JP2006075155A - New non-human animal - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new non-human animal useful for screening therapeutic and/or prophylactic drugs for psychiatric disorders including mania, depression, manic-depressive, hyperkinetic disorders, autism, schizophrenia and post-traumatic stress disorders or dysmnesia including learning one and ones associated with aging, cerebrovascular disorders, dementia, Parkinson's disease and depression. <P>SOLUTION: The non-human animal that is transferable is such that activin gene is overexpressed at least in its brain, and has at least either one phenotype among activity elevation, emotion abnormality, information processing mechanism abnormality and learning dysmnesia. The offsprings thereof are also provided. Another version of this non-human animal that is transferable is such as to be transferred with activin gene under the control of a promoter activated at least in its brain. The offsprings thereof are also provided. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a novel non-human animal. Specifically, the present invention relates to a non-human animal in which the activin gene is overexpressed.

Activin is a secretory factor belonging to the TGFβ (Transforming growth factor β) superfamily. Activin plays an important role in determining differentiation in the early stages of development, and it is widely known that normal development is not possible when these signals are disturbed.
The activin signal starts from the release of activin, which is a secretory factor (see, for example, Non-Patent Documents 1 to 3). When the released activin binds to the type II activin receptor, the type II activin receptor is activated, and the type I activin receptor is activated. The activated type I activin receptor then phosphorylates and activates the transcription factor Smad2 / 3. Activated Smad2 / 3 translocates to the nucleus by binding to Smad4 and regulates the transcription of genes downstream of the activin signal system.

  Activin signal is widely known to play an important role in development and differentiation, but also has various actions in skin formation, bone formation, hormone secretion, etc. (for example, Non-Patent Documents 4 and 5). In addition, involvement in learning / memory is also suggested (see, for example, Non-Patent Documents 6 and 7). In animals, there are four types of genes encoding activin β subunits: activin A gene, B gene, C gene, and E gene, which code for activin βA, βB, βC, and βE, respectively. . There are many parts that have not been elucidated about the difference in these functions, but activin is a dimer composed of two of these β subunits. For example, in the brain, activin consisting mainly of a combination of βA / βA It is known to exist. Among the four types of genes, only the activin A gene increases the expression level of mRNA in nerves depending on nerve activity (see, for example, Non-Patent Documents 6 and 7).

  In order to elucidate the functions of such a wide variety of activins, various genetically modified animals of the activin signal system have been created, but it is suitable for elucidating the effects of activins on the cranial nervous system, especially the expression of activins in the brain No animal has been reported that has changed and is causing some abnormality in the cranial nervous system. For example, in order to analyze the function of activin in the whole body including the brain, a knockout mouse of an activin or a factor related to the activin signal system has been prepared. Among these, the activin βA knockout mice, in which the mRNA expression level in the nerve increased in a nerve activity-dependent manner, had no cheek beard and incisors, and abnormal palate formation. However, although this mouse is born, it will die within 24 hours after birth, so the effect of activin on the cranial nervous system cannot be analyzed (see, for example, Non-Patent Document 8). In addition, type II activin receptor knockout mice (see, for example, Non-Patent Document 9) are rarely abnormal in bone and face formation, but many are reported to grow normally. However, this knockout mouse has no fertility due to a decrease in follicle-stimulating hormone, cannot be subcultured, and is not capable of sufficient analysis over a long period of time. It was.

Thus, although several genetically modified animals related to activin have been prepared so far, none of them are sufficient to analyze the function of activin, and learning / memory and other diseases involving activin No animal that could be used as a model animal was obtained.
On the other hand, an appropriate model animal for mental illness is desired for the treatment and / or development of preventive drugs for mental illness. If there is an appropriate model animal, the initial screening of the drug can be easily performed by, for example, a general animal behavior test. However, at present, it is hard to say that a model animal for mental illness is still established. For example, model animals in which disease states are induced by drugs such as narcotics are frequently used as model animals for mental illness such as depression, depression, and schizophrenia. However, in such animals, accurate and homogeneous screening is performed, such as the interaction between the drug used for screening and the drug administered to induce the disease state becomes a problem, or the induction state of the disease state varies depending on the individual. It was difficult. In view of the above, it has been desired to produce a genetically homogenous animal model of mental illness that stably exhibits a medical condition of mental illness and can be used for screening of psychiatric illness and / or preventive drugs. In addition, an appropriate model animal for memory impairment is desired for the development of therapeutic and / or preventive drugs for memory impairment. If there is an appropriate model animal, the initial screening of the drug can be easily performed by, for example, a general animal behavior test. In view of the above, it has been desired to produce a genetically homogenous memory impairment model animal that stably exhibits a pathological condition of memory impairment and can be used for treatment of memory impairment and / or screening of preventive drugs.
Nature, 425, 577-584, 2003 Cell, 103, 295-309, 2000 Trends Endocrinol. Metab., 11, 309-314, 2000 Proc. Sc. Exp. Biol. Med., 214, 114-122, 1997 Proc. Sc. Exp. Biol. Med., 215, 209-222, 1997 FEBS Lett., 382, 48-52, 1996 Neuroscience, 69, 781-796, 1995 Nature, 374, pp354-356, 1995 Nature, 374, pp356-360, 1995

  The present invention has been made to provide a non-human animal in which the activin gene is overexpressed. More specifically, the activin gene is at least overexpressed in the brain and has at least a phenotype of any of increased activity, emotional abnormality, information processing mechanism abnormality, and learning memory abnormality It was made to provide possible non-human animals or their progeny. In addition, an activin gene placed under the control of a promoter activated at least in the brain has been introduced, and it has been made to provide a non-human animal or its progeny characterized in that it can be passaged It is. Furthermore, the present invention provides a method for producing the animal, a method for screening a drug for treating and / or preventing a mental illness or memory disorder involving activin, and the like.

  As a result of diligent studies to achieve the above-mentioned problems, the present inventors have introduced at least an activin gene under the control of a promoter activated in the brain, according to at least It was found that a non-human animal that overexpresses the activin gene in the brain and that can be stably passaged can be produced. Moreover, when this animal was analyzed, it discovered that it had phenotypes, such as an active mass increase, emotional abnormality, information processing mechanism abnormality, and learning memory abnormality. The present invention has been accomplished based on these findings.

That is, according to the present invention,
(1) A non-passable non-passive gene in which the activin gene is overexpressed at least in the brain and has at least a phenotype of increased activity, emotional abnormality, information processing mechanism abnormality, or learning memory abnormality A human animal, or progeny thereof is provided. Also,
(2) a non-human animal, or a progeny thereof, wherein an activin gene placed under the control of a promoter activated at least in the brain is introduced and can be passaged;
(3) The animal according to (2) above, wherein the promoter is further not activated in the reproductive organs,
(4) The animal according to (2) or (3) above, wherein the promoter is activated after the developmental period of the cranial nerve network
(5) The animal according to any one of (2) to (4) above, wherein the promoter is activated specifically in the forebrain.
(6) The animal according to any one of (2) to (5), wherein the promoter is a CaMKII promoter,
(7) The animal according to any one of (2) to (6) above, wherein the animal has a phenotype of at least an increase in activity, emotional abnormality, information processing mechanism abnormality, or learning memory abnormality An animal according to any of the
Is provided. further,
(8) The method for producing an animal according to any one of (2) to (6) above, which comprises the following steps (i) to (iii):
(I) introducing an activin gene placed under the control of at least a brain-activated promoter into a non-human animal fertilized egg,
(Ii) generating the fertilized egg, analyzing the obtained non-human animal, and selecting an individual into which the activin gene has been introduced;
(Iii) confirm that the selected individual can be passaged;
(9) The method for producing an animal according to (1) or (7) above, comprising the following steps (i) to (iii):
(I) introducing an activin gene into a non-human animal fertilized egg,
(Ii) generating the fertilized egg, analyzing the obtained non-human animal, and selecting an individual in which the activin gene is overexpressed,
(Iii) Confirm that the selected individual has at least one of the phenotypes of increased activity, emotional abnormality, information processing mechanism abnormality, and learning memory abnormality and can be passaged. To
Is provided.

