-) mice
crossed with heterozygous males:
[0215] all the newborns, derived from primiparous STOP KO (-/-)
mothers die within 24 h following birth due to lack of attention
from the mother (FIG. 11a), whatever their genotype (genetic
background BALBc/129Sv or 129Sv). By comparison, a 93% survival
rate is observed in the newborns derived from a primiparous mother
carrying the wild-type allele in the homozygous state [(+/+)
wild-type mice], which exhibit normal maternal behavior comprising,
in particular, preparation of a nest and reinstallation of the
young mice in the nest (FIG. 11a). It was also shown that the
maternal behavior of the STOP KO mice was not improved by repeated
gestations (multiparous mothers);
[0216] the newborns of a STOP KO (-/-) mother are never
cannibalized and they are raised until weaning when they are
adopted by wild-type mothers, which demonstrates that the death of
the young mice is directly linked to the genotype of the mother. To
determine the causes of death, a deficiency in suckling at the teat
associated with an absence of olfactory signal at the level of the
teats of the STOP KO (-/-) females was investigated. When the young
mice derived from a STOP KO (-/-) mother are left in the presence
of their mother but repeatedly re-placed in position to be
mothered, through a human intervention, all the young mice show
behavior consisting of searching for and attaching to the teat. The
presence of this guided behavior indicates that the STOP KO (-/-)
females possess the olfactory signals essential to suckling. In
addition, under these conditions, the presence of milk in the
stomach of the young mice was observed. These results demonstrate
that the death of the young mice was not linked to lactation
deficiencies in the STOP KO (-/-) mice;
[0217] the deficiency in reinstallation in the nest is not due to a
deficiency in olfactory recognition of the young mice in the STOP
KO (-/-) mice, given that the STOP KO (-/-) females placed close to
their progeny sniff the young mice and, in addition, show normal
behavior in an olfaction test (hidden food test);
[0218] in order to verify whether the deficiency in maternal
behavior observed in the STOP KO (-/-) mice was associated with
hormonal status, complementary tests were carried out in the
nulliparous females and in the young males. The results of the
"reinstallation in the nest" tests, carried out in these two groups
of mice, show a deficiency in maternal behavior of the nulliparous
female or male STOP KO (-/-) mice (FIGS. 10b and 10c).
[0219] All of these results indicate that the deficiency in
maternal behavior observed in the STOP KO (-/-) mice is independent
of an obvious organic deficiency and of the hormonal status of
these mice, which indicates that there is only one manifestation of
the multiple behavioral deficiencies observed in these STOP KO
(-/-) mice.
[0220] 2-2 Other Behavioral Disorders of the STOP KO (-/-) Mice
[0221] Although examination of the general condition of the STOP KO
(-/-) mice reveals no apparent deficiency, they exhibit a strange
behavior with phases of intense activity with no apparent purpose,
accompanied by frequent changes in activity, occurring randomly.
Occasionally, the mice exhibit a period in which they have an
attack, of approximately 20 min, during which the animals turn in
circles or dig in the cage, compulsively. These mice also go
through periods of apparent prostration during which they remain
immobile, do not sleep and do not react to the environment. Such
attacks have never been observed in the wild-type mice and do not
resemble epileptiform events. The acute attacks are difficult to
study systematically, but they represent paroxysmal manifestations
of a continuous background noise of behavioral abnormality. The
video recording was used to evaluate the behavior of the mice
quantitatively. The time spent by the mice in eating, sleeping,
grooming, walking and remaining immobile while awake was measured
for a period of 3 hours, and the results are given in FIG. 11a.
Compared to the wild-type mice, the STOP KO (-/-) mice spend more
time moving around in the cage or remaining immobile, although they
are awake, to the detriment of the time spent feeding and sleeping.
The mutant mice exhibit greater changes in activity, largely due to
a significantly greater number of phases of movement without
purpose and of phases of immobility (FIG. 11a). The changes in
activity of the STOP KO (-/-) mice often break a period of
characteristic activity. For example, in the wild-type mice, 71% of
the sleep phases are preceded by a grooming phase with or without a
tranquility phase being intercalated. The corresponding frequency
in the STOP KO (-/-) mice is 47%, a value just above the expected
background noise in the case of random sequences of activity (35%,
FIG. 12a). These quantitative results confirm the impression of
non-organized activity which is not directed toward a purpose,
given by the observation of the STOP KO (-/-) mice.
