WO2005038027A1

WO2005038027A1 - Transgenic nonhuman mammal

Info

Publication number: WO2005038027A1
Application number: PCT/JP2004/015872
Authority: WO
Inventors: Tadafumi Kato; Takaoki Kasahara
Original assignee: Riken
Priority date: 2003-10-21
Filing date: 2004-10-20
Publication date: 2005-04-28
Also published as: JP2005124412A; JP3817638B2

Abstract

It is intended to provide an animal model of manic depression which spontaneously shows depressive conditions and manic conditions. Namely, a transgenic nonhuman mammal characterized by having a gene encoding a mutant mtDNA synthase (polymerase Ϝ) having been transferred thereinto and thus brain-specifically expressing this gene, or a part of the same.

Description

Genetic non-human mammals Technical field

The present invention relates to transgenic non-human mammals. More specifically, the present invention relates to a transgenic non-human mammal of a manic-depression model into which a gene encoding a mutant mtDNA synthetase (polymerase γ) has been introduced, and a method of using the same. . Background art

Manic Depression (Bipolar Disorder) (Since the word manic depression is ambiguous whether or not to include depression, it has recently been often referred to as bipolar disorder according to the American Psychiatric Association diagnostic criteria. ) Is a mental illness that develops in adulthood and repeats two phases, depression and manic. Approximately 20% of manic-depressed patients die from suicide, and social problems in a manic state rarely lose their social lives. Depression, on the other hand, is often stress-induced and ends in a single episode, but the-part can repeat independently of stress, and because of its power in manic-depressed families, Such recurrent depression is also considered a related disorder of manic depression. In manic-depressive illness, drugs called mood stabilizers (three include lithium, valproic acid, and carbamazepine) are known to have preventive effects. Many patients are ineffective, and the development of new drugs is required. Tools for the development of new drugs include the use of animal models. The following models are known as models of depression due to behavioral and pharmacological loads, and models of manic state due to pharmacological loads.

(1) Model for determining the effect of antidepressants

There are two behavioral tests used in rodents (rats) to test the effects of antidepressants: tail suspension test and forced swimming test. In the tail suspension test, the rat's tail Hang around. In forced swimming, rats are placed in a cylindrical water tank. Animals are desperate and immobile when subjected to rappelling and forced swimming. However, administration of existing antidepressants prolongs this immobility time. For this reason, when a new antidepressant is developed, this test is often used to determine whether it is effective. However, this puts stress on healthy mice and only looks at the behavior during stress, not a model of depression.

(2) Depression model

In a typical model of depression, a model of learning helplessness, a rat is first placed in a box with two compartments connected by a tunnel. Initially, both boxes are shocked. The rat then escapes to another box, but finds that there is an electric shock there. The next day, an electric shock is applied to only one compartment. In this way, many rats (while knowing that the opposite box is also shocked?) Will escape to the opposite compartment anyway. However, some rats (knowing that the opposite box is also electrically shocked and giving up from the beginning) never try to escape. Rats that do not try to escape in the second experiment are called learned helpless rats, and this is a model of depression. Administering antidepressants to these helpless rats will avoid them again. Since this model looks at long-term behavioral changes after stress, it can be said to be a depression model. There are several other models that are depressed, such as models that have been forced to run for a long time and those that have become immobile due to drug administration.

(3) Manic model

Animals are overactive when psychostimulants such as amphetamines and cocaine are administered. For this reason, there is a concept that this is a manic animal model. However, after long-term administration of amphetamine-cocaine, abnormal behaviors such as stereotypy appear, which is also considered an animal model for schizophrenia (schizophrenia). Therefore, it is not a specific model.

As described above, behavioral and pharmacological load models of depression, pharmacological load There is a model of manic state due to However, there is no report of a model in which spontaneous depressive state ゃ manic state appears, and the absence of an animal model of manic depressive disorder has hindered research on the pathological state of manic-depressive disorder and development of new drugs. Disclosure of the invention

An object of the present invention is to solve the above-mentioned problems of the conventional technology. That is, an object of the present invention is to provide an animal model of manic-depressive disorder in which a spontaneous depressive state ゃ manic state appears. Further, the present invention has an object to provide a method for screening for a therapeutic agent and / or a prophylactic agent for manic depression using the above animal model.

The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, have found that a transgenic mouse that specifically expresses a mutant mt DNA synthetase (a gene encoding a polymerase by introducing the gene into the brain) is obtained. The transgenic mice were successfully produced, and the activity of the transgenic mice produced was recorded over a long period of time, and behavioral analysis was performed.As a result, the transgenic mice were useful as an animal model for manic depression. The present invention has been completed based on these findings.