Further, from another aspect of the present invention,
(10) A test substance is administered to the animal according to (1) or (7) above, and the effect of the test substance on the animal is changed by a phenotype change induced by overexpression of activin in the animal. A method for screening a therapeutic and / or prophylactic agent for a mental disorder or memory disorder involving activin, characterized by
(11) Activin and a test substance or only a test substance are added to a cell expressing activin receptor, and the influence of the test substance on the cell is detected using the change in the activin signal system as an index. A screening method for a therapeutic and / or prophylactic agent for psychiatric disorders or memory disorders involving activin,
(12) A test substance is added to activin-expressing cells, and the influence of the test substance on the cells is detected using the change in activin expression as an index, A method for screening a drug for treating and / or preventing memory impairment;
(13) Any of the above (10) to (12), wherein the psychiatric disorder involving activin is depression, depression, depression, hyperactivity disorder, autism, schizophrenia, or posttraumatic stress disorder The method according to
(14) A memory disorder involving activin is a learning memory disorder, a memory disorder associated with aging, a memory disorder associated with cerebrovascular disorder, a memory disorder associated with dementia, a memory disorder associated with Parkinson's disease, or a memory disorder associated with depression The method according to any one of the above (10) to (12),
(15) A substance selected by the method described in (13) or (14) above is formulated, and depression, depression, depression, hyperactivity disorder, autism, integration Treatment of memory impairment associated with ataxia, posttraumatic stress disorder, learning memory disorder, memory disorder associated with aging, memory disorder associated with cerebrovascular disorder, memory disorder associated with dementia, memory disorder associated with Parkinson's disease, or depression / Or a method for producing a prophylactic agent,
Is provided. further,
(16) In the animal according to (1) or (7) above, the follistatin gene is overexpressed, and at least in any of the indicators of activity, emotionality, information processing mechanism, and learning memory, A test substance is administered to an animal having a phenotype opposite to that of the animal described in 1) or (7), and either of the following substances (i) or (ii) is selected: A method of screening for a therapeutic and / or prophylactic agent for depression or schizophrenia,
(I) in the animal according to (1) or (7) above, the value is at least normal in any of the indicators of activity, emotionality, and information processing mechanism, the follistatin gene is overexpressed, and A substance that does not affect the value of the indicator in an animal having a phenotype opposite to that of the animal described in (1) or (7) above in at least one of activity, emotionality, information processing mechanism, and learning memory ,
(Ii) The follistatin gene is overexpressed and has a phenotype opposite to that of the animal described in (1) or (7) above, at least in any of activity, emotionality, information processing mechanism, and learning memory Substances in which at least one of the indicators of activity amount, emotionality, and information processing mechanism is close to normal in animals and does not affect the value of the indicator in animals described in (1) or (7) above ,
Is provided.

  According to the present invention, a non-human animal in which an activin gene is overexpressed at least in the brain is provided. The animal is useful for the treatment and / or screening of preventive drugs for psychiatric disorders or memory disorders involving activin genes.

Hereinafter, the present invention will be described in more detail. However, the following description of the constituent elements is a representative example of the embodiment of the present invention, and the present invention is not limited only to these contents.
In this specification, molecular biological techniques such as DNA and vector preparation, and general techniques related to the production of genetically modified animals, for example, “Molecular Cloning, A Laboratory Manual, 3rd edition”, unless otherwise specified. (Sambrook and Russell, published by Cold Spring Harbor Laboratory Press (2001)) '' and `` Manipulating the Mouse Embryo. A Laboratory Manual, third edition (Cold Spring Harbor Laboratory Press published (2003)) '' etc. It can carry out according to the method of it or it.

1. Non-human animal of the present invention and method for producing the same The non-human animal of the present invention has an activin gene that is at least overexpressed in the brain and at least increased activity, affective abnormalities, abnormal information processing mechanisms, and learning memory It has a phenotype of any of the above abnormalities, and the first feature is that it can be passaged. In particular, the relationship between overexpression of activin and increased activity, emotional abnormalities, information processing mechanism abnormalities, etc. has not been known so far, and is the first time that the non-human animal of the present invention has been produced. It has become clear.

  As a result of the overexpression of the activin gene in at least the brain, the non-human animal of the present invention has at least increased activity, emotional abnormality, information processing mechanism abnormality, and learning memory abnormality (hereinafter referred to simply as “behavior”). The activin gene may be overexpressed by any method as long as it has any phenotype of “abnormal” (sometimes referred to as “abnormal”) and can be passaged.

  The term “activin gene is overexpressed” means that activin, which is a translation product of the above activin gene, is present in a larger amount in any tissue of the animal than in the wild type animal of the same species. To do. For example, when any tissue of the animal is analyzed using a normal ELISA method, Western blot method, etc., the amount of activin detected is greater than the amount of activin present in the same tissue of the wild type animal. means. Further, comparison may be made according to the expression level of mRNA encoding activin. Analysis of the mRNA expression level can be performed by a known method such as Northern blotting. In the present specification, “activin gene is expressed” means production of mRNA encoding activin or production of activin.

  Here, the activin gene is a nucleic acid carrying genetic information of activin, which is a protein, and means a gene encoding activin. Examples of the gene encoding activin include activin A gene, activin B gene, activin C gene, activin E gene, or combinations thereof. Although it is considered that there is no significant difference in these functions, since it is known that activin βA mainly exists in the brain, the activin A gene is preferably used in the present invention.

  In the non-human animal of the present invention, the tissue in which the activin gene is overexpressed includes at least the brain, and may be any tissue as long as the animal has the behavioral abnormality described above. Among the brain, it is particularly preferable that it is overexpressed in the forebrain. When using an artificially overexpressed activin gene as an animal of the present invention, a DNA in which the activin gene is under the control of an appropriate promoter activated in the above tissue is prepared and introduced. The above-mentioned animal can be produced by Examples of methods for producing such animals include the methods described in detail below.

  Moreover, as a non-human animal, it is an animal other than a human, and what is necessary is just to express this when an activin gene is introduced. Preferred are mammals, and examples include mice, rats, guinea pigs, hamsters, rabbits, goats, pigs, dogs, cats and the like. More preferred are rodents, further preferred are mice or rats, and most preferred are mice.

  Said "behavioral abnormality" should just have at least 1 or more phenotype among these, Preferably it is 2 or more, More preferably, it has 3 or more. Any of these behavioral abnormalities can be detected by a commonly used animal behavior test or the like known per se. For example, results obtained using the animals of the present invention and control individuals (preferably wild-type wild type) When a significant difference is found by comparing the results obtained using the (animal), it can be determined as abnormal. Specifically, in the animal of the present invention, the amount of activity is increased compared to the control individual. Emotionality, information processing mechanism, and learning memory are increasing or decreasing. Preferably, both are rising. The determination of the significant difference can be performed by a known general statistical analysis method. Specifically, it is as detailed below. Further, the non-human animal of the present invention may have a phenotype different from that of the control individual in addition to the behavioral abnormality.

  Animal behavior tests include, for example, “Current Protocol in Neuroscience (John Wiley & Sons, Inc.)”, “Animal Behavior Function Test (Biochemistry, published by Medical School, Vol. 45, No. 5 (1995)”, etc. In addition to the above-described methods, any method can be used as long as it is applicable to the non-human animal. It can be analyzed by a general activity test such as “test” etc. Emotionality can be analyzed by a light and dark test, a forced swimming test, an elevated plus maze test, etc. The information processing mechanism can be analyzed by the prepulse inhibition test, etc. The learning memory is Morris water maze, fear conditioning, object recognition test. st) etc. It is preferable to use some of these in combination.