[0222] Complementary analyses of the behavior of the STOP KO (-/-)
mice were carried out using conventional tests.
[0223] State of Anxiety of the STOP KO (-/-) Mice
[0224] The STOP KO (-/-) mice are frightened by an
anxiety-generating stimulus and the wild-type (+/+) mice have a
normal behavior, as shown by the results of the light stimulation
test (light/dark test) given in FIGS. 12a and 12b. In addition, it
was shown, by prior tests, that the spontaneous locomotor activity
of the mutant (-/-) mice was not modified compared to the wild-type
(+/+) mice.
[0225] FIG. 12a shows that the wild-type (+/+) mice spend much more
time in the lit box than the STOP KO (-/-) mice, which remain in
the darkened box throughout almost the entire test. The differences
observed between the KO (-/-) mice and the wild-type (+/+) mice are
statistically significant: p<0.01, Mann and Whitney U test.
[0226] FIG. 12b shows that the wild-type mice enter the lit box
more frequently than the STOP KO (-/-) mice. The differences
observed between the STOP KO (-/-) mice and the wild-type (+/+)
mice are statistically significant: p<0.01, Mann and Whitney U
test.
[0227] Short-term Memory of the STOP KO (-/-) mice
[0228] The STOP KO (-/-) mice exhibit short-term memory disorders,
as shown by the results of the recognition test, given in FIG.
13:
[0229] the recognition index, measured in the wild-type mice (65%)
is significantly higher than the index of 50% observed in the STOP
KO (-/-) mice (p=0.004 for the (+/+) mice, Student's test);
[0230] the time spent exploring objects A and B, recorded during
the second test (recognition test) shows that the wild-type mice
explore the new object in preference to the familiar object;
[0231] the STOP KO (-/-) mice explore neither of the objects during
the two tests (learning and recognition). They move around in the
open space but show no interest in the objects. This behavior might
be explained by their state of anxiety, as shown by the results of
the light/dark test. This state of anxiety is revealed in
particular by the fact that the mice move along the walls and that
they do not cross the open space, since they appear to be
frightened by the environment and by the objects which represent an
anxiety-generating stimulus.
[0232] Social Behavior
[0233] The STOP KO (-/-) mice exhibit disorders of social
investigation (FIGS. 14a, 14b and 14c).
[0234] FIG. 14a shows that the time spent by the residents in
exploring the intruder is significantly reduced when the resident
is a STOP KO (-/-) male, (p<0.05).
[0235] FIG. 14b shows that, in the inter-male aggression test, the
number of attacks carried out by the STOP KO (-/-) residents is
less than the number of attacks carried out by the wild-type males
(p<0.01).
[0236] FIG. 14c shows that the time spent fighting by the resident
STOP KO (-/-) males is reduced in comparison to the wild-type males
(p<0.01).
EXAMPLE 4
Effect of Anxiolytics and of Neuroleptics on the Maternal Behavior
of the STOP KO (-/-) Mice
[0237] 1 - Materials and Methods
[0238] The effect of anxiolytics (diazepam) and of neuroleptics
(chlorpromazine, haloperidol or clozapine) on the behavioral
disorders of the STOP KO (-/-) mice was evaluated in the maternal
behavior test as defined in example 3 ("reinstallation in the nest"
test).
[0239] Haloperidol (Haldol.RTM., JANSSEN-CILAG), chlorpromazine
(Largactil.RTM., RHONE-POULENC) and diazepam (Valium.RTM., ROCHE)
were administered to the mice in the drinking water, at a dose of
0.5 mg/kg/day.
[0240] 2 - Results
[0241] a) Short-term Treatment
[0242] The effect of short-term administration (for 6 to 8 days
from the 6th day preceding the birth) of anxiolytics (diazepam) and
of neuroleptics (chlorpromazine, haloperidol or clozapine) on the
behavioral disorders of the STOP KO (-/-) mice was evaluated in the
"reinstallation in the nest" test.