That is, according to the present invention, a transgenic non-human gene characterized in that a gene encoding a mutant mtDNA synthetase (polymerase γ) is introduced into the brain by introducing the gene. A human mammal or a portion thereof is provided. Preferably, in the transgenic non-human mammal of the present invention, the deleted short mt DNA is accumulated in the brain. Preferably, the transgenic non-human mammal of the invention is capable of spontaneously exhibiting periodic behavioral changes. Preferably, the transgenic non-human mammal of the present invention has a recombinant in which a gene encoding a mutant mtDNA synthase (polymerase _y ) is incorporated under the control of a promoter that enables brain-specific expression. DNA has been introduced. Preferably, the promoter enabling brain-specific expression is calmodulin kinase IIa (CAMKI la) promoter or neural specific enolase (NSE) promoter. Preferably, the gene encoding the mutant mtDNA synthetase (polymerase _y ) is obtained by substituting alanine for aspartic acid, the 18 th amino acid residue, in the gene encoding mouse mtDNA synthase. Is a gene that has been Preferably, the non-human mammal is a mouse.

According to another aspect of the present invention, there is provided a method for screening a therapeutic and / or prophylactic agent for manic depression, comprising using the above-mentioned non-human mammal of the present invention or a part thereof. You. According to still another aspect of the present invention, there is provided a substance obtained by the above-described screening method. According to yet another aspect of the present invention, there is provided a therapeutic agent and / or a preventive agent for manic-depressive disease, comprising as an active ingredient a substance obtained by the above-described screening method. Brief Description of Drawings

FIG. 1 shows the structure of the recombinant vector for gene transfer used in the present invention. Asparaginic acid, which is the 18th amino acid residue of mouse Ροΐγ, was replaced with alanine by site-specific mutagenesis (adenine, the 580th base in the coding region, was replaced with cytosine). did) . The construct shown in FIG. 1 was prepared by inserting the coding region of this mutant Poly into the I site of the pMM403 vector [Mayford M. et al., Science 274, 1678-1683 (1996)].

FIG. 2 shows the results of examining the gene expression of Poly in each tissue of the two transgenic mice prepared (line B and C).

Figure 3 shows PCR-Southern plots of short, abnormal mitochondrial DNA (mtDNA) in each tissue of transgenic (Tg) and wild-type (non-Tg) mice from littermates B and C, respectively. The detection result is shown.

Figure 4 shows the results of measuring the activity (number of rotations) of the mice on line B one by one in cages equipped with a rotation device over a period of about one month. The activity of a representative individual is shown in a notation called a double plot. The vertical direction (arrow direction) is the number of days, The horizontal axis is time, and displays 48 hours of data per horizontal line. The first line shows the data for the first and second days, and the second line shows the data for the second and third days. A double plot is a drawing method that makes it easy to see the rhythmicity of the amount of activity by displaying such overlapping. The black and white strips on each panel indicate the light conditions of the breeding box, the white time zone is lit, and the black time zone is off. Mice are active during the black hours due to nocturnal activity.

FIG. 5 shows the results of plotting the total amount of wheel rotation per mouse for all the measured individuals (5 each for Tg mice and non-Tg mice).

Figure 6 shows the results of using the Lomb-Scarge periodic analysis method to determine the periodicity of wheeling behavior. Each curve represents the rhythm of each mouse. It can be seen that there is a periodicity of 110 to 130 hours as well as a periodicity of 24 hours. The vertical axis indicates the strength of the periodicity (probability), and the dotted horizontal line in the graph indicates the probability corresponding to p = 0.02.

Figure 7 shows the measurement results of the open field test, the elevated plus maze test, and the forced swimming test. BEST MODE FOR CARRYING OUT THE INVENTION

First, the history of the development of the transgenic non-human mammal of the present invention and the results thereof will be described.

(i) Mitochondrial hypothesis and its verification

The present inventors have found that the energy bill Hi is reduced in the brain of patients with manic-depressive illness, and is a type of mitochondrial disease, in which multiple deletions of mitochondrial DNA (mtDNA) accumulate throughout the body. Chromosomal dominant chronic progressive external ophthalmoplegia (adCPE0) '' and Wolfram disease, both exhibit recurrent depression ゃ manic depression.Manic depression often affects the maternal. In consideration of the previously reported facts, the authors have conducted a hypothesis that the impairment of mitochondrial function due to abnormal mitochondrial DNA (mtDNA) is involved in the etiology of manic depression. As a result, in the postmortem brain of patients with manic depression, The authors reported that mtDNA was frequently deleted and that manic depression was associated with specific mtDNA polymorphisms, and much evidence was obtained that mtDNA abnormalities cause manic depression. (ii) Preparation of model animals

adCPEO has three causative genes, mtDNA synthase (polymerase y), Twinkle (mtDNA helicase), and ANT1 (adenine-knuleotide translocator-1), all of which are recurrent depression or bipolar. It has been shown to present sexual disorders. Thus, a point mutation was introduced into the mouse mtDNA synthase gene, and a mutant gene encoding a mtDNA synthase (polymerase γ) having a mutation that would synthesize abnormal mtDNA was created. The same strategy was used to create an animal model of cardiomyopathy by accumulating abnormal mtDNA using the same strategy (Zhang D, et al: Construction of transgenic mice with tissue-specific acceleration of mitochondria 丄 DNA mutagenesis. Genomics. 2000 Oct 15; 69 (2): 151-61), and it was thought that a similar strategy could be used to create an animal model in which brain-specific mtDNA deletions accumulate. is there.