  Among the behavioral abnormalities of the non-human animal of the present invention, “increased amount of activity” generally means that the degree of activity, curiosity and aggressiveness are stronger than those of the control. Examples of such phenotypes include those in which the action time, action distance, number of rises, and rise time are significantly increased compared to the control individual in a search action test (Open field test). In addition, it includes that the occupation time of the middle part in the test is significantly longer than that of the control.

  “Emotional abnormality” generally means having an abnormality in anxiety behavior, despair behavior, behavior against fear, and the like. Such phenotypes include, for example, a light and dark test in which light occupancy time is significantly higher than control and anxiety is reduced, forced swimming test (Forced swimming test) The immobility time in the test) is significantly shorter than the control and the resistance to despair is increased, or the existence time in the open arm in the elevated plus maze test is controlled And the like are significantly increased and the fear of high places is reduced.

“Abnormality of information processing mechanism” generally means that there is a different phenotype in the behavior of ignoring unnecessary information compared to control. As such a phenotype, for example, a prepulse inhibition test (Prepulse inhibition test) in which the degree of inhibition by prepulse is increased as compared to control.
“Learning / memory abnormality” generally means that learning ability and memory ability increase or decrease. Such phenotypes include, for example, increased learning and memory abilities compared to controls in behavioral tests such as Morris water maze, fear conditioning, and object recognition test. Or the thing etc. which are falling are mentioned.
The formation of memory consists of three phases: acquisition, retention, and recall, but the molecular mechanism is different in each phase. Recently, in addition to these, it has been clarified that memory formation has a fourth phase called refixation. Refixation means that memory is temporarily destabilized when recalled, and then the memory is further strengthened by the action of a molecular cascade including protein synthesis. “Learning / memory abnormalities” include those resulting from abnormalities in these phases.

  Further, “passable” in the animal of the present invention means that normal mating can be repeated, and preferably, the characteristics of the animal of the present invention can be stably maintained even after repeated mating. It is. Specifically, for example, it is preferable that fertility is normal and normal male and female mating is possible. The mating can be stably repeated for at least one generation or more, preferably two generations or more, more preferably three generations or more. The genotype may be either homotype or heterotype as long as it is stably maintained. By having such properties, a homogeneous population can be obtained continuously, which is very useful when, for example, the non-human animal of the present invention is used as a disease model animal.

  For example, an animal in which the activin gene has been overexpressed artificially is usually crossed with a wild-type animal of the same strain, and while selecting the animal into which the gene has been introduced from the resulting offspring, Will be maintained. At this time, if the reproductive ability is abnormal and normal mating is impossible, it is difficult to obtain a litter and it is difficult to maintain the strain. Since it takes a long time and labor to produce the animal, if a sufficient number of offspring cannot be obtained by stably maintaining the strain, for example, analysis / experiment can be performed using this. It will also be difficult to perform stably.

  As such a non-human animal of the present invention, an artificially overexpressed activin gene may be used, or an overexpressed activin gene may be selected by induction of mutation or the like, but it is preferable. As an example, a non-human animal into which an activin gene placed under the control of a promoter activated at least in the brain is introduced and can be passaged (hereinafter sometimes referred to as “activin-introduced animal”) can be mentioned. It is done.

  Any promoter can be used as long as it is activated at least in the brain, overexpresses the activin gene in the introduced non-human animal body, and the introduced animal has the ability to be passaged. . That is, it may be activated only by the brain, or may be activated at a site other than the brain in addition to the brain. By selecting and using such a promoter, a non-human animal in which the activin gene is overexpressed at least in the brain and the function of activin in the brain is enhanced can be produced.

  More preferably, a promoter that is not activated in the reproductive organ is used. By selecting such a promoter, the produced non-human animal can be stably maintained without affecting fertility. More preferably, a promoter that is activated after the developmental period of the brain neural network is used. The term after the developmental period of the cranial nerve network means after 3 weeks, preferably after 4 weeks. By using such a promoter, the function of activin in the cranial nerve activity in the normally developed cranial nerve network can be analyzed because it does not affect the development of the cranial nerve network itself. It is also preferable to use a promoter that is activated specifically in the forebrain. The forebrain is a part mainly composed of the cerebral hemisphere and diencephalon, and includes complex parts such as cerebral cortex, thalamus, olfactory bulb, hypothalamus, pituitary gland, basal ganglia, hippocampus, amygdala, etc. It is considered as a part that plays an important role in memory, emotions, etc. If this forebrain-specific promoter is used, the function of activin related to memory, sensation, emotion, etc. can be analyzed in the forebrain.

  Specific examples of promoters used for producing activin-introduced animals of the present invention include, for example, βCaMKII promoter, nerve cell-specific enolase promoter, Thi-1 promoter, etc. . An example of a promoter that is activated after the developmental period of the cranial nerve network and is activated specifically in the forebrain is the αCaMKII promoter (Science, vol 274, pp1678-1683, 1996). In the present invention, this αCaMKII promoter is particularly preferably used.

  Here, the activin gene is a nucleic acid carrying genetic information of activin, which is a protein, and means a gene encoding activin. Examples of the gene encoding activin include activin A gene, activin B gene, activin C gene, and activin E gene. Although it is considered that there is no significant difference in these functions, since it is known that activin βA mainly exists in the brain, the activin A gene is preferably used in the present invention. The activin gene may be any as long as it can produce activin having the ability to function normally in the activin signaling system in the animal when it is expressed. For example, a region encoding activin on the genome may be obtained and used, but cDNA is preferably used in the present invention. Examples of such an activin gene include an activin A gene encoding activin βA and having the base sequence described in SEQ ID NO: 1 in the sequence listing. The gene may be derived from an animal of the same species as the non-human animal to be produced or may be derived from a different species, but is preferably derived from the same species. In addition, for example, a gene in which a mutation is introduced into the gene using a point mutagenesis method or the like can be used as long as the generated activin maintains the function as activin as described above. In addition, in order to confirm that the gene was expressed in an animal, a base sequence for co-expressing the tag protein with the gene (hereinafter sometimes referred to as “tag sequence”) is added. It is also preferable to use this. Such a configuration is convenient because the presence or absence and degree of expression of the gene can be detected by the presence or absence and degree of expression of the tag protein. Examples of the tag protein include Myc tag, HA tag, and FLAG tag. In the present specification, “activin gene is expressed” means production of mRNA encoding activin or production of activin.

  Further, “being under the control of” the promoter means that the promoter is activated in the introduced tissue to induce the expression of the activin gene. Specifically, for example, Then, a DNA (hereinafter sometimes referred to as “transgene”) in which the activin gene is linked downstream of the promoter and the tag sequence, terminator and other expression control regions are appropriately linked, is prepared. This state can be induced by introducing the compound into a non-human animal by a method described in detail below. Preparation of each component of the transgene and a technique for linking them can be performed by a commonly used method known per se.

“Introducing” an activin gene means introduction into any of the genomes of the non-human animal to be introduced. Here, a DNA (transgene) containing the activin gene in the genome is preferably introduced as 1 or more copies, preferably 2 or more copies, more preferably 3 or more copies, as the non-human animal of the present invention.
One of the characteristics of the activin-introduced animal is that it can be passaged. For example, in general, a foreign gene-transferred animal is usually crossed with a wild-type animal of the same strain, and the strain is maintained while repeating selecting the animal into which the gene has been introduced from the resulting offspring. At this time, if the reproductive ability is abnormal and normal mating is impossible, it is difficult to obtain a litter and it is difficult to maintain the strain. Since it takes a long time and labor to produce such an animal, if it is not possible to stably maintain the strain and obtain a sufficient number of pups, for example, this can be used for analysis and experimentation. It becomes difficult to carry out the above and the like stably.

