JP2023006214A - Model animal of timothy syndrome - Google Patents

Abstract

To provide a model animal of Timothy syndrome.SOLUTION: A model animal of Timothy syndrome expresses the mutated CACNA1C gene including a causative mutation for Timothy syndrome symptoms, only in tissue other than the heart.SELECTED DRAWING: None

Description

The present invention relates to a model animal of Timothy's syndrome and the like.

Timothy syndrome (TS) is a systemic disorder caused by a single-nucleotide mutation (G406R) in the voltage-gated Ca ²⁺ channel (Cav1.2, gene name CACNA1C). Known characteristic symptoms include cardiac QT prolongation and autism. TS1 and TS2 are classified according to the exon into which the mutation is introduced, and the mutation in Cacna1c Exon8A is named TS1, and the mutation in Cacna1c Exon8 is named TS2. This Exon8 and Exon8A are mutually exclusive Splicing variants, and it is known which Exon is more expressed depending on the organ. G402S mutation is known as another mutation in Exon8 (Non-Patent Document 1).

In addition, in Non-Patent Document 2, as a result of screening patients showing cardiac QT prolongation, several mutations in the CACNA1C gene were identified, and these mutations, like TS, caused changes in current amplitude, inactivity It has been reported that it brings about changes in activation rate, voltage dependence of activation/inactivation, etc., and is likely to cause QT prolongation.

2018 Feb 1;20(2):377-385. Journal of Molecular and Cellular Cardiology Volume 80, March 2015, Pages 186-195. Circulation: Arrhythmia and Electrophysiology. 2015;8:1122-1132. 2021; 9: 668546. Proc Natl Acad Sci U S A. 2011 Sep 13;108(37):15432-7.

In the existing TS model mouse TS2-neo, expression of the mutant channel is suppressed because the drug-resistant gene expression cassette is located near the mutant channel expression cassette. According to Non-Patent Document 3, the expression rate of mutant channels (=mutant channel expression level/(mutant channel expression level + wild-type channel expression level)) is about 25%, which was confirmed by the present inventor to be low. . However, in actual TS patients, one of the alleles is mutated, so the expression rate of the mutated channel is considered to be about 50%. However, simply changing the position of the drug-resistant gene expression cassette, for example, in the mouse model described above in order to increase the expression ratio of the mutated channel will increase the mortality rate as the mutated channel is expressed in the heart above a certain level, leading to increased breeding. It is thought that it will be difficult to keep them alive until their working age.

An object of the present invention is to provide an animal model of Timothy's syndrome. Preferably, an object of the present invention is to provide an animal model of Timothy's syndrome in which the expression rate of mutant channels can be increased.

As a result of intensive research in view of the above problems, the present inventors focused on expressing a mutant CACNA1C gene containing a causative mutation of Timothy's syndrome only in tissues other than the heart. Although the use of the Cre-loxP system is conceivable for the production of such mutants, the desired mutants could not be obtained by conventional methods. As a result of further intensive research, the present inventors succeeded in producing animals in which the mutant CACNA1C gene containing the causative mutation of Timothy's syndrome is expressed only in tissues other than the heart, thereby completing the present invention. That is, the present invention includes the following aspects.

Section 1. An animal model of Timothy syndrome that expresses a mutant CACNA1C gene containing a mutation that causes Timothy syndrome symptoms only in tissues other than the heart.

Section 2. Item 2. The model animal according to Item 1, which has the mutant CACNA1C gene on only one of a pair of chromosomes.

Item 3. Item 3. The animal model according to Item 1 or 2, wherein the tissue comprises at least one selected from the group consisting of nerves, gastrointestinal tracts, skin, eyes, nose, teeth, lungs, endocrine organs, muscles, and immune systems.

Section 4. Item 4. The model animal according to any one of Items 1 to 3, wherein the tissue comprises nerves.