With adCPEO, it is difficult to determine whether or not a person is depressed, even if muscle symptoms appear, but in order to express this mutant gene in the brain-specific manner, calmodulin kinase IIa (CAMKIIa) Promoter (Used with permission from Mark Mayford, who cloned and characterized this promoter region. Mayford M, et al. The 3'-untranslated region of CaMKII alpha is a cis-acting signal for the localization and translation. of mRNA in dendrites.Proc Natl Acad Sci

U.S.A. 1996 Nov 12; 93 (23): 13250-5) or the neuromotor-specific enolase (NSE) motor (Professor Kenji Sakimura who cloned and characterized this promoter region)

(Niigata University Brain Research Institute) Used with permission. Sakimura K, et al: Upstream and intron regulatory regions for expression of the rat neuron-specific enolase gene.Brain Res Mol Brain Res. 1995 Jan; 28 (1): 19-28) It was specifically expressed in the brain (Fig. 1). As a result, 3 lines of CAMKII and 7 lines of NSE were established. Among them, two lines of CAMKIIα transform into the brain It was confirmed that atypical polymerase γ was expressed (Fig. 2). Furthermore, in two of these lines, CAMKII o ;, it was confirmed by PCR-Southern blotting that the deletion type mtDNA had accumulated in the brain (FIG. 3).

(i i i) Behavior analysis

It was examined whether the animals prepared as described above exhibited behavioral abnormalities similar to manic-depressive illness. First, long-term activity was measured by the number of rotations, and it was found that the activity of these mice changed periodically with a cycle of about 5 days (Fig. 4). It was thought that this alone would extend the circadian rhythm and appear to be a pattern that appeared due to a steady light-dark cycle and swell, but since this behavioral rhythm pattern was maintained even under a steady 喑 condition, it was simply a circadian rhythm It was not a disability, but a 5-day cycle. Open field studies have shown that locomotor activity is lower in wild and novel environments than in wild-type mice. In the elevated plus maze, the time spent in the closed arm was prolonged, suggesting that the mouse was highly anxious. On the other hand, in the forced swim test, the immobility time was shorter than expected. From the above results, it was considered that the transgenic animal of the present invention had characteristics different from those of the existing depression model. Hereinafter, embodiments of the present invention will be described more specifically.

(1) Characteristics of the transgenic non-human mammal of the present invention

As described above, the transgenic non-human mammal of the present invention is capable of brain-specifically expressing a gene encoding a mutant mtDNA synthase (polymerase) by introducing the gene. Characterized transgenic animal. More preferably, the transgenic non-human mammal of the present invention is characterized in that the deleted short mtDNA exhibits a force accumulated in the brain and / or a spontaneous periodic behavioral change. I do.

In the transgenic non-human mammal of the present invention, the expression level of the mutant mtDNA synthase can be determined by Northern plot, RT-PCR, immunohistochemical staining, or the like. Can be measured or analyzed. In addition, the accumulation of the deleted mitochondrial DNA can be detected by a PCR-Southern plot method (specific examples are described in Example 3 of the present specification). Furthermore, for spontaneous periodic behavioral changes (ie, bipolar disorder-like abnormalities), long-term recording of the activity of transgenic non-human mammals and behavioral analysis (for specific examples, see (Described in Examples 4 and 5 of the book).

As used herein, the term “gene encoding a mutant mtDNA synthase (polymerase γ)” refers to a gene obtained by introducing a mutation into the mtDNA synthase (polymerase γ) gene, which is a known gene. Regarding the base sequence and amino acid sequence of known mt DNA synthetase (polymerase γ) gene, for example, GeneBank shows human (Satsu-002693), mouse (Marine _017462, or AB121698, BAC98463), rat (Marine-053528), It is registered as a budding yeast mother (NC-001147) (http: // www. Ncbi. Nlm. Nih. Gov / UniGene / clust. Cgi? 0RG = Mm & CID = 3616). The polymerase γ gene predicted by the computer from the mouse genome sequence is registered as Mm7-39468-30-112-1.

The mutated gene referred to in the present invention means a gene in which a mutation (for example, mutation) has occurred in the DNA sequence of the mtDNA synthase (polymerase γ) gene. Use a gene in which a part of the base sequence in the gene is deleted, a gene in which a part of the base sequence of the gene is replaced by another base sequence, a gene in which another base sequence is inserted in a part of the gene, etc. be able to. The number of bases to be deleted, substituted or added is not particularly limited, but is generally about 1 to 50, preferably about 1 to 15, and more preferably about 1 to 6. Deletion, substitution or addition of such a base sequence may result in deletion or substitution of mtDNA synthase (preferably about 1 to 5, more preferably about 1 or 2 amino acids in the amino acid sequence of a polymerase). Or an addition will occur.