  That is, being able to be subcultured means that normal mating can be repeated, and preferably, the characteristics of the animal of the present invention are stably maintained even when mating is repeated. Specifically, for example, it is preferable that fertility is normal and normal male and female mating is possible. The mating can be stably repeated for at least one generation or more, preferably two generations or more, more preferably three generations or more. The genotype may be either homotype or heterotype as long as it is stably maintained. By having such properties, a homogeneous population can be obtained continuously, which is very useful when, for example, the non-human animal of the present invention is used as a disease model animal.

The thus obtained non-human animal of the present invention has at least one of the phenotypes of increased activity, emotional abnormality, information processing mechanism abnormality, and learning memory abnormality, and can be subcultured. However, those having other phenotypes are also included in the activin-introduced animals of the present invention.
The activin-introduced animal of the present invention can be prepared using the above-described promoter, activin gene, and the like in accordance with a generally known method for preparing a foreign gene-introduced animal that is known per se. Specifically, for example,
(1) introducing an activin gene placed under the control of at least a brain-activated promoter into a non-human animal fertilized egg,
(2) Generate the fertilized egg, analyze the obtained non-human animal, and select an individual into which the activin gene has been introduced.
(3) Confirm that the selected individual can be passaged.
Can be produced.

Or
(1) introducing an activin gene into a non-human animal fertilized egg;
(2) Generate the fertilized egg, analyze the obtained non-human animal, and select an individual in which the activin gene is overexpressed.
(3) Confirm that the selected individual has at least one of the phenotypes of increased activity, emotional abnormality, information processing mechanism abnormality, and learning memory abnormality and can be passaged. To
Can be produced.

In the above production method, for example, preparation of a non-human animal fertilized egg, introduction of an activin gene into the fertilized egg, generation of the fertilized egg, acquisition of a litter derived from the fertilized egg, selection of an individual into which the activin gene has been introduced Etc. can be carried out by a commonly used method known per se.
In addition, the non-human animal may be any animal other than a human, and any animal can be used as long as the activin gene is introduced together with the promoter and the like. Preferred are mammals, and examples include mice, rats, guinea pigs, hamsters, rabbits, goats, pigs, dogs, cats and the like. More preferred are rodents, further preferred are mice or rats, and most preferred are mice.

In the following, an activin-introduced mouse is prepared using an activin βA cDNA and an αCaMKII promoter (Science, vol 274, pp1678-1683, 1996) etc. explain.
For activin βA cDNA, for example, a method of preparing mRNA by extracting mRNA from a tissue expressing mouse activin, preparing a cDNA library, and screening it by a conventional method is used. As the base sequence of the Myc tag protein, for example, a known sequence synthesized from the base sequence is used. As the SV40 poly A sequence, for example, a commercially available base sequence generally widely used can be appropriately selected and used. These connections can be performed by a commonly used method known per se. For example, a DNA in which these are easily linked can be prepared by performing a PCR reaction using a primer containing the base sequence of Myc tag protein downstream of activin βA cDNA. Further, by inserting this DNA into a plasmid containing the αCaMKII promoter and the SV40 poly A sequence, a DNA (transgene) in which these are all linked can be prepared.

  Next, the obtained DNA is introduced into a mouse fertilized egg. Examples of mouse strains include C57BL. Mouse fertilized eggs may be collected by natural mating, but fertilized eggs prepared by in vitro fertilization of unfertilized eggs collected from superovulated female mice and sperm collected from male mice in a conventional manner. Alternatively, a fertilized egg collected from a female mouse after mating a superovulated female mouse and a male mouse can also be used. The DNA can be introduced into this by introducing the DNA by a microinjection method or the like, and the resulting activin gene-introduced fertilized egg can be transplanted into the oviduct of a female mouse pseudo-pregnant to generate and give birth.

Individuals into which the activin gene has been introduced are selected from the pups thus obtained. For example, the tip of the mouse is cut off, genomic DNA is extracted using a commercially available kit such as a known DNA extraction method, etc., and the presence or absence of the activin gene introduced into the genome using Southern blotting, PCR, etc. And the number of copies can be analyzed.
It is also preferable to confirm that the activin gene is actually expressed in the individual and that activin is generated. Furthermore, the expression of activin can be indirectly confirmed using an antibody against Myc tag protein expressed together with activin. Such activin gene expression can be confirmed by, for example, ELISA, Western blotting, or the like. Further, for example, the expression level of mRNA encoding activin may be analyzed. Alternatively, the expression of the activin gene can be indirectly confirmed by using, as a probe, the SV40 poly A sequence constituting the transgene together with the activin gene. Such activin gene expression can be confirmed by, for example, in situ hybridization method, Northern blot method and the like. Specifically, these analyzes may be performed using, for example, various organs, tissues, cells, and the like of the animal, and extracts prepared from these as samples. As the activin-introduced mouse of the present invention, an individual in which at least the activin gene is overexpressed in the brain is preferably selected. Preferably, the expression level of the activin gene in the brain of the mouse of the present invention is analyzed, and this is compared with the expression level of the activin gene in the brain of the wild-type mouse, so that the expression level higher than that of the wild-type mouse is selected. It is preferable. However, especially when trying to analyze activin itself, the amount of activin produced in the body of wild-type animals is usually very small, so that the activin is below the detection limit in the commonly used ELISA method, Western blot method, etc. There is. On the other hand, when activin is detected in the animal of the present invention, it can be said that the non-human animal of the present invention contains more activin than the wild-type animal of the same species.

The activin-introduced mouse of the present invention thus obtained is preferably crossed with a syngeneic wild-type mouse to maintain the strain. Each time a cross is performed, an individual overexpressing the activin gene is selected from the obtained offspring. Moreover, a homozygous individual can also be obtained by mating male and female heterozygous individuals obtained.
More preferably, the obtained activin-introduced mouse is used to conduct a behavioral test, and has at least one of a phenotype of increased activity, emotional abnormality, information processing mechanism abnormality, and learning memory abnormality Select. Any behavior test may be used as long as it can be applied to the mouse, and it is preferable to perform a comprehensive judgment by combining a plurality of behavior tests. The obtained results are preferably compared with the results obtained using wild-type mice of the same litter, and can be determined to be abnormal when there is a significant difference. For example, analysis of variance (ANOVA) or the like can be used to determine the result. The phenotypic selection criteria in each behavior test are as described in detail above.

2. Method for screening for therapeutic and / or prophylactic agent for psychiatric disorders or memory disorders involving activin of the present invention
2-1. Screening method using the non-human animal of the present invention Among the non-human animals of the present invention, the activin gene is at least overexpressed in the brain, and at least increased activity, emotional abnormality, information processing mechanism abnormality, and learning Animals that have any phenotype of memory abnormalities and can be passaged are very useful for screening for therapeutic and / or prophylactic drugs for psychiatric or memory disorders involving activins that exhibit pathologies similar to these behavioral abnormalities Useful for. Examples of mental disorders in which activin is involved include depression, depression, depression, hyperactivity disorder, autism, schizophrenia, post-traumatic stress disorder, and the like. In addition, memory impairment involving activin includes learning memory impairment, memory impairment associated with aging, memory impairment associated with cerebrovascular disorder, memory impairment associated with dementia, memory impairment associated with Parkinson's disease, memory impairment associated with depression, etc. Is mentioned.