Item 5. Item 5. The model animal according to any one of items 1 to 4, wherein the causative mutation is a mutation in the transmembrane domain or intracellular domain of the CACNA1C protein.

Item 6. wherein the mutation in the transmembrane domain is a mutation in the vicinity of the intracellular domain within the transmembrane domain and/or the mutation in the intracellular domain is a mutation in the vicinity of the transmembrane domain in the intracellular domain 5. The model animal according to 5.

Item 7. The causative mutations are from G406, G402, A28, R860, I1166, I1475, E1496, E407, G419, R518, S643, R1115, A1473, G1911, A582, K834, P857, R858, R860, and R1906 in the human CACNA1C protein Item 7. The model animal according to any one of items 1 to 6, wherein the mutation causes an amino acid mutation corresponding to at least one amino acid selected from the group consisting of:

Item 8. Item 8. The model animal according to any one of Items 1 to 7, wherein the causative mutation is a mutation that causes an amino acid mutation corresponding to G406 in the human CACNA1C protein.

Item 9. Item 9. The model animal according to any one of items 1 to 8, wherein the causative mutation is a mutation that causes a mutation corresponding to the G406R mutation in human CACNA1C protein.

Item 10. A fertilized egg, sperm or embryo developed in the model animal according to any one of Items 1 to 9.

Item 11. A cell derived from the model animal according to any one of Items 1 to 9.

Item 12. An agent for preventing, improving, or exacerbating Timothy syndrome using the model animal according to any one of Items 1 to 9, or the fertilized egg, sperm or embryo according to Item 10, or the cell according to Item 11. screening method.

Item 13. (a) administering or contacting a test substance to the model animal according to any one of items 1 to 9, or the fertilized egg, sperm or embryo according to item 10, or the cell according to item 11;
(b) assessing the Timothy syndrome phenotype of the model animal, the fertilized egg, sperm or embryo, or the cell administered or contacted with the test substance; and (c1) the phenotype is cured or alleviated. (c2) selecting the test substance as a preventive or ameliorating agent for Timothy syndrome if the sorting as
Item 13. The screening method according to Item 12, comprising

According to the present invention, a model animal and model cells of Timothy's syndrome can be provided.

Schematic representation of the allele structure of FLEx mice. 2 shows the measurement results of the expression ratio of the mutant channel (G406R mutant) in Test Example 3. FIG. 4 shows the measurement results of grooming time in Test Example 4-1. The outline of the method of the 3-chamber test of Test Example 4-2 is shown. The measurement results of the 3-chamber test of Test Example 4-2 are shown.

1. Definitions As used herein, the expressions "contain" and "include" include the concepts "contain", "include", "consist essentially of" and "consist only of".

Amino acid sequence “identity” refers to the degree of amino acid sequence identity of two or more comparable amino acid sequences to each other. Therefore, the higher the identity or similarity between two amino acid sequences, the higher the identity or similarity between those sequences. The level of amino acid sequence identity is determined, for example, using the sequence analysis tool FASTA, using default parameters. or Algorithm BLAST by Karlin and Altschul (Karlin S, Altschul SF. "Methods for assessing the statistical significance of molecular sequence features by using general scoringschemes" Proc Natl Acad Sci USA. 87:2264-2268 (1990) Karlin S, Altschul SF. "Applications and statistics for multiple high-scoring segments in molecular sequences." Proc Natl Acad Sci USA. 90:5873-7 (1993)). A program called BLASTX based on such a BLAST algorithm has been developed. Specific methods of these analysis methods are known, and the website of the National Center of Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/) can be referred to. "Identity" of base sequences is also defined according to the above.

As used herein, "conservative substitution" means that an amino acid residue is replaced with an amino acid residue having a similar side chain. For example, substitutions between amino acid residues having basic side chains such as lysine, arginine, and histidine correspond to conservative substitutions. In addition, amino acid residues having acidic side chains such as aspartic acid and glutamic acid; amino acid residues having uncharged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine and cysteine; alanine, valine, leucine, isoleucine, amino acid residues with nonpolar side chains such as proline, phenylalanine, methionine, tryptophan; amino acid residues with β-branched side chains, such as threonine, valine, isoleucine; and aromatic side chains, such as tyrosine, phenylalanine, tryptophan, histidine. Substitutions between amino acid residues are also conservative substitutions.