[0243] The reinstallation of the young mice in the nest is
dramatically altered in the untreated STOP KO (-/-) mothers and is
slightly improved by the administration of diazepam (FIG. 15A). On
the other hand, the STOP KO (-/-) mothers treated with neuroleptics
behave as well as the wild-type mice (FIG. 15A). However, no
survival of the young mice was observed in either the treated or
untreated STOP KO (-/-) mice. These results indicate a specific but
limited beneficial effect of short-term administration of
neuroleptics, on the behavior of the STOP KO (-/-) mice.
[0244] b) Long-term Treatment
[0245] Seven STOP KO (-/-) mice and seven wild-type mice were given
a daily administration of a mixture of chlorpromazine and
haloperidol, for 4 months, starting from weaning and continuing
during growth, coupling with males, gestation, birth and the
post-partum period. The seven wild-type mice exhibited normal
maternal behavior and all their young mice survived (FIG. 15B).
Notably, in four of the seven STOP KO (-/-) mice, an improvement in
the maternal behavior was sufficient to allow survival of the young
mice, with survivor/newborn ratios of, respectively, 3/11, 4/8, 2/4
and 1/5 in these four mice.
[0246] The proportion of female STOP KO (-/-) mice with surviving
young mice is significantly higher in the mice given long-term
treatment with neuroleptics (4/7), compared to the untreated mice
(0/20) or to the mice given short-term treatment with neuroleptics
(0/6, FIG. 15B).
[0247] These results indicate that long-term administration of
neuroleptics is capable of re-establishing a normal behavior in the
STOP KO (-/-) mice, compatible with survival of the young mice.
[0248] As emerges from the above, the invention is in no way
limited to its methods of implementation, preparation and
application which have just been described more explicitly; on the
contrary, it encompasses all the variants thereof which may occur
to a person skilled in the art, without departing from the context
or the scope of the present invention.
Sequence CWU 1
1
6 1 7133 DNA Mus musculus 1 gaattcaaag atgccattat cttgctttct
ttcgtcccta cttctgatct cacggcagcc 60 ccagcagagg tcctatgagt
cctccaagga ctacgtagct tcaacactga agcctgtaac 120 tttgtcccat
ggtcccctaa tgcgtaagga aaaagcccag catttcacga taaagcagga 180
agcagagtga gtgcctttga tataataggg aaggtctaac ttacaggcta ttttaagaat
240 tgattgctaa gtatcaatat gatcacgctt ttaagatgac tcatgatctt
ccacattaat 300 ctgttgtgtg tggaattata tctgtacatt gctggctatt
aaaccatggc ttgctcctaa 360 cataaacact actaaatatc agatttttct
atctatctat ctatctattg atctatctat 420 ctatctatct atctatctac
ctacctacct acctatcatc tatctatcta tcaatcatgt 480 atgtatgtat
gtatgtatgt atgtatctat ctatctatct atctatctaa gtgtgtatga 540
gtgtagctgg gcataacatc acatgcttgt atgtagaggt aggaggacaa tcccttgaac
600 agatccctca tcaacaagtt ggatctggcc aaattggcat aagaaaaaaa
aagctcaatg 660 tattatgttg ctaaagactt gcaaattaac aacaaatgta