Mutant mtDNA synthase used in the present invention (a specific example of a polymerase gene is mouse: 18 1st in the gene encoding mtDNA synthase). A gene in which aspartic acid, which is an amino acid residue of the above, is substituted with alanine can be mentioned. Such a mutant mtDNA synthase (polymerase γ) gene DNA is, for example, a genomic DNA encoding a known mtDNA synthase (polymerase γ) gene, and a base at the mutation site. It can be obtained by PCR or the like using a missense mutation-introducing primer substituted so as to encode the above amino acid. In addition, methods such as PCR, preparation of primers, preparation of genomic DNA, cloning, and enzyme treatment can be carried out by conventional methods well known to those skilled in the art.

(2) Preparation of transgenic non-human mammal of the present invention

The method for producing the transgenic non-human mammal of the present invention is not particularly limited. For example, an expression vector having a mutant mt DNA synthetase (polymerase γ) gene incorporated under the control of a promoter is introduced into a fertilized egg or the like. A method for producing a transgenic non-human mammal that specifically expresses the mutant mtDNA synthase (polymerase γ) gene, particularly in the brain, by introducing the gene is described below. explain.

As a transgene used for producing a transgenic non-human mammal, a mutant mt DNA synthetase (a recombinant gene in which a polymerase gene is ligated downstream of an appropriate mammalian promoter as described above) is used. A polyA signal can be ligated downstream of the mutant mt DNA synthetase (polymerase V) gene, if desired.

The type of mammalian promoter used for the construction of the transgene is not particularly limited. A promoter capable of expressing a mutant mtDNA synthase (polymerase) gene in the brain (particularly, neurons of the brain) may be used. I like it. Specific examples of such a promoter include, but are not limited to, a calmodulin kinase IIa (CAMKIIa) promoter or a nerve-specific enolase (NSE) promoter. In addition, any other mammalian (eg, human, A promoter of a gene derived from a heron, a dog, a cat, a monorekit, a hamster, a rat, a mouse, etc.) can be used as appropriate.

Further, a terminator required for expression of a mutant mtDNA synthase (polymerase T /) gene may be linked to the recombinant gene used in the present invention. The terminator is used as a sequence that terminates transcription of a target messenger RNA (so-called poly A) in a transgenic animal, and the sequence of each gene derived from a virus, various mammals or birds may be used. it can. Specifically, SV40 terminator of Simian virus can be used. In addition, a splicing signal of a known gene and an enhancer region can be ligated to further enhance the expression of a mutant mt DNA synthase (polymerase gene. It is also possible to connect the region 5 'upstream of one promoter region, between the promoter region and the translation region, or 3 or downstream of the translation region.

When the mutant mtDNA synthase (polymerase _γ ) gene incorporated into the transgene is expressed in the cell, the mutant mtDNA synthase (polymerase) is produced in the cell.

Transgenic non-human mammals that express a mutant mt DNA synthetase (polymerase 0 /) by introducing a mutant mt DNA synthase (polymerase V) gene can be used, for example, in fertilized eggs of non-human mammals. The mutant mtDNA synthase (polymerase gene is introduced, the fertilized egg is transplanted into a pseudopregnant female non-human mammal, and the mutant mtDNA synthase (polymerase 0; Non-human mammals into which the gene has been introduced can be produced by delivery, for example, rodents such as mice, hamsters, guinea pigs, rats, and rabbits, and the like. Dogs, cats, goats, sheep, pigs, pigs, monkeys, etc. can be used, but mice, hamsters, guinea pigs, etc. Rodents such as rats, rats, and egrets are preferred, with mice being most preferred. In the transgenic non-human mammal used in the present invention, the germinal cells and the germ cells or somatic cells are transferred to the non-human mammal or an ancestor of the animal at the stage of embryonic development (preferably, a single cell or a fertilized egg cell). In this stage, and generally before the 8-cell stage), it is produced by introducing a recombinant gene containing an exogenous mutant mtDNA synthase (polymerase γ) gene. The construction of the mutant mt DNA synthase (polymerase V) gene is as described above.

The introduction of the mutant mtDNA synthase (polymerase) gene at the fertilized egg cell stage can be carried out so as to be maintained in all germ cells and somatic cells of the target mammal. The mutated mtDNA synthase (the presence of the polymerase gene indicates that all the progeny of the produced animal have mutant mtDNA synthase (germ cells and somatic cells) in the germ cells of the animal after gene transfer. Means that the polymerase γ) gene is present The offspring of this type of animal that inherits the gene have a mutant mtDNA synthase (polymerase V) gene in all of its germinal and somatic cells.

After confirming that the transgenic animal of the present invention stably retains the gene by mating, the transgenic animal can be subcultured in a normal breeding environment as the gene-bearing animal. By obtaining a homozygous animal having the transgene on both homologous chromosomes and mating the male and female animals, it is possible to breed the offspring so that all offspring have the gene in excess. To identify the expression site of the mutant mtDNA synthase (polymerase _y ) gene, the expression of the mutant mtDNA synthase (polymerase V) gene must be observed at the individual, organ, tissue, and cell levels. Can be done. It is also possible to measure the expression level of the mutant mtDNA synthetase (polymerase γ) by an enzyme immunoassay using an antibody to the enzyme.