2. Model animal, cell In one aspect of the present invention, a model animal of Timothy syndrome expressing a mutant CACNA1C gene containing a causative mutation of Timothy syndrome symptoms only in tissues other than the heart (herein, "the present invention (sometimes referred to as "model animal of"). This will be explained below.

Animals include, but are not limited to, mammals such as monkeys, mice (mus musculus), rats, dogs, cats, rabbits, pigs, horses, cows, sheep, goats, and deer. Among these, animals that can be used as experimental animals are preferred, Mus musculus is more preferred, and Mus musculus is even more preferred. In addition, mammals may be lineage species or hybrids. In the case of mouse strains, for example, C57BL/6, C3H, ICR, and BALB/c can be mentioned, but C57BL/6 is preferred. When humans are used as model animals, cells derived from healthy individuals or diseased patients, iPS cells, and the like are assumed to be used.

CACNA1C (Calcium Voltage-Gated Channel Subunit Alpha1 C) gene is a voltage-gated Ca ²⁺ channel gene. Nucleotide sequences and amino acid sequences of CACNA1C genes are known in various animals such as mammals, or can be easily determined based on the known nucleotide sequences and amino acid sequences of CACNA1C genes (for example, by identity analysis, etc.). be able to. As an example, the mouse CACNA1C gene is a gene identified by NCBI Gene ID: 12288, and its amino acid sequence includes the amino acid sequence (SEQ ID NO: 3) identified by NCBI RefSeq accession number: NP_001153006.1. , and its mRNA sequence includes the nucleotide sequence (SEQ ID NO: 5) specified by NCBI RefSeq accession number: NM_001159534.2.

The CACNA1C gene targeted by the present invention includes functionally normal mutants that can occur in nature. The CACNA1C gene targeted by the present invention may have nucleotide mutations such as substitutions, deletions, additions, and insertions as long as the enzymatic activity of the encoded protein is not significantly impaired. The mutation is preferably a mutation that does not cause amino acid substitution or a mutation that causes conservative amino acid substitution in the protein translated from the mRNA.

The CACNA1C gene targeted by the present invention has, for example, an amino acid sequence of a protein encoded by it that is 95% or more, preferably 98% or more, of the amino acid sequence of a protein encoded by a wild-type CACNA1C gene of a homologous animal. , more preferably a gene having an identity of 99% or more. In addition, the CACNA1C gene targeted by the present invention is, for example, the amino acid sequence of the protein encoded by it is identical to the amino acid sequence of the protein encoded by the wild-type CACNA1C gene of the homologous animal, or 1 or more (for example, 2 to 10, preferably 2 to 5, more preferably 2 to 3, still more preferably 2) are substituted, deleted, added, or inserted into the amino acid sequence of the gene is.

The model animal of the present invention has a mutant CACNA1C gene containing a causative mutation for Timothy's syndrome symptoms. That is, in the model animal of the present invention, the CACNA1C gene is replaced with the mutant CACNA1C gene in one or both of the paired chromosomes. From the viewpoint of ensuring a constant life span of the model animal, the model animal of the present invention preferably has the mutant CACNA1C gene on only one of the paired chromosomes.

Symptoms of Timothy syndrome are not particularly limited as long as they are recognized as symptoms of patients with Timothy syndrome.・Frequently found in the lower limbs), baldness from birth, microdontia, cardiac malformations (patent foramen ovale, patent ductus arteriosus, ventricular septal defect, etc.), mental retardation, facial malformation, Myopia, immunodeficiency, intermittent hypoglycemia, hypothermia, muscle disease, language development delay, etc. Among these, QT interval prolongation is preferred.