acccaaacag acacagcacc 720 aaacactggc aagggggaga gaaacaggag
ttctcattag ttactactga tcactcaaaa 780 ctggtacaac tatggtggaa
gacagtttgt tttgttacaa aactaactca tcagttacta 840 ctgagtactc
aaaactggta caattatggt ggaagacagt ttgctttgtt acaaaactag 900
ctattctttt ggccacataa tcacaatcct gctccttgat attacccaaa aagagcccaa
960 agcttatgtc agcacagaaa cctgaacact gttttagcag ctttgtttat
aattcccccc 1020 aattgaagca accagcatgc cattcagcag gtgacggata
aataatctat gggaacacgt 1080 aatattacag aacaagagaa atgagatacc
agccatgaaa agatatgatg gatccttgag 1140 cttattacta agtcagagct
aatctgaaac actgcttact atctgatgga tcaaaaagga 1200 ggaatctacc
tcaggtgaca agatgaacct tgtcacctcc ttaagggtta atccagtgag 1260
acaggtgagg ggactgagag gatccacccc aggaaagaca tattcacctt acaataacgt
1320 actcttccct tacagaatta tggcctccta atgcagttcc cttgctatgc
aagtgtgagc 1380 acatgagttc aagtcctgca cagtgaagca caaatcaggg
tgtggtcagg tgagcacctg 1440 ggacctcagc actgtgaggg gcagagccag
gaaaactgca ggggcttgct agccagtgag 1500 cctagcttca gattcagtga
gaaaccttgt ttcatgggga aaaaggcaga gaatggtaga 1560 gcagaatacc
cagcatcctc ccttagacgt acacatgtgc atatatgcca caaataaatt 1620
aattaagagt aaatatttgg gacttttaag aaaatgaact ctttcttttc cttgtgaagt
1680 agcagatttc caagtagcag gcttctcagt ggcttatctg ggaggctctg
cctgtgagac 1740 ttttggataa gcttttgaat attaacaggt tctgctgaag
aagccatttc tatatgccag 1800 gagctgcagc taatatttct tgtgaattgt
gtcacttcag tcttcagaaa aactttgtaa 1860 gttcagaata gatacccttg
ctcagagaca aagttaaggg tcagaggttg ccacaatttg 1920 tctatgcctt
agagccaaaa gtgcagaagc caggatccaa atccctgagt atggactcca 1980
ggcttctgct ctgggtgctt aactttcctc atgaaaccac cccgaggcat ttattagctc
2040 tactaccctc ccccccccca ccccccgccc cgacccaggg gactgcagcc
cagagacaac 2100 ccaagtcaca aagagagtat cagagccaaa gcaggtccat
tagattccaa aatctgttca 2160 accccaaagt agaaaagaag gcagaaacac
aaccgagaat cccctcaaag aggtctccct 2220 tttcctaggg aactgtctct
catccagccg cttacctcct cccttgggtc tctttccaga 2280 atcttccttt
ttctcccttt cccccagtgc agcatctggg ctggagaaga accaatttta 2340
tcctgggaga gtagactcac tgcttcccag ttgcttaaag aaggaacagc cagagtatgg
2400 ctaggccggc ttcccaggcc ggcctctccc aatctcatac aactggggaa
gcccctgtac 2460 acataggctt aggtcaagaa ccagcaacac ggagcataag
tacagcctga tagctgacca 2520 attaactgca gctctccctt ttaaagtatc
tattcagggg ctgagtggtt aaaaacttgt 2580 attggtgttc cagaggaccc
aagtccgggt cccagcattc acatgggggt ggggtgggag 2640 ctcatagtcg
cctgcacagc accagggaaa tctacttctg accctctggc ctcagcaggc 2700
accagagccc atgtgcatat acccaacaca gacaaaagca ctaattaaaa ggtggcgcaa
2760 ccctttaatc ccagcacttg ggaggcaaag gcaagcggct tttctgagtt
caaggccagc 2820 ctggttacag actgagttcc aggacagcca agactacaga
gagaaaccct gttcgaaaaa 2880 acaaaagcca aaaaccaaaa aaaaaaaaaa
gtaaaaaaga tttttaaaag aagccaaagc 2940 atttagtctg aaacagacat
ttcatggagg cggggcggtc ttacacttaa aaaattagta 3000 ttcattggtt
ggggcgattt tacgaaagag aggagactcg gttttaggac atgaagctgg 3060
caactcaagg acagcaagtc ctcaggagct tgggacttga acctatcaag gacccagttc
3120 tgctgttgcc tgggaacaga gaagggagga gctgccagag agaagggagg