  Non-human animal behavioral tests have already been used for screening for therapeutic and / or preventive drugs for various mental illnesses or memory disorders in various psychiatric or memory disorder model animals. There is a lot of knowledge about what psychiatric disorders or memory disorders are considered to be similar symptoms. For example, if there is an increase in activity in a general activity test such as an open field test, the animal is considered to have symptoms similar to human mania or hyperactivity disorder. . Similarly, if the value decreases in the light and dark test or the forced swimming test, which is an emotional behavior test, or if the value increases in the elevated plus maze test, the animal Is understood to have symptoms similar to human mania and hyperactivity disorder. Conversely, if each behavior test yields the opposite result, the animal is considered to have symptoms similar to human depression. In addition, for example, in the prepulse inhibition test, which is an action test of the information processing mechanism, when the degree of inhibition decreases, it is considered to be related to human schizophrenia. Furthermore, if the results of Morris water maze, fear conditioning, object recognition test, etc., which are behavioral tests for learning memory, decline, human learning memory impairment and aging Memory impairment associated with dementia such as Alzheimer's disease, memory impairment associated with cerebrovascular disorder, memory impairment associated with neurological diseases such as Parkinson's disease, memory impairment associated with depression, etc. Yes. On the other hand, if an increase or decrease in memory re-fixation is observed in fear conditioning, it is considered to be related to post-traumatic stress disorder or the like. Furthermore, if there is a decrease in working memory in the Morris water maze, the animal is considered to be associated with human schizophrenia.

Thus, activin is prepared by administering a test substance to the non-human animal of the present invention and detecting the influence of this test substance on the animal by using the phenotypic change as described above as an index. For the treatment and / or prevention of psychiatric disorders or memory disorders involving
Examples of the test substance include peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, etc., and these compounds are novel compounds. It may also be a known compound. Specifically, the test substance is administered to the non-human animal of the present invention, the behavior test is performed, and the influence of the test substance on the animal is analyzed. Preferably, a behavioral test is performed using an untreated control animal, and the result is compared with the analysis. As a method for administering the test substance to these non-human animals, for example, oral administration, intravenous injection, and the like are used, and may be appropriately selected in view of the type of non-human animals, symptoms, properties of the test substance, and the like. . Similarly, the dose of the test substance can be selected as appropriate.

  Specifically, for example, as described in detail in 1 above, the non-human animal of the present invention preferably has an increased amount of activity and emotion as compared to a wild-type animal. An increase in activity or emotionality means that the animal has symptoms similar to human mania or hyperactivity disorder. Therefore, for example, when a non-human animal of the present invention administered with a certain test substance is subjected to an activity amount or emotional behavior test and a decrease in the activity amount or emotional activity is confirmed, the test substance is not It can be seen that it can be used as a therapeutic and / or prophylactic agent for hyperactivity disorder. On the other hand, if a further increase in the amount of activity or emotion is confirmed, it can be seen that the test substance can be used as a therapeutic and / or prophylactic agent for depression. In addition, for example, the information processing mechanism of the non-human animal of the present invention is preferably enhanced as compared to a wild-type animal. When the test substance administration shows a change in the results of the information processing mechanism behavior test, it can be seen that the test substance can be used as a therapeutic and / or prophylactic agent for schizophrenia. Furthermore, for the learning memory function, similarly, when a change in the results of the learning memory behavior test is observed due to the administration of the test substance, the test substance is used for the treatment of dementia such as learning memory disorder or Alzheimer's disease. And / or can be used as a prophylactic. The phenotypic selection criteria for each behavioral test is as detailed above.

2-2. Screening method using cells expressing activin receptor Analysis of behavioral abnormalities in activin-introduced animals of the present invention revealed an association between activin overexpression and mental illness or memory impairment. Thus, these diseases can be treated and / or prevented. That is, screening for therapeutic and / or preventive drugs for mental disorders or memory disorders involving activin is also performed using cells expressing activin (hereinafter sometimes referred to as “activin-expressing cells”). Can do. Specifically, a test substance can be added to activin-expressing cells, and the influence of the test substance on the cells can be detected using changes in the activin expression level as an index.

  Screening for therapeutic and / or prophylactic agents for psychiatric disorders or memory disorders involving activin can also be performed using cells expressing activin receptors. Specifically, activin and the test substance or only the test substance are added to cells expressing activin receptor, and the effect of the test substance on the cell is measured using changes in the activin signal system as an index. This can be done by detecting.

  Activin binds to the activin receptor, activates the activin signal system, and expresses its function. Thus, for example, a substance that enhances a factor present in this signal system is thought to enhance the function expressed by activin. Therefore, if cells expressing the activin receptor are used, a substance that affects the activin signal system can be screened easily using a cultured cell system or the like. The cells to be used may be any cells expressing activin receptor, but preferably cells expressing not only activin receptor but also activin. Examples of such cells that can be used include fibloblasts and keratinocytes that express activin receptors. Further, from the viewpoint of reflecting the function of activin in the brain, for example, primary cultured cells of cerebral nerve cells prepared from the hippocampus and the like are preferable. Cells expressing the activin receptor derived from the non-human animal of the present invention described in 1 above are also preferably used. Furthermore, even cells that originally do not express activin receptor can be used cell lines in which activin receptor is introduced and expressed by known techniques.

  The addition of the test substance to the cells can be easily performed by, for example, a method of directly adding to the medium in which the cells are cultured. As the test substance, the same substances as those detailed in 2-1 can be used. If the cells express activin receptor but do not express activin, activin is administered together with the test substance. The dose of activin may be an amount that can activate the activin signal system in the cells to which it is added.

  Thus, in the cells in which the activin signal system is activated, the influence of the test substance on the signal system may be analyzed. The analysis is preferably performed in comparison with cells not added with the test substance. The influence of the test substance on the signal system may be analyzed, for example, by analyzing any of various factors existing downstream of activin in the activin signal system. Examples of such factors include activin receptor and Smad2 / 3. For example, in the case of activin receptor, Smad2 / 3, etc., the influence of the test substance on the activin signal system can be analyzed by analyzing changes in the phosphorylation state of these factors by Western blotting etc. .

  As a result of analysis, for example, when phosphorylation of the activin receptor or Smad2 / 3 is decreased, it is determined that the test substance is useful as a therapeutic and / or prophylactic agent for mania or hyperactivity disorder Can do. Conversely, when phosphorylation is enhanced, it can be determined that the test substance is useful as a therapeutic and / or prophylactic agent for depression. The phenotypic selection criteria for each behavioral test is as detailed above.

2-3. Screening method using cells expressing activin Analysis of behavioral abnormalities in activin-introduced animals of the present invention revealed an association between activin overexpression and mental illness or memory impairment. Therefore, by controlling the amount of activin expression Treatment and / or prevention of these diseases can be performed. That is, screening for therapeutic and / or preventive drugs for mental disorders or memory disorders involving activin is also performed using cells expressing activin (hereinafter sometimes referred to as “activin-expressing cells”). Can do. Specifically, a test substance can be added to activin-expressing cells, and the influence of the test substance on the cells can be detected using changes in the activin expression level as an index.

When the amount of activin increases, the activin signal system is more activated. For example, a substance that increases the amount of activin involved in the activin gene is a substance that enhances the function of activin. Therefore, if activin-expressing cells are used, a substance that affects activin expression can be screened.
The activin-expressing cells may be any cells that can express activin, and examples thereof include primary cultured cells of brain neurons expressing activin prepared from the hippocampus. Here, activins include activins βA, βB, βC, and βE. Although it is considered that there is no significant difference in these functions, since it is known that activin βA mainly exists in the brain, cells expressing activin βA are preferably used in the present invention. It is done. A test substance is added to these cells, and the influence of the test substance on the cells can be detected using the change in the activin expression level as an index. As the test substance, the same substances as those detailed in 2-1 can be used. The test substance can be easily added by, for example, a method of directly adding the test substance to the medium in which the cells are cultured. Thus, the change in the activin expression level in the cells to which the test substance is added is analyzed by a known protein analysis method such as ELISA or Western blotting. The analysis is preferably performed in comparison with cells not added with the test substance.