Hereinafter, the transgenic non-human mammal will be specifically described with reference to a transgenic mouse as an example. A transgene containing a mutant mt DNA synthetase (cDNA encoding the polymerase gene downstream of the promoter) was constructed, and the transgene was microinjected into the male pronucleus of a mouse fertilized egg. After culturing the obtained egg cells, the cells are transplanted into the oviduct of a pseudopregnant female mouse, and thereafter, the recipient animal is bred, and the pups having the cDNA are selected from the pups laid. Nick mice can be produced. The fertilized egg of the mouse, for example, 129ZS V, C 57 BL / 6 N B ALB / c, C 3H, those arbitrary if those obtained by crossing mice derived from SJL / Wt or the like can be used.

The appropriate number of transgenes to be injected is 100 to 3000 molecules per fertilized egg. In addition, selection of pups having cDNA is performed by extracting DNA from the tail of the mouse, etc., and introducing a hybrid of the mutant: mtDNA synthase (polymerase γ) gene as a dot hybrid probe. It can be carried out by the Zession method or the PCR method using specific primers.

The transgenic non-human mammal of the present invention is characterized in that a mutant mt DNA synthetase (which is characterized by overexpressing a polymerase gene, and can be used for screening tests for therapeutic or preventive drugs for manic depression. This model is also useful in research fields such as elucidation of the mechanism of development of manic depression.

In addition, the transgenic non-human mammal of the present invention includes, as a part thereof, cells, intracellular organelles, tissues and organs of the non-human mammal, head, fingers, hands, feet, abdomen, And the like, all of which fall within the scope of the present invention.

(3) Screening for therapeutic and / or prophylactic agents for manic-depression

The method for screening a therapeutic and / or prophylactic agent for manic depression according to the present invention can be performed using the transgenic non-human mammal of the present invention that overexpresses a variant mtDNA synthase (polymerase V). . That is, a test substance is administered to the transgenic non-human mammal (eg, transgenic mouse) of the present invention, and the physiological data, exercise ability, and the like of the transgenic non-human mammal are evaluated. By examining it, the therapeutic and preventive effects of the test substance on manic depression can be evaluated. Alternatively, a test substance is administered to the transgenic non-human mammal of the present invention, and the expression state of the mutant mtDNA synthetase (polymerase) after administration and the accumulation state of the deleted short mtDNA in the brain after administration. In addition, the therapeutic and preventive effects of the test substance on manic depression can be evaluated by analyzing the dynamics of the administered test substance in vivo.

That is, by using the transgenic non-human mammal of the present invention, it is possible to evaluate the therapeutic effect and the preventive effect of manic-depressive disease. It can be used as a disease state evaluation model animal. For example, by using the transgenic non-human mammal of the present invention, it is possible to determine the recovery of these conditions and the severity of the disease, and to examine a method for treating this disease.

Furthermore, by using the transgenic non-human mammal of the present invention, the expression of the mutant mt DNA synthetase (polymerase 0;) and the progress of the above-mentioned disease are analyzed to determine The mechanism of disease onset and progression can be elucidated.

The test substance to be subjected to the screening method of the present invention includes, for example, peptides, proteins, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, Examples thereof include plasma, and these compounds may be novel compounds or known compounds. Libraries containing a large number of molecules, such as peptide libraries and compound libraries, can also be used as test substances.

As a method of administering a test substance to the transgenic non-human mammal of the present invention, for example, oral administration, intravenous injection and the like are used. The dose of the test substance can be appropriately selected according to the administration method, the properties of the test substance, and the like.

The substance obtained by using the screening method of the present invention is a substance selected from the test substances described above, and has a preventive / therapeutic effect on manic-depressive phenomena. It can be used as a medicine such as a prophylactic agent. Further, a compound derived from the substance obtained by the above screening can also be used as a medicine. The substance obtained by the screening method may form a salt, and the salt of the substance may be a physiologically acceptable acid (eg, an inorganic acid or an organic acid) or a base (eg, an alkali metal). And particularly preferably a physiologically acceptable acid addition salt.

Examples of the salt include a salt with an inorganic acid (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or an organic acid (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, Salts with tartaric acid, cunic acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) are used.

The substance obtained by the screening method can be used, for example, as a sugar-coated tablet, capsule, elixir, microcapsule, etc., orally, or water or any other pharmaceutically acceptable substance. It can be used parenterally in the form of an injection, such as a sterile solution with the resulting solution or a suspension.

For example, a pharmaceutical preparation can be produced by mixing the substance with a physiologically acceptable carrier, flavoring agent, excipient, vehicle, preservative, stabilizer, binder and the like. Examples of additives that can be mixed with tablets, capsules, and the like include binders such as gelatin, corn starch, tragacanth, and gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, and alginic acid. Useful bulking agents, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, and flavoring agents such as peppermint, cocoa oil or cherry are used. A sterile composition for injection can be formulated according to a conventional method such as dissolving or suspending an active substance in a vehicle such as water for injection, or a naturally occurring vegetable oil such as sesame oil or coconut oil. Examples of the aqueous solution for injection include physiological saline, isotonic solution containing glucose and other auxiliary agents (eg, D-sorbitol, D-mannitol, sodium salt, etc.), and the like. Suitable dissolution aids, such as alcohols (eg, ethanol), polyalcohols (eg, propylene glycol, polyethylene glycol), non-ionic surfactants (eg, For example, polysorbate 80 ™, HCO-50) can be used in combination. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and a dissolution aid such as benzyl benzoate or benzyl alcohol may be used in combination.