aggggacagg 3180 gagagggcaa cctttcagct gcgcgccctg acgacagcag
gtgatttttt ttttttttct 3240 gcagcgcgca tactctcagc atcttttctt
gatcacccca cctcccttgc tgagccgcaa 3300 aaaagggtgg ggccgcctct
ctgcagcaga agacgacagc ggcagcggct gcggcatcac 3360 cgggggtata
gatagaggcc gttttgctct ctgctctgtc tgggttggag gtgaccgctg 3420
ccaagcctcg ctaggcggcc ggctgaacca gacagaaagg agataaaagc ctgcttgcga
3480 tccttcctgc gccatggctt aagcccacag cctctttgcc aagcatctcc
ttgctctgcc 3540 ggggtctgct agacaccgca gtcgcagaga gggcgcgccc
agacgcccta ggcctggact 3600 ctgggacgct gagcctcgct cctattcttc
actgcccaca gcagctcctc tgcagcaggc 3660 gtttgcagcc ggcaatcgag
ggactttacg gactttatct cagcggtacc ttgtccccgg 3720 gtgctctttg
agggtggagg acgaggcaaa gggcttctaa gggaaggaag cggtgggaac 3780
cacattggcg ggtctgggtt ggggttaaag ggagattgga gatttgattt aggaccacaa
3840 aaaggctttg tggctaacat ggcgtggccg tgcatcacaa gggcctgctg
catcgcccgc 3900 ttctggaacc agctggacaa ggcggacatt gcggtgccgc
tggttttcac caagtactcg 3960 gaggccaccg aacacccagg cgcccctccg
cagccgccag ctccgctgca gcccgcgtta 4020 gcgcccccct cgcgtgctgt
cgccatagag acgcagccag cccagggaga gtcggatgca 4080 gttgcccggg
caacagggct tgcgcccggg cccagcgtcg accgcgagac tgtagccgcc 4140
cccggccgga gcgggctggg cttgggcgcg gcctcagcct ccacttccgg ctcaggcccc
4200 gcggactcgg tgatgcgaca ggactaccgc gcctggaaag tgcagcggcc
cgagccaagc 4260 tgccggccgc gcagcgagta ccagccgtcc gacgcgccct
tcgagcgcga gacccagtac 4320 cagaaggact tccgcgcctg gccgctgccc
cggcgcgggg accatccctg gatccccaag 4380 ccggtgcaaa tccctgcgac
ttcgcagcct tcccaacctg ttctcggggt gcccaagcgt 4440 cggcctcaga
gccaagagcg cgggcccatg caactttctg ctgatgcccg ggacccggag 4500
ggtgctggag gagccggggt gctggcggca ggaaaggcgt ccggtgtaga ccagcgcgac
4560 acacgtagga aggcagggcc agcatggatg gtgactcgca acgaagggca
cgaagagaag 4620 cctctgcccc cagcccaatc ccagacccag gagggtggtc
ctgcagctgg aaaggcgtcc 4680 ggtgcagatc agcgtgacac acgcaggaag
gcagggccag catggatggt gactcgcagc 4740 gaagggcacg aagagaagcc
tctgccccca gcccaatccc agacccagga gggtggtcct 4800 gcagctggaa
aggcgtccgg tgcagatcag cgtgacacac gcaggaaggc tggacccgcg 4860
tggatggtga ctcgcacgga agggcacgag gagacgccgc tgccacccgc ccagtctcag
4920 acccaggagg gcggccccgc agctggaaag gcatctggtg cagacgagcg
cgacacgagg 4980 aggaaggcgg ggccggcctg gatggtgcgt cgctcggagg
ggcacgaaca gacacccgct 5040 gcccatgccc aaggcacagg gcctgaagga
agcaaggggc gcgcggtggc agatgccctc 5100 aacaggcaaa tccgggagga
ggtggcgagt acagtaagca gctcttacag gtgagactgg 5160 ggcagcaggt
gatgctggtc accctcatcc cctcgcgagg accacccatt ctacccccac 5220
accgaaagct tcgcattcag cttcttctcc agggccagac cacacctctt cagccacatt
5280 ccagaaccct ttcaacccag actttactgc ccaccctgtc ggaaagccct
taacagtttc 5340 cacactggtt ttcccagctt gttttttgtg ccacccctaa
gccacatttt cctcttggct 5400 gcctagctca gctccctatc tgccccacag
agaccctgtc agtttccccc tgtctcatca 5460 gttcgcctgc tgtcccagcc
tggctcccgt ccctagtcgc ctccactcat tcacttctca 5520 cttacttccc
cgtgagaccc tttctcctcc ccagtctcac attgtcctgg tctcttccta 5580
ttgattcctc ccccagttct ggccgacact caagcgccac acccttttcc agatttctct