The causative mutation of Timothy syndrome symptoms is not particularly limited as long as it can cause Timothy syndrome symptoms. Causative mutations preferably include mutations within the transmembrane domain or mutations within the intracellular domain of the CACNA1C protein. In addition, which region is the transmembrane domain and intracellular domain of the CACNA1C protein is already known, or using a transmembrane domain prediction program based on known sequence information, and / or the transmembrane domain and the intracellular domain can be easily determined based on a comparison with the known amino acid sequence of the CACNA1C protein.

In a more preferred embodiment of the present invention, the mutation within the transmembrane domain is a mutation near the intracellular domain within the transmembrane domain, and/or the mutation within the intracellular domain is a mutation near the transmembrane domain within the intracellular domain. can be a mutation in Here, the "near the intracellular domain within the transmembrane domain" is, for example, the half region of the transmembrane domain on the intracellular domain side, preferably consisting of 1 to 30 amino acids on the intracellular domain side of the transmembrane domain. It can be a region, a region consisting of 1-20 amino acids, or a region consisting of 1-10 amino acids. Similarly, the term "near the transmembrane domain within the intracellular domain" is, for example, a region consisting of 1 to 30 amino acids from the transmembrane domain side of the intracellular domain, preferably 1 to 30 amino acids from the transmembrane domain side of the intracellular domain. It can be a region consisting of 20 amino acids, or a region consisting of 1-10 amino acids.

Specific examples of causative mutations include G406, G402, A28, R860, I1166, I1475, E1496, E407, G419, R518, S643, R1115, A1473, G1911 in human CACNA1C protein (amino acid sequence: SEQ ID NO: 6), Examples include mutations that cause amino acid mutations corresponding to at least one amino acid selected from the group consisting of A582, K834, P857, R858, R860, and R1906. The above amino acid notation is amino acid + the amino acid number from the N-terminus. That is, G406 means the 406th amino acid (glycine) from the N-terminus of SEQ ID NO:6. Amino acids corresponding to the above amino acids of the human CACNA1C protein, that is, corresponding amino acids in other CACNA1C proteins (for example, CACNA1C proteins of other species) can be easily identified by identity analysis with the human CACNA1C protein. More specifically, for example, the amino acid sequence of the human CACNA1C protein (SEQ ID NO: 6: amino acid sequence 1) is compared with the amino acid sequence of the CACNA1C protein (amino acid sequence 2) different from the amino acid sequence, and the identity is maximized. When both are arranged in parallel, the residue in amino acid sequence 2 corresponding to amino acid residue G406 in amino acid sequence 1 can be determined to be the “corresponding amino acid”.

The causative mutation is preferably an amino acid substitution. In this case, preferred amino acids after substitution in specific examples of the causative mutations described above are as follows:
G406: preferably arginine, lysine, histidine, more preferably arginine
G402: preferably serine, threonine, more preferably serine
A28: Preferably serine, threonine, more preferably threonine
R860: preferably glycine, alanine, more preferably glycine
I1166: preferably serine, threonine and valine, more preferably threonine and valine
I1475: preferably methionine, cysteine, more preferably methionine
E1496: preferably arginine, lysine, histidine, more preferably lysine
E407: preferably alanine, glycine
G419: preferably arginine, lysine, histidine, more preferably arginine
R518: preferably arginine, lysine, histidine, cysteine, methionine, more preferably histidine, cysteine
S643: preferably phenylalanine, tryptophan, tyrosine, more preferably phenylalanine
R1115: preferably arginine, lysine, histidine, more preferably lysine
A1473: preferably glycine
G1911: preferably arginine, lysine, histidine, more preferably arginine
A582: Preferably aspartic acid, glutamic acid, more preferably aspartic acid
K834: preferably aspartic acid, glutamic acid, more preferably glutamic acid
P857: preferably arginine, lysine, histidine, leucine, isoleucine, valine, more preferably arginine, leucine
R858: preferably arginine, lysine, histidine, more preferably histidine
R860: preferably glycine, alanine, more preferably glycine
R1906: preferably glutamine, asparagine, more preferably glutamine.