The therapeutic and / or prophylactic agent for manic depression includes, for example, a buffer (eg, a phosphate buffer, a sodium acetate buffer), a soothing agent (eg, benzalconium chloride, pro-chloride). And a stabilizer (eg, human serum albumin, polyethylene glycol, etc.), a preservative (eg, benzyl alcohol, phenol, etc.), an antioxidant, etc.

The preparation thus obtained is safe and has low toxicity, and thus can be administered, for example, to humans and mammals. The dose of the substance varies depending on the target disease, the administration subject, the administration route, and the like. For example, in the case of oral administration, in general, in adults, the compound should be used in an amount of about 0.1 to about L 0 per day. 0 mg, preferably about 1.0 to 50 mg is administered. When administered parenterally, the single dose of the compound varies depending on the target of administration, target disease, etc.For example, when administered to an adult in the form of an injection, the compound may be administered once a day. About 0.01 to 100 mg, preferably about 0.1 to 50 mg is administered by intravenous injection.

The present invention will be described more specifically with reference to the following examples, but the present invention is not limited to the examples. Example

Example 1: Production of transgenic mice

To create mice with neuronal-specific abnormalities in mitochondrial DNA (mtDNA), we decided to use genetically engineered mtDNA synthase (DNA synthase /). By introducing the mutation, the proofreading ability of DNA synthase γ can be lost, that is, a high frequency of errors occurs when replicating mtDNA (incorporating the wrong base) [Zhang D. et al., Genomics 69, 151-161 (2000)]. If the replication reaction proceeds as it is, the error part will remain in mtDNA as a point mutation. Also, In many cases, the replication reaction stops at the same time that an error occurs. When mtDNA whose replication has stopped halfway becomes recircular, it becomes a short mtDNA, that is, a deleted mtDNA. Thus, by expressing the DNA synthase ^, which is liable to cause errors, in nerve cells, it is possible to accumulate abnormal nrtDNA in a nerve-specific manner. Therefore, transgenic mice (Tg mice) that express mutant DNA synthase γ were produced using a promoter that induces expression in nerve cells. First, mouse DNA synthase γ (mPol y) cDNA was cloned from mouse whole brain mRNA. When both chains were sequenced by the dideoxynucleotide method, a difference was observed in both the base sequence and the deduced amino acid sequence from the standard sequence registered in the database (Martian 017462). . Therefore, we compared and analyzed the Pol cDNA of other species and concluded that the sequence of the cDNA cloned above was the correct mPol sequence.

The 181st amino acid residue, aspartic acid, was changed to alanine using the QuikChange site-directed mutagenesis kit from Stratagene. This aspartic acid is an essential residue for binding Mg ^2+, and it has been reported that mutating it to alanine significantly reduces its proofreading ability [Zhang D. et al., Genomics 69, 151-161. (2000)]. The sequences of the primers used at this time are as follows.

mut-mPolg-F, 5 '-GCCCTGGTGTTCGCCGTGGAGGTCTGC-3' (SEQ ID NO: 1)

mut-mPolg-R, 5'-GCAGACCTCCACGGCGAACACCAGGGC-3 '(SEQ ID NO: 2)

In addition, due to the experimental procedure of adding a promoter, two Not I restriction enzyme sites existing in the coding region (I is cut immediately after the 115th and 407th bases from the start codon). The sequence was altered, eliminating all Not I sites. At that time, care was taken not to change the amino acid sequence. This procedure was also performed using the QuikChange site-directed mutagenesis kit, and the sequence of the primer was used as follows.

mPolg-xNotll-F, 5 '-GTCCCCAGCGACGGTCGGCCGCCGTC-3' (SEQ ID NO: 3) mPolg-xNotll-R, 5 '-GACGGCGGCCGACCGTCGCTGGGGAC- 3' (SEQ ID NO: 4) mPolg-xNotI2-F, 5,-CCTTACTTGGAGGCTGCCGCCTCG-3, (SEQ ID NO: 5)

mPolg-xNotI2-R, 5, -CGAGGCGGCAGCCTCCAAGTAAGG-3 '(SEQ ID NO: 6)

Only the nucleotide region of the mutated mPoly cDNA was extracted by PCR using the following primers, and was once subcloned into the pCR Blunt II-TOP0 vector (Invitrogen).