5640 acatgctcct taataactgg ctctagtact tgaagtcatt cccctcctgc
ctcttcagta 5700 gctttgacac attgggcgag ctttttaacc ttcctgggcc
ttcacattct tatctgtaac 5760 tgggatcaat aatagtcaac ttagaactat
ccacacagga ctgatttaag ctgagatatt 5820 ctgtacagtt ttgaggatag
actaaatgcc aaacatggct ctagctgatg gaaagtggaa 5880 agagatgact
cgggatgact ggccatgcct gagatgtagt gagaggtctt gtttccattt 5940
atactctcct atgtgatgcc cccttattct tgattctgct tcctggatgt ccctagccct
6000 cttccctgtt gaggcagctc ctttcttcct ttgtgttcag atgtttctat
tttttctttt 6060 aattaattag ttaattaatt aatttactta ttccctttac
atcctgctca ctgcccccta 6120 cagtccttct ccccatgccc cacttcttct
ctaagtgggt ggggtctccc cctgagtatc 6180 tccctaccct ggtacatcaa
gtttctgcag ggctagtcta atcctctccc actcaggcca 6240 aacaagtcaa
cccagctaga agaacatatt ccacgaacag ccaacagctt tttggggtag 6300
cccccattcc agttgtttgg gacccacatg aagaccaagc tgcacatctg ctgtatatgt
6360 gcagagaggt ctaggtccag cccatgtatg ctctttggtt tgtggttcag
tctctgagaa 6420 cccctaaggg ttccaggtta gttgactctg ttgatcttcc
tgtggagttc ctatctcctt 6480 aggggcctac aatccttctc cctatacttc
cacaagactc cccaagctcc atccactgtt 6540 tggccatggg tgtctgtatc
tgtctgagtc agctgctggg tggagcttct caaaggtcag 6600 acattaatgt
gtcccttatc ctctggcaca ttcacgttcc ttgaaaatga acgctttcaa 6660
acttagaaac atcccccatc ttcctccacg cacaccctgt ttatacaatt ctccctgtgc
6720 tgcatgcagc cttggcctgc tttctcacct cacatccatc cagtgcacat
ctctgaatgt 6780 ttagcttttg ggccttcctt tatgtttcta ccaacatcag
ctagctgata gatcctagtt 6840 gacttgaaca tgtagcagca ccgggcttgg
gtactgagga agcagatgtg tgtgataatg 6900 atggagctgg ccttgtgcag
ctgtgacttc tgaggtcttc attcagctcc acagtcagtt 6960 aggtgccctt
ttcctaagtg tggaaggaag ggctacagag ttagtgtctt ctgctaaccc 7020
tgtgtgtggg cgcttcagta ggctctgcag gcactcctct catgcagcca gaagactggc
7080 ataaatagag actctattgt taaggcaaaa gtcagagccc tgcctcagaa ttc
7133 2 1881 DNA Mus musculus 2 ctgtgagttg gtggccagct cggtttacat
agtgagttcc aggacagcca gggcagaaaa 60 aaaaaaaaag tacctatata
aaacttatga ggggctggtg agatggctca gtgggttaga 120 gcacccgact
gctcttccga aggtccagag ttcaaatccc agcaaccaca tggtggctca 180
caaccatccg taatgagatc tgactccttc ttctggagtg tctgaagaca gctacaatgt
240 acttacatat aataaataaa taaataaata aataaatctt aaaaaaaaaa
acttatgaga 300 acatcggcgg agggacagtc ccttaaagag ccatttgtta
tagctgaaga gatggtttca 360 ctggatagca gcacttgctg ctcttgcaga
gaacccaggc tcagttcctc atccctatat 420 gatggttcgc aaccatttgt
aactccagtt ccaggggaac caacgatacg tgtacataca 480 agcaggcaaa
acacccacac tcataaaatg aaatacacaa ctcttaactg tttttgttca 540
cctgtctcca cccccagtgt tggggtagca gacacatgca gctatgccta acttttacgt
600 aagttctggg ggcctgaatt ccaatcttca tcccgatcca gcaagcactt
ttacccctgt 660 agccatctcc ttggccttga gaaagcttct taagggttga
tatcattccg aggaaaatat 720 tgaatacata tttactatgg agtcatgtat
atttaagaaa tgcacacagc atgcatttat 780 gcctccttcc acgttctgga
aacactggag gaaggtaggc agggagtgtg tttaggatgg 840 aatttaaaat
ttatccgggc cattttattt aagggcatct cagatctatc accaaggatt 900