The causative mutation is preferably a mutation that causes an amino acid mutation corresponding to G406 in the human CACNA1C protein, more preferably a mutation that causes a mutation corresponding to the G406R mutation in the human CACNA1C protein.

The animal model of the present invention expresses the mutant CACNA1C gene only in tissues other than the heart. That is, the model animal of the present invention does not express the mutant CACNA1C gene in the heart, but expresses it in tissues other than the heart. The term "express" means that mRNA and protein, which are gene expression products, are produced.

Tissues other than the heart are not particularly limited, and include, for example, nerves, gastrointestinal tracts, skin, eyes, noses, teeth, lungs, endocrine organs, muscles, and the immune system. Among these, nerves are preferred, central nerves are more preferred, and brains are even more preferred.

The expression ratio of the mutant CACNA1C gene in tissues expressing the mutant CACNA1C gene (=mutant CACNA1C gene expression level/(mutant CACNA1C gene expression level+wild-type CACNA1C gene expression level)) is, for example, 15% or more. The expression ratio is preferably 20% or higher, more preferably 25% or higher, still more preferably 30% or higher, and even more preferably 35% or higher. The upper limit of the ratio is not particularly limited, but is, for example, 70%, 60%, 55%, or 50%. The expression ratio can be measured according to or according to the method of Test Example 3 described later.

The animal model of the present invention has symptoms of Timothy's syndrome, such as autism, syndactyly (simple syndactyly, bilaterally symmetrical, often found on both upper and lower limbs), baldness from birth, and microdontia. , intellectual developmental disorder, facial malformation, myopia, immunodeficiency, intermittent hypoglycemia, hypothermia, muscle disease, language development delay, etc. The animal model of the present invention can be used, for example, for pathological analysis of Timothy's syndrome, screening of therapeutic agents, and the like.

In one aspect, the present invention relates to cells derived from the animal model of the present invention (cells of the present invention). The cells of the present invention can be obtained by harvesting cells from tissue expressing the mutant CACNA1C gene in the model animal of the present invention. The cells of the present invention can be primary cultured cells, or can be cell lines of the primary cultured cells.

The cells of the present invention can be used as model cells of Timothy's syndrome, for example, for pathological analysis of Timothy's syndrome, screening of therapeutic agents, and the like. Cells are not particularly limited, whether they are primary cultured cells or cell lines. For example, nerve cells, blood cells, hematopoietic stem/progenitor cells, gametes (sperm, ovum), fibroblasts, epithelial cells , vascular endothelial cells, hepatocytes, keratinocytes, muscle cells, epidermal cells, endocrine cells, ES cells, iPS cells, tissue stem cells, cancer cells and the like. The organisms from which the cells are derived are not particularly limited, and examples thereof include mammals such as humans, monkeys, mice (mus musculus), rats, dogs, cats, rabbits, pigs, horses, cows, sheep, goats, and deer. .

3. Method for producing model animals and cells The model animals of the present invention can be produced by homologous recombination using a targeting vector and recombination with Cre.

As shown in the upper part of FIG. 1, the targeting vector is a mutant exon in which the mutation of interest (causative mutation of Timothy syndrome symptoms) is introduced in the CACNA1C gene, and the corresponding wild-type exon is arranged face-to-face. has a structure in which loxP and lox2272 are arranged as shown in Fig. 1. That is, if one side is direction 1 and the opposite side is direction 2, from direction 1 side, lox2272 (or loxP) in direction 1, wild-type exon, loxP (or lox2272) in direction 1, opposite to wild-type exon The structure is arranged in the order of orientation mutant exon, orientation 2 orientation lox2272 (or loxP), orientation 2 orientation loxP (or lox2272).