XhoI-mPolg-F, 5, -CTCGAGCCATGAGCCGCCTGCTCTGGAAGAAG-3 '(SEQ ID NO: 7) Sfil-mPolg-R, 5, -GGCCAATTGGGCCTTCCGTTAGGAGGAC-3' (SEQ ID NO: 8)

After checking the sequence, the promoter of the mouse CaMKII o; (calcium-calmodulin-dependent protein kinase II α;) gene and the 5,-and 3, -intron regions were ligated. Specifically, the restriction region of the coding region of the mutant ηι 制限 οΙγ. After excising with I and blunt ends, it was inserted into the RV site of pNN265 vector [Choi T. et al., Mol. Cell. Biol. 11, 3070-3074 (1991)]. , Sfi I and Eco RV sites will disappear). From there, Not I fragment (5, -intron 'mutant mPoly coding region · 3, -intron' poly-A additional sequence linked structure) was cut out and the PMM403 vector [Mayford M. et al., Science 274]. , 1678-1683 (1996)] at οί I site. This construct (Fig. 1) was found to be extremely unstable in Escherichia coli (XLI Blue-MRF,, T0P10, SURE, etc.) used in ordinary molecular biology experiments, and the STBL2 strain (Invitrogen) Was constructed at 30 ° C for the first time.

After deleting the vector portion from the completed construct with restriction enzyme I, the construct was injected into the pronucleus of a fertilized egg of a C57BL / 6J mouse and transplanted into the fallopian tube of a foster parent. A total of 894 fertilized eggs were injected, of which 431 embryos were transferred. Forty litters were obtained, and 27 grew to weaning. Three of them had a transgene. The genotype was determined by PCR using genomic DNA extracted from a part of the mouse tail. The primers used at this time are as follows.

raPolgTg-F, 5, -TTGAGGTTTTCCAGCAGCAG-3 '(SEQ ID NO: 9) mPolgTg-R, 5'-AAGGACTCGATGGCTCTGTAGG-3 '(SEQ ID NO: 10) However, among the three lines (A, B, C) of the obtained Tg mice, line A hardly produces Tg mice (about 70 Since only two of the offspring were Tg), no further analysis was performed. Example 2: Expression of transfected mutant Pol

The remaining two lines (lines B and C) were examined for ιηιοΙγ gene expression in each tissue. Total RNA was extracted using Trizol reagent (Invitrogen), and reverse transcription was performed to produce cDNA. Real-time quantitative PCR was performed using primer sets that specifically amplify mutant or normal (endogenous) mPoly cDNA, respectively (SYBR Green Master Mix Reagent · SDS7000; Applied Biosystems) . Normalization was performed using the GAPDH mRNA amount measured at the same time.

The following primer sets amplify only the mutant mPol · cDM.

mutPolg-TMF, 5,-CTGCCTTACTTGGAGGCT-3 '(SEQ ID NO: 11)

mutPolg-TMR, 5'-CAAGCAGACCTCCACGG-3 '(SEQ ID NO: 12)

Primer sets that amplify only normal mPol · cDNA are as follows.

wtPolg-TMF, 5, -CTGCCTTACTTGGAGGCG-3 '(SEQ ID NO: 13)

wtPolg-TMF, 5'-CAAGCAGACCTCCACGT-3 '(SEQ ID NO: 14)

As a result, it was found that the mutant mPol y mRNA had strong expression in the nerve tissue of line B, but had low expression in other tissues and mice of line C (Fig. 2). Example 3: Accumulation of deleted mitochondrial DNA

Short abnormal mitochondrial DNA (mtDNA) in each tissue of transgenic (Tg) and wild-type (non-Tg) mice of lines L and C, respectively, was detected by PCR-Southern plot. Line B mice were 12 weeks old and line C mice were 8 weeks old. Specifically, first, a DNA purifier ( DNA (including nuclear and mitochondrial DNA) was extracted from each tissue using Labo. Next, if the extension time of the PCR was sufficiently long, PCR was performed with a "short extension reaction time" using a primer set capable of amplifying the entire length of mtDNA (16.6 kbp). Only short Natsuta mtDNA to include deletion is amplified and detected the PCR product ⁽³ ~8kbp) by Southern blot. The D loop portion of mtDNA, which is crucial for mtDNA replication and should always contain the deleted mtDNA, was used as a probe for Southern blot analysis. The PCR conditions for “short extension reaction time” are as follows.

Composition of reaction solution (25 L): DNA 0.1 / g, 0.8 mM dNTP, 1 1τΕτ¾ buffer, 0.4 μ 各 each primer, Ex Taq 0.03U.

Temperature and time: 94 ° C x 1 min (94 ° C x 20 sec-65 ° C x 30 sec-72 ° C x 2.5 min) x 33 cycles.

ramtDNAdel-F, 5, -GAGGTGATGTTTTTGGTAAACAGGCGGGGT-3 '(SEQ ID NO: 15) mmtDNAdel-R, 5' -GGTTCGTTTGTTCAACGATTAAAGTCCTACGTG-3 '(SEQ ID NO: 16) It turned out that mtDNA was more accumulated. No clear mtDNA accumulation was observed in line C Tg mice. This result was considered to reflect the expression level of the transfected mutant Pol y (Fig. 3). Example 4: Long-term recording of activity in Tg mice

The basic feature of bipolar disorder (of course in humans) is one or more manic episodes, often with one or more major depressive episodes. That is, the manic episode and the major depression episode are repeated. The diagnostic criteria for manic episodes are clearly present, but none are biochemical data (eg, blood levels of some substances). Because it is a standard such as "Mood is abnormal and persistently uplifted, open or irritable ..." I can't. Therefore, based on the basic pathology of repetition of manic episode and major depressive episode, mouse activity was We decided to examine whether the dose increased or decreased repeatedly to determine if the mice had bipolar disorder-like abnormalities.