cccacttaga aaatttggac aacataagcc tcaaaatgaa aagtgaaaaa aatgggttaa
960 taccacagta aaaatattca tgaatccgac agtgtataac aaagaggcca
gctactaagt 1020 gtagaaagaa taaaggattc tgagaaatca tcatgtgaca
aacatcatca tagcgagtgt 1080 gctgggcaag ggacgccagc agttggtaaa
tgatcgggcg acaattcctt gggtaacaaa 1140 caaacgagtt gcagagtgtc
aggtgacctt gcttagtgta gtagttggac tggataatga 1200 atagcagagc
accttgaaat ctgaggacgt tatagttgtt ttaaaaatgt cttcacaagg 1260
ccctcatgtt gtgtaaagga gattttaaaa ttttagttca acaaaaagaa gacatgttta
1320 ctgtgtatct tagcctgttt tagttctggg acagacacca tagataccga
gctgaaacaa 1380 caagtgctct ttacccacag ttccagggcg tgggaagtac
aacatcaaag ctctgatgca 1440 agtggtgtcc gtgaggatcc atttcctgct
atgcagatgc tgcaaacata tgactccggc 1500 ctcttcctct tctttaaggg
catgtgtctg aagtggggtg gggtggcatc ctcatgactt 1560 catctgtgtt
ttccctagag atagcccctt tggttagggt tttaacatag atgtttggtg 1620
cacacataca tttagtctat gatagtctct agcaacagcc agttgggagg gaaggagagg
1680 gagagggaga gagagagaga gagagaacac atgtatgcga gtctgtgatt
tctagcttct 1740 tagcttctta tatttgatct aattttcatt tattttatga
ttctggaact ctgttgtttt 1800 attactttga aattctagga ctccaaataa
atatttttga tcttaaaata ttatcattat 1860 tttattctga tcccagaatt c 1881
3 27 DNA Artificial sequence Description of artificial sequence
Synthetic primer 3 agagtcggat gcagttgccc ggcaaca 27 4 27 DNA
Artificial sequence Description of artificial sequence Synthetic
primer 4 ggctcctcca gcaccctccg ggtcccg 27 5 21 DNA Artificial
sequence Description of artificial sequence Synthetic primer 5
aacagggtgt gcgtggagga a 21 6 1183 DNA Mus musculus 6 gatatcattc
cgaggaaaat attgaataca tatttactat ggagtcatgt atatttaaga 60
aatgcacaca gcatgcattt atgcctcctt ccacgttctg gaaacactgg aggaaggtag
120 gcagggagtg tgtttaggat ggaatttaaa atttatccgg gccattttat
ttaagggcat 180 ctcagatcta tcaccaagga ttcccactta gaaaatttgg
acaacataag cctcaaaatg 240 aaaagtgaaa aaaatgggtt aataccacag
taaaaatatt catgaatccg acagtgtata 300 acaaagaggc cagctactaa
gtgtagaaag aataaaggat tctgagaaat catcatgtga 360 caaacatcat
catagcgagt gtgctgggca agggacgcca gcagttggta aatgatcggg 420
cgacaattcc ttgggtaaca aacaaacgag ttgcagagtg tcaggtgacc ttgcttagtg
480 tagtagttgg actggataat gaatagcaga gcaccttgaa atctgaggac
gttatagttg 540 ttttaaaaat gtcttcacaa ggccctcatg ttgtgtaaag
gagattttaa aattttagtt 600 caacaaaaag aagacatgtt tactgtgtat
cttagcctgt tttagttctg ggacagacac 660 catagatacc gagctgaaac
aacaagtgct ctttacccac agttccaggg cgtgggaagt 720 acaacatcaa
agctctgatg caagtggtgt ccgtgaggat ccatttcctg ctatgcagat 780
gctgcaaaca tatgactccg gcctcttcct cttctttaag ggcatgtgtc tgaagtgggg
840 tggggtggca tcctcatgac ttcatctgtg ttttccctag agatagcccc
tttggttagg 900 gttttaacat agatgtttgg tgcacacata catttagtct
atgatagtct ctagcaacag 960 ccagttggga gggaaggaga gggagaggga
gagagagaga gagagagaac acatgtatgc 1020 gagtctgtga tttctagctt
cttagcttct tatatttgat ctaattttca tttattttat 1080 gattctggaa
ctctgttgtt ttattacttt gaaattctag gactccaaat aaatattttt 1140
gatcttaaaa tattatcatt attttattct gatcccagaa ttc 1183
* * * * *