  As a result of the analysis, for example, when the activin expression level is decreased, it can be determined that the test substance is useful as a therapeutic and / or prophylactic agent for mania or hyperactivity disorder. Conversely, when the expression level is increased, it can be determined that the test substance is useful as a therapeutic and / or prophylactic agent for depression.

2-4. Formulation method of the substance obtained by the screening method of the present invention Thus, the substance selected by the screening method of the present invention described in 2-1 to 3 and 3 below is formulated by a commonly used method known per se, Depression, depression, depression, hyperactivity disorder, autism, schizophrenia, post-traumatic stress disorder, and other mental disorders, or learning memory impairment, memory impairment associated with aging, A therapeutic and / or prophylactic agent for memory disorders such as memory disorders associated with cerebrovascular disorders, memory disorders associated with dementia, memory disorders associated with Parkinson's disease, and memory disorders associated with depression can be prepared.

  Substances contained as an active ingredient in the medicament of the present invention may be physiologically acceptable salts, hydrates, solvates and the like. Examples thereof include pharmaceutically acceptable salts such as salts with inorganic bases, salts with organic bases, salts with inorganic acids, salts with organic acids, and salts with basic or acidic amino acids. Preferable examples of the salt with an inorganic base include alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as calcium salt and magnesium salt, aluminum salt and ammonium salt. Preferable examples of the salt with an organic base include, for example, trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N, N′-dibenzylethylenediamine. And salt. Preferable examples of the salt with inorganic acid include salts with hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid and the like. Preferable examples of the salt with organic acid include formic acid, acetic acid, propionic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, benzoic acid. And salt. Preferable examples of the salt with basic amino acid include salts with arginine, lysine, ortinine and the like, and preferable examples with acidic amino acid include salts with aspartic acid, glutamic acid and the like.

  The substance contained as an active ingredient in the drug of the present invention is itself a mental disorder such as depression, depression, depression, hyperactivity disorder, autism, schizophrenia, post-traumatic stress disorder, or Can be administered to patients with memory impairment such as learning memory impairment, memory impairment associated with aging, memory impairment associated with cerebrovascular disorder, memory impairment associated with dementia, memory impairment associated with Parkinson's disease, and memory impairment associated with depression However, it can also contain two or more active ingredients. Moreover, it is preferable to prepare and administer a pharmaceutical composition using a pharmacologically acceptable additive for pharmaceutical preparation. For example, tablets, capsules, granules, fine granules, powders, pills, microcapsules, liposome preparations, troches, sublinguals, liquids, elixirs, emulsions, suspensions, etc. As an injection, a suppository, an ointment, a patch or the like manufactured orally as a sterile aqueous liquid or oily liquid. These include, for example, the compound in admixture in a unit dosage form required for accepted pharmaceutical practice, along with physiologically recognized carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, and the like, It can be produced using methods well known in the art such as filling or tableting. The amount of active ingredient in these pharmaceutical compositions is such that an appropriate volume within the indicated range is obtained.

  For example, additives that can be mixed into tablets, capsules and the like include binders such as gelatin, corn starch, tragacanth and gum arabic, excipients such as crystalline cellulose, fillers such as cellulose, mannitol, and lactose. , Starch, polyvinylpolypyrrolidone, starch derivatives, disintegrating agents such as sodium starch glycolate, lubricants such as magnesium stearate, wetting agents such as sodium lauryl sulfate, sucrose, lactose Alternatively, a sweetening agent such as saccharin, a flavoring agent such as peppermint, red mono oil or cherry is used. In addition, tablets can be coated with an enteric coating agent or the like as necessary. As for the capsule, a liquid carrier such as fats and oils can be further contained in the additive.

In addition, for example, a sterile pharmaceutical composition used as an injection should be formulated in accordance with normal pharmaceutical practice such as dissolving or suspending active substances in vehicles such as water for injection, naturally produced vegetable oils such as sesame oil and coconut oil, etc. Can do. As an aqueous solution for injection, for example, an isotonic solution (D-sorbitol, D-mannitol, sodium chloride, etc.) containing physiological saline, glucose and other adjuvants, etc. are used. , Alcohol (ethanol etc.), polyalcohol (propylene glycol, polyethylene glycol etc.), nonionic surfactant (polysorbate 80 ^™ , HCO-50 etc.) and the like can be used in combination. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and can be used in combination with solubilizing agents such as benzyl benzoate and benzyl alcohol. The above pharmaceutical composition includes, for example, a buffer (phosphate buffer, sodium acetate buffer, etc.), a soothing agent (benzalkonium chloride, procaine hydrochloride, etc.), a stabilizer (human serum albumin, polyethylene, etc.). Glycol, etc.), preservatives (benzyl alcohol, phenol, etc.), antioxidants and the like may be incorporated. The aqueous liquid for injection thus prepared is usually produced by dissolving the compound in a medium as described above and sterilizing by filtration, and then filling it into a suitable sterilized vial or ampoule. To enhance stability, the water in the vial may be removed under vacuum after the pharmaceutical composition has been frozen. An oily liquid can be produced in substantially the same manner as in the case of an aqueous liquid, but can be preferably produced by suspending the compound in the medium and then sterilizing with ethylene oxide or the like.

  Since the pharmaceutical composition thus obtained is safe and has low toxicity, for example, depression, depression, depression, hyperactivity disorder, autism, schizophrenia for humans and non-human mammals , Post-traumatic stress disorder, learning memory disorder, memory disorder associated with aging, memory disorder associated with cerebrovascular disorder, memory disorder associated with dementia, memory disorder associated with Parkinson's disease, or memory disorder associated with depression and / or Or it can be administered as a prophylactic agent. The dose of the substance, which is an active ingredient contained in the composition, varies depending on the target disease, symptoms, target organ, administration target, administration method, and the like. (With a body weight of 60 kg), it is about 0.01 to 10 mg, preferably about 0.1 to 5 mg, more preferably about 0.1 to 2 mg per day. In addition, when administered parenterally, for example, when administered to an ordinary adult (as 60 kg) in the form of an injection, about 0.01 to 3 mg per day, preferably about 0.01 to 2 mg, more preferably about It is preferable to administer about 0.01 to 1 mg by intravenous injection. Moreover, it is desirable to administer the daily dose in one to several times. In the case of other animals, an amount converted per 60 kg can be similarly administered.

3. Screening method for therapeutic and / or prophylactic agent for depression and schizophrenia of the present invention Among the non-human animals of the present invention, at least increased activity, emotional abnormality, information processing mechanism abnormality, and learning memory Animals that have any of the abnormal phenotypes and can be passaged are very useful for the screening of therapeutic and / or prophylactic drugs for psychiatric disorders involving activins that exhibit pathologies similar to these behavioral abnormalities. Specifically, for example, depression, depression, depression, hyperactivity disorder, autism, schizophrenia, post-traumatic stress disorder, learning memory impairment, memory impairment associated with aging, memory associated with cerebrovascular disorder As described in detail in 2 above, it is useful for screening for therapeutic and / or preventive drugs such as disorder, memory disorder associated with dementia, memory disorder associated with Parkinson's disease, or memory disorder associated with depression.

  Among these diseases, depression and schizophrenia are known as diseases in which treatment and drug development are very difficult because they have biphasic symptoms. For example, a patient with depression is characterized by repeating such a state and a depressed state, and drug administration is performed while carefully observing the cycle and degree of the repetition. In addition, patients with schizophrenia develop delusions, hallucinations, emotional disorders, etc., but are known to repeat the onset and recovery of these symptoms. In these biphasic symptoms, if a drug that exerts a strong effect on only one symptom is administered unnecessarily, it may adversely affect the other symptom and eventually worsen the condition. Therefore, it is necessary to consider the development of drugs. In addition, for example, many currently used drugs for depression and depression are effective only in about 30 to 40% of administered patients, and are currently known to be effective. There is also a demand for the development of drugs with a new mechanism of action different from the mechanism.