Line B Tg mice and their littermate wild-type mice (non-Tg mice), each 5 (13-14 weeks old), are placed in a cage with a rotation device (Ohara Medical Sangyo) one by one, and are rotated. The amount was measured. 12 hours: Under a 12-hour light / dark cycle, food and water were freely available.

As a result, both Tg mice and non_Tg mice showed 24-hour activity rhythms synchronized with the light-dark cycle, but in addition to this one-day cycle rhythm (the so-called dian rhythm), Tg mice also had very long rhythms. It was found to show a periodic rhythm. As shown in Fig. 4, the night after the active movement, the next night did not move much, and the activity was low for (the night) several days thereafter. And then move again and again. Figure 5 plots the number of wheel rotations per day. Thus, it was found that the increase and decrease in the activity of the Tg mice was repeated about every 5 days. To determine the length of this cycle accurately, we used the Lomb-Scargle periodic analysis method to determine the periodicity of the wheel rotation amount, and found that the cycle was 110 to 130 hours (Fig. 6). These results suggest that this Tg mouse is a mouse that repeatedly increases and decreases in activity spontaneously and can be a model animal for human bipolar disorder. Example 5: Routine behavioral analysis

As a usual behavioral analysis, open field, elevated plus maze, and forced swimming tests were performed on line B Tg mice and non-Tg mice of the same litter (Fig. 7). In open field tests to determine activity in the new environment, Tg mice showed low activity and often remained in one location for long periods of time and did not move. In an elevated plus-maze test for fear and anxiety, the Tg mouse stayed on the closed arm for a long time with closed arms (there is no danger of falling down). From this, it was presumed that Tg mice had strong anxiety.

A forced swimming test, widely used as a screen for antidepressants, was performed. The mouse swims desperately when the mouse is put into a pool where there is no place to catch, but eventually (probably) in despair it stops swimming and leaves it floating and becomes immobile. As a result of the measurement, Tg mice took longer to become immobile than non-Tg. This result is contrary to the tendency of other animal models, which are commonly referred to as a depression model, to become immobile sooner, indicating that the present Tg mouse is a unique mouse. In humans, antidepressants improve the depression of bipolar disorder (symptomatically), but are known to enhance the repetitiveness of manic and depression by antidepressant administration. Industrial applicability

In the present invention, a transgenic non-human mammal was prepared by introducing a mutation into a causative gene of a neuromuscular disease adCPEO presenting depression and expressing it in a nerve-specific manner. In the transgenic non-human mammal of the present invention, accumulation of mtDNA deletion in the brain, which is considered to be associated with manic depression, was confirmed. In addition, the transgenic non-human mammal of the present invention spontaneously exhibited periodic behavioral changes. Therefore, the transgenic non-human mammal of the present invention is useful as an animal model for recurrent depression or manic depression. The transgenic non-human mammal of the present invention is used for research and development of therapeutic drugs for mood depression and depression (mood stabilizers, antidepressants), research of pathological conditions of manic depression and depression, and research of diagnostic methods. It is possible.

Claims

The scope of the claims

1. A mutated mtDNA synthase (a transgenic non-human mammal or a part thereof characterized in that the gene encoding a polymerase is introduced into the brain specifically when the gene is introduced).

2. The transgenic non-human mammal or part thereof according to claim 1, wherein the deleted short mtDNA is accumulated in the brain.

3. The transgenic non-human mammal or part thereof according to claim 1 or 2, which exhibits spontaneous periodic behavioral changes.

4. A recombinant DNA comprising a gene encoding a mutant mt DNA synthetase (polymerase γ) incorporated under the control of a promoter that enables brain-specific expression. Or the transgenic non-human mammal or a part thereof.

5. The transgene according to any one of claims 1 to 4, wherein the promoter capable of brain-specific expression is a calmodulin kinase II α (CAMKII α) promoter or a neuron-specific enolase (NSE) promoter. Nick non-human mammals or parts thereof.

6. Mutant mtDNA synthase (The gene encoding the polymerase is a gene in which aspartic acid, which is the 181st amino acid residue in the gene encoding the mouse mtDNA synthase, is substituted with alanine. Item 6. The transgenic non-human mammal according to any one of Items 1 to 5, or a part thereof.

7. The transgenic non-human mammal or a part thereof according to any one of claims 1 to 6, wherein the non-human mammal is a mouse.

8. A method for screening for a therapeutic and / or prophylactic agent for manic depression, comprising using the non-human mammal according to any one of claims 1 to 7 or a part thereof.

9. A substance obtained by the screening method according to claim 8.

10. A substance obtained by the screening method according to claim 8 as an active ingredient As a therapeutic and / or prophylactic agent for manic depression.