A spacer sequence is preferably introduced between the mutant exon and the wild-type exon. This allows higher expression rates of mutant channels. The base length of the spacer sequence is, for example, 300 or more, preferably 600 or more, more preferably 800 or more, and still more preferably 1000 or more. As the spacer sequence, any sequence that does not affect gene expression or genome stability (eg, transcription terminator sequence, polyadenylation sequence, etc.) can be used in combination.

A targeting vector is a DNA containing a nucleotide sequence in which a 5' homology arm, the structure described above, and a 3' homology arm are arranged from the 5' side.

The 5'-side homology arm and 3'-side homology arm refer to the base sequences of the regions adjacent to the 5'-side and 3'-side of the genomic region that is the target site to be replaced with the above structure. Homologous recombination via these homology arms can replace the above structure.

The targeting vector may have other sequences besides the above. Other sequences include, for example, expression cassettes of negative selection markers for homologous recombination (eg, DTA (Diptheria Toxin A) cassettes, etc.).

Homologous recombination is usually carried out by introducing a targeting vector into ES cells. The introduction method is not particularly limited, and an example thereof is electroporation. After introduction of the targeting vector, drug selection is usually performed using drug resistance genes. ES cells in which substitution to the above structure has occurred are aggregated with early embryos, transplanted into pseudopregnant mice to produce chimeric animals, and animals having the above structures heterozygously or homozygously can be obtained by mating between chimeric animals. .

Subsequently, an animal having the above structure is used to specifically express Cre in tissues other than the heart (specifically, for example, an expression cassette in which the Cre coding sequence is linked under the control of a specific promoter for tissues other than the heart). in the genome) to obtain animals that have the above structure and express Cre specifically in tissues other than the heart. The animal expresses Cre specifically in tissues other than the heart, and the enzymatic activity of the Cre causes recombination specifically in the tissues other than the heart, as representatively illustrated in FIG. An animal of the present invention can be obtained in this manner.

A fertilized egg, sperm and embryo (eg, frozen embryo) developed in the model animal of the present invention obtained in the process of the production method described above is also an aspect of the present invention. In addition, a fertilized egg obtained by fertilizing the sperm and/or egg of the model animal of the present invention is also a fertilized egg that can develop in the model animal of the present invention, and such a fertilized egg is also an aspect of the present invention. . These fertilized eggs are not particularly limited as long as they can be generated in vivo from a cryopreserved state, and include eggs immediately after fertilization, pronuclear stage embryos, early embryos, blastocysts, and the like.

Four. Use of Model Animals and Cells The model animals of the present invention, the fertilized eggs, sperm and embryos developed in them, and the cells of the present invention can be used for analysis of Timothy's syndrome pathology, development of preventive methods, development of therapeutic methods, etc. .

In one aspect, the present invention relates to a screening method for Timothy syndrome preventive agents, ameliorating agents, or exacerbation causative agents using the model animal of the present invention, the fertilized egg or embryo developed in this, or the cell of the present invention. .

The screening method is not particularly limited as long as the model animal of the present invention or the cell of the present invention is used. For example, steps (a), (b), and (c1) or (c2):
(a) a step of administering or contacting a test substance to the model animal of the present invention, or the fertilized egg, sperm or embryo of the present invention, or the cell of the present invention;
(b) assessing the Timothy syndrome phenotype of the model animal, the fertilized egg, sperm or embryo, or the cell administered or contacted with the test substance; and (c1) the phenotype is cured or alleviated. (c2) selecting the test substance as a preventive or ameliorating agent for Timothy syndrome if the sorting as
including.

The type of test substance is not particularly limited as long as it can be a candidate for a therapeutic drug. Examples include proteins, peptides, non-peptide compounds (nucleotides, amines, carbohydrates, lipids, etc.), organic low-molecular-weight compounds, inorganic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and the like. .

Administration can be performed according to known methods. For example, enteral administration such as oral administration, tube feeding, and enema administration; mentioned.