  Accordingly, the present inventors analyzed the results of these two non-human animals using the non-human animals exhibiting the opposite symptoms together with the non-human animals of the present invention, thereby treating these diseases and / or It was conceived that screening of preventive drugs can be effectively performed. In activin, a protein called follistatin that inhibits its action is known (Science, 247,836-838, 1990, GenBank accession number: CR541813 of human follistatin cDNA), and this follistatin is overexpressed (Molecular Endocrinology, 12, pp96-106, 1998), and non-human animals having an expression system opposite to that of the animal of the present invention (Japanese Patent Application No. 2004-233619) can be used for such screening. In addition, psychiatric disorders involving activin are considered to be different from the conventionally known mechanism of action for depression and depression, and thus the substances selected in this way are problematic for conventional drugs. There is a possibility that can be solved.

That is, the method for screening for a therapeutic and / or prophylactic agent for depression and schizophrenia according to the present invention comprises at least an increase in activity, emotional abnormality, information processing in the non-human animal of the present invention detailed in 1 above. An animal having any of the phenotypes of abnormalities in mechanism and abnormalities in learning memory, overexpression of follistatin gene, and at least one of activity level, emotionality, information processing mechanism, and learning memory The test substance is administered to an animal having a phenotype opposite to that of the non-human animal of the present invention, and the substance (1) or (2) below is selected.
(1) In the non-human animal of the present invention, at least any of the indicators of activity, emotionality, and information processing mechanism has a value close to normal, the follistatin gene is overexpressed, and at least activity, emotionality A substance that does not affect the value of the index in an animal having a phenotype opposite to that of the non-human animal of the present invention in any one of information processing mechanism and learning memory.
(2) The follistatin gene is overexpressed, and at least the amount of activity in an animal having a phenotype opposite to that of the non-human animal of the present invention in any of activity amount, emotionality, information processing mechanism, and learning memory, A substance whose value is close to normal in any of the indicators of emotionality and information processing mechanism and does not affect the value of the indicator in the non-human animal of the present invention.

By conducting such screening, treatment of depression or schizophrenia that shows an ameliorating effect on one symptom and does not adversely affect the other symptom and exacerbate the condition and / or Alternatively, prophylactic drugs can be screened.
The test substance, behavior test, and result analysis method used in the above method can all be performed in the same manner as described in detail in 1 and 2 above.
EXAMPLES Hereinafter, although an Example demonstrates this invention, this invention is not limited at all by these Examples.

Production of ActivinβA forebrain-specific overexpression mice In order to analyze the function of activin in the brain, the animal of the present invention does not cause any abnormalities in the early stages of development, and is specific for the forebrain Create a transgene (see Fig. 1) so that mouse ActivinβA is expressed under the control of the αCaMKII promoter (Science, vol 274, pp1678-1683, 1996), which begins expression after 4 weeks). Produced. The mouse strain used is C57BL.

  Activin βA cDNA (SEQ ID NO: 1) was obtained by extracting mRNA from the hippocampus of the brain expressing mouse activin and amplifying it by RT-PCR. A known base sequence (SEQ ID NO: 2) was used as the base sequence of the Myc tag protein. First, PCR was performed using a primer containing the base sequence of Myc tag protein downstream of activin βA cDNA to prepare a DNA in which these were linked. Further, this DNA was inserted into a plasmid (Invitrogen) containing an αCaMKII promoter and an SV40 poly A sequence to prepare a DNA (transgene) in which all of them were linked (FIG. 1).

  Next, the obtained DNA was introduced into a mouse fertilized egg. C57BL was used as a mouse strain. As a fertilized mouse, a fertilized egg collected from a female mouse after mating a female mouse and a male mouse subjected to superovulation treatment was used. The transgene was introduced into this by the microinjection method, and the obtained activin gene-introduced fertilized eggs were transplanted into the oviducts of pseudo-pregnant female mice to give birth and give birth.

As a result, activin transgenic mice (activin transgenic mice; Ac tivin- M yc transgenic mice: hereinafter, sometimes abbreviated as "ACM") was a total of two lines Preparation of ACM3 the ACM4 as independent lines. The expression state of the transgene in the obtained individuals was analyzed by in situ hybridization. As a probe, the SV40 polyA signal sequence contained in the transgene is used. As a non-human animal of the present invention, a mature 100-day-old mouse is used, and as a control individual, a wild-type mouse born from the same litter is used. did. As a result, it was confirmed that the expression in the hippocampus was significantly increased in ACM3 and ACM4 as compared with the wild type mouse. In addition, ACM4 was found to have higher mRNA expression than ACM3.

  Next, in order to confirm that the activin gene was actually expressed in the obtained individual and activin was generated, analysis by ELISA was performed. The analysis was performed using an activin assay kit (MCA1426KZZ: manufactured by Cellotec). The hippocampus was excised from one side of a 100-day-old mouse brain, homogenized with Tris buffer (0.32 M sucrose, 5 mM Tris-HCl pH 7.0, Protease inhibitor cocktail (Sigma)), and the extract was used as a sample. did. As a result, in the hippocampus, the expression of activin could not be confirmed in wild type mice and ACM3, but in ACM4, the presence of activin could be confirmed. FIG. 2 shows the results of analyzing the amount of activin βA produced in the hippocampus of ACM3, ACM4, and wild type mice by ELISA. In the figure, “Wild” represents a control wild-type mouse, and ACM3 and ACM4 represent activin-introduced mice of the present invention. “N.D.” means not detectable. The vertical axis of the graph represents the amount of activin βA (ng) per hippocampus obtained from one side of one mouse brain.

  In the hippocampus, a significant difference in the amount of activin was observed between wild-type mice and ACM4, which clearly showed that activin was overproduced specifically in the forebrain under the control of the αCaMKII promoter. In addition, since there was very little endogenous activin, it was suggested that analysis of endogenous activin in wild-type mice was difficult with this ELISA kit. Although ACM3 could not detect activin expression in the hippocampus itself, it was thought to be below the detection limit due to weaker transgene mRNA expression than ACM4. However, in various behavioral tests described in detail below, ACM3 also showed behavioral abnormalities similar to ACM4, so although it was below the detection sensitivity of the kit, a large amount of activin was expressed compared to wild type mice. It was inferred that

  ACM3 and ACM4 grew normally, and there were no abnormal physical characteristics (appearance, weight, height, fur, arm strength), and normal reproduction was possible in both males and females.

General activity test
The general activity of ACM was measured by an open field test. In the exploratory behavior test, the mouse is left in a 30 cm x 30 cm square (Muromachi Kagaku Co., Ltd.) surrounded by a high wall for 30 minutes, and the behavioral distance, behavioral time, number of rises, and rise of the mouse in a normal state This test measures time every 5 minutes.

  When this experiment was performed on ACM (100 to 150 days old, male), the action time and action distance increased significantly (ANOVA, p <0.05) in ACM3 compared to control individuals, and the number of rises and rises in ACM4. Time increased (ANOVA, p <0.05) (Figure 3). FIG. 3A shows the results of the open field test performed on the activin transgenic mice of the present invention. In the figure, the value indicated by a circle (◯) is the result of the activin-introduced mouse of the present invention, and the value indicated by a square (□) is the result of the control wild-type mouse. In each graph, the horizontal axis represents the experiment time (minutes), and the vertical axis represents the action time (sec), action distance (cm), number of rises, and rise time (sec) at each time.

  On the other hand, [B] in FIG. 3 is a graph showing the results of measuring the time taken to occupy the middle part in the same test. The vertical axis represents the occupation time (sec). In general, since mice are very alert in the first place, they prefer the edge of the wall and tend not to stay in the middle open area. However, especially in ACM4, the occupation time of the middle part was found to be significantly higher than that of the control individuals. The occupancy time in the middle is called risk-taking behavior, and is said to indicate a high degree of aggressiveness, and the amount of standing up behavior indicates a high degree of curiosity.