Phenotypic evaluation can be performed according to or according to known methods.

Useful therapeutic drugs can be selected by proceeding with further analysis using the selected test substances as therapeutic candidate substances.

EXAMPLES The present invention will be described in detail below based on examples, but the present invention is not limited by these examples.

Test example 1. Generation of recombinant mouse The FLEx system was used as a new mouse model. In the CACNA1C gene, wild-type Exon8 (nucleotide sequence: SEQ ID NO: 1) and mutant Exon8 (nucleotide sequence: SEQ ID NO: 2) are used as appropriate spacer sequences (sequences that do not affect gene expression or genome stability can be candidates. In this model, 1300 bases (SEQ ID NO: 7) in which the yeast His3 gene transcription terminator sequence and the SV40 polyadenylation sequence are combined are used. As a result (FIG. 1), the CACNA1C gene (amino acid sequence: SEQ ID NO: 3) having wild-type Exon8 is expressed in the absence of Cre enzyme, and the CACNA1C gene (amino acid sequence: A recombinant mouse (heterozygous mouse of FLEx Allele (FIG. 1)/WT Allele) in which SEQ ID NO: 4, G406R mutation) was expressed was produced according to a conventional method. This new strain was named FLEx mice.

Test example 2. Mice that express the Cre enzyme specifically in the mouse mating nervous system (mouse containing an expression cassette (Cre Allele) with a Cre coding sequence under the control of the SOX1 promoter, Cre Allele + / Cre Allele - heterozygous mice) and FLEx mice. They were mated to obtain offspring mice. There are four genotypes of offspring mice: WT mice, Cre mice, FLEx mice, and cKI mice.

Test example 3. Measurement of mutant channel expression ratio The expression ratio of the mutant channel (G406R mutant) was confirmed by the same method as in Non-Patent Document 5. Female mice were bred until 14 weeks of age, mouse brains were excised, and RNA was extracted from a portion of the brains using the PureLink RNA Mini kit (ThermoFisher Scientific). cDNA was prepared from RNA using PrimeScrip RT Master Mix (Takara). Using the cDNA as a sample, PCR was performed using primers targeting Exons (Exon7, 9) on both sides of the target Exon (Exon8 or Exon8A), and a plasmid was constructed using the product. This plasmid was introduced into E. coli, each sample was spread on each plate, and 30-32 colonies were recovered from each plate. The plasmid sequence of each colony was sequenced, and the expression ratio was calculated from the colony counts of wild-type Exon8, Exon8A, and mutant-type Exon8. The mutation rate was calculated as the sum of mutant Exon8 (G406R) and wild-type Exon8 and the rate of mutant Exon8 (G406R) in the same manner as in Non-Patent Document 1.

The results are shown in FIG. cKI-Nii (Test Example 2: cKI mice (KI Allele (Fig. 1) / WT Allele heterozygous and Cre Allele + / Cre Allele - heterozygous mice)) expressed the mutant channel (G406R mutant) expression rate was 41%, which was higher than that of TS2-Neo (model mouse produced in Non-Patent Document 5).

In addition, cKI-Nii was confirmed to survive up to about 16 weeks after birth, with the exception of some individuals.

Test example 4. behavioral experiment
Behavioral experiments were performed on WT, Cre, FLEx and cKI mice. Before the behavioral experiment, each animal was handled for 5 minutes a day for 3 consecutive days. All behavioral experiments were conducted after the mice were allowed to wait in the waiting room for 1 hour or longer.

<Test Example 4-1. Grooming＞
Seven-week-old male mice were used. The cage was placed in a soundproof box adjusted to 100 lux and the mouse was placed in the corner of the cage. First, they were acclimatized for 10 minutes, then a video was recorded for 10 minutes as a test, and the experimenter confirmed the grooming time of the video.

The results are shown in FIG. As a result of testing in 4 groups (one way anova, post hoc tukey), cKI significantly increased grooming time compared to WT.

<Test example 4-2.3 chamber test>
9-10 week old male mice were used. Two days before the experiment, the experimental mice were changed from group housing to individual housing. A 60*40 cm box (10 lux) was divided into a left chamber, a center chamber and a right chamber every 20 cm. A quadrant-shaped cage with a radius of 10 cm was placed in the corner of the box in the left and right chambers, the stimulus mouse was placed therein, and moving images were taken and analyzed using automatic analysis software TimeCSI (O'Hara & Co). A total of two trials were performed. As shown in FIG. 4, trial 1 was with the left and right cages empty, and trial 2 was with stimulus mouse 1 in the right cage, and the experimental mice were allowed to explore freely for 12 minutes.

The results are shown in FIG. In Trial 1, there was no difference between the four groups in the stay time near the left and right cages. In trial 2, in WT, Cre, and FLEx, the staying time near the right cage (the cage containing the stimulated mouse 1) was significantly longer than that in the left cage (empty cage). there was no difference.

Increased stereotyped behavior (increased grooming time) and social abnormalities (abnormal responses to stimulus mice in the 3-chamber test) indicated that cKI mice (Test Example 2) exhibited autism-like behaviors, and neuronal It was found to be valid as a model of systemic Timothy syndrome.

Claims (13)

An animal model of Timothy syndrome that expresses a mutant CACNA1C gene containing a mutation that causes Timothy syndrome symptoms only in tissues other than the heart. 2. The model animal according to claim 1, which has the mutant CACNA1C gene on only one of a pair of chromosomes. 3. The model animal according to claim 1 or 2, wherein said tissue comprises at least one selected from the group consisting of nerves, gastrointestinal tracts, skin, eyes, nose, teeth, lungs, endocrine organs, muscles, and immune system. . The model animal according to any one of claims 1 to 3, wherein said tissue contains nerves. The model animal according to any one of claims 1 to 4, wherein the causative mutation is a mutation in the transmembrane domain or mutation in the intracellular domain of CACNA1C protein. The mutation in the transmembrane domain is a mutation in the vicinity of the intracellular domain within the transmembrane domain, and/or the mutation in the intracellular domain is a mutation in the vicinity of the transmembrane domain in the intracellular domain. Item 5. The model animal according to item 5. The causative mutations are from G406, G402, A28, R860, I1166, I1475, E1496, E407, G419, R518, S643, R1115, A1473, G1911, A582, K834, P857, R858, R860, and R1906 in the human CACNA1C protein 7. The model animal according to any one of claims 1 to 6, wherein the mutation causes an amino acid mutation corresponding to at least one amino acid selected from the group consisting of: The model animal according to any one of claims 1 to 7, wherein the causative mutation is a mutation that causes an amino acid mutation corresponding to G406 in the human CACNA1C protein. The model animal according to any one of claims 1 to 8, wherein the causative mutation is a mutation that causes a mutation corresponding to the G406R mutation in human CACNA1C protein. A fertilized egg, sperm or embryo developed in the model animal according to any one of claims 1 to 9. A cell derived from the model animal according to any one of claims 1 to 9. Timothy syndrome using the model animal according to any one of claims 1 to 9, or the fertilized egg, sperm or embryo according to claim 10, or the cell according to claim 11, an agent for preventing or improving Timothy syndrome, or Screening method for exacerbation-causing substances. (a) a step of administering or contacting a test substance to the model animal according to any one of claims 1 to 9, or the fertilized egg, sperm or embryo according to claim 10, or the cell according to claim 11; ,
(b) assessing the Timothy syndrome phenotype of the model animal, the fertilized egg, sperm or embryo, or the cell administered or contacted with the test substance; and (c1) the phenotype is cured or alleviated. (c2) selecting the test substance as a preventive or ameliorating agent for Timothy syndrome if the sorting as
13. The screening method of claim 12, comprising:

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