These results indicate that ACM has significantly higher activity, curiosity, and aggressiveness than the control individuals. ANOVA (analysis of variance) is a numerical value obtained by an analysis of variance.

Light and dark test
In the light and dark test, a quadrilateral surrounded by a high wall is divided into two areas, a light place and a dark place (made by Muromachi Kagaku Co., Ltd.). There is a hole in the middle of the wall so that the mouse can move freely. Anxiety behavior can be measured by leaving the mouse in the device for 30 minutes and recording the behavior. Since mice usually prefer dark areas and do not tend to appear in bright areas, the degree of anxiety of the mice can be determined by measuring the respective occupation times. In other words, the longer the occupied time of the bright part, the lower the degree of anxiety, which means active and bold, and the shorter the time, the stronger the degree of anxiety.

As a result of conducting this experiment on ACM, there was no significant difference between ACM3 and control individuals, but it was found that ACM4 significantly occupies the bright area compared to control individuals (ANOVA). , p <0.05) (Figure 4). The results of the light and dark test are shown in FIG. The horizontal axis of the graph is the experiment time (minutes), and the vertical axis is the dark area occupation time (%).
This result shows that the degree of anxiety is decreasing in ACM4.

Forced swimming test
In the forced swimming test, water (water temperature: 25 ° C) is placed in a cylindrical pool (25 cm in diameter) to a height (20 cm) at which the mouse's feet do not reach the floor, and the mouse is placed in it. This is a test that measures the activity of mice (15 minutes). Normally, when a mouse is forced to swim without this escape, it gradually stops swimming and becomes immobile (so-called despair behavior). By measuring this immobility time, it is possible to know the behavior pattern of the mouse in despair. That is, the shorter the immobility time, the higher the resistance to despair, and the longer the non-moving time, the lower the resistance to despair, the easier it is to despair. This test is frequently used for screening for depression drugs.

  A 15 minute test was conducted once a day for 2 days. The activity of the mouse was measured with an infrared sensor (Muromachi Kagaku Co., Ltd.). As a result, in ACM3, the immobility time on the second day was significantly reduced compared to the control individuals (ANOVA, p = 0.02) (FIG. 5). The results of this forced swimming test are shown in FIG. The horizontal axis of the graph is the experimental time (minutes), and the vertical axis is the immobility time (sec) for each one minute.

  This result indicates that ACM3 is highly resistant to despair.

Elevated plus maze test The elevated plus maze test was conducted with a device (made by Ohara Medical Sangyo Co., Ltd.) that had four passages assembled in a cross shape at a height of 30 cm. The book is an open arm and the remaining two passages are surrounded by a transparent plastic wall (closed arm). Usually, the mouse is not good at high places, so the existence time in the open arm tends to be shorter than that in the closed arm. Therefore, by measuring the existence time of the mouse in each of the open arm, the closed arm, and the intersection (center), it is possible to know the behavior of the mouse with respect to fear. That is, it means that it has resistance to fear when the presence time in the open arm is long, and conversely, it means that fear is strong.

  The result of the elevated plus maze test is shown in FIG. The duration of the mouse in open arm, closed arm, and center was measured, and the ratio of each was shown in a graph. The vertical axis of the graph represents the ratio (%) of the occupation time in each part. As a result, in ACM3, there was no significant difference from the control individual at each time of existence. On the other hand, in ACM4, a statistically significant difference was not obtained (ANOVA, p = 0.06), but the time in the open arm tended to be slightly longer than in the control individuals.

  These results suggest that ACM4 has a slightly reduced fear of heights compared to controls.

Prepulse inhibition test
The prepulse inhibition test is a behavioral test used for screening for schizophrenic drugs. Normally, mice give a big startle response when they hear loud sounds. However, if a small sound (hereinafter sometimes referred to as “prepulse”) is heard immediately before a loud sound, the startle response when the loud sound is heard decreases (this phenomenon is referred to as “prepulse inhibition (PPI) ) "). The mechanism of PPI is considered to reflect the function of the neuron circuit to prevent the processing of sensory information input first from being disturbed by the immediate stimulus. Using this mechanism, abnormalities in information processing functions in the brain can be analyzed by PPI tests. It is known that when the information processing is incomplete (for example, human schizophrenia patients, etc.), the degree of suppression is reduced or does not occur.

  The loud sound was set to 120 dB, and three small sounds (pre-pulses) of 68, 71, and 77 dB were heard 100 msec before that. The startle response when a loud sound was heard was measured by the change in body weight that occurred when the mouse jumped up (San Diego Instruments). The results of this behavior test on ACM3 are shown in FIG. It is a graph which shows the result of having measured the startle response with respect to the sound of 120 dB of a mouse | mouth, when a prepulse (PP) is 68, 71, and 77 dB, respectively. The vertical axis represents the prepulse inhibition rate (PPI).

ACM3 significantly increased the degree of suppression even with a prepulse as small as 68 dB (ANOVA, p = 0.0016) (FIG. 7). At this level of prepulse, the degree of suppression is low even in normal control individuals. Although the meaning of the increase in reactivity was not clear, it was found that at least the degree of suppression in PPI increased and no failure of the information processing mechanism was observed.
From the above results, it was found that the activin-introduced mice produced by the present inventors had a greater activity, increased curiosity, and resistance to anxiety and despair compared to wild-type mice. Moreover, the failure of the information processing mechanism was not seen. From these results, this activin-introduced mouse was shown to be a model mouse for mania and hyperactivity disorder.

Fear conditioning learning test
A fear conditioning learning test was performed on ACM mice. When a mouse is placed in a box and a fear condition (electric shock) is given, the mouse learns that there is a connection between the box and the electric shock (associative learning). By measuring the time during which a freezing reaction (a frightening reaction in which movements other than breathing stopped) was measured, it was determined whether or not the fear condition was remembered. For the freezing reaction, the movement of the mouse was recorded by a video camera, and it was later determined whether or not it was a freezing reaction by image analysis using a personal computer.
Conditional stimulation was performed by applying electric shock (training) at 0.5 mA-0.5 sec 4 times at 3 second intervals 2 minutes after placing the mouse in the box, and taking the mouse out of the box 4 minutes after the completion of stimulation, Was returned to the normal cage. On the next day, the mouse was again placed in the box (test-1) and the freezing reaction was analyzed. At this time, no electric shock is given. The next day (test-2), mice were placed in the box and the freezing reaction was observed without applying an electric shock.

The freezing response during training increased significantly more rapidly with ACM4 than with wild (FIG. 8). This indicates that learning ability is higher than wild.
In Test-1, ACM4 did not show a significant difference compared to wild, but Test-2 showed a significant increase in freezing response (FIG. 8). This result indicates that activin has the effect of promoting memory re-fixation.

  The non-human animal of the present invention can be used for depression, depression, depression, hyperactivity disorder, autism, schizophrenia, post-traumatic stress disorder, etc. It is used as a model animal for memory impairment such as memory impairment associated with memory impairment, memory impairment associated with cerebrovascular disorder, memory impairment associated with dementia, memory impairment associated with Parkinson's disease, memory impairment associated with depression, etc. Screening for therapeutic and / or prophylactic agents for disease or memory impairment can be performed.

It is the figure which showed the structure of the transgene used for preparation of the activin introduction | transduction mouse | mouth of this invention. It is the result of having analyzed the production amount of the activin (beta) A in a hippocampus by ELISA method about the activin introduction | transduction mouse | mouth of this invention. [A] is a graph showing the results of an open field test performed on the activin-introduced mice of the present invention. [B] is a graph showing the results of measuring the time taken to occupy the middle part in the same test. The vertical axis represents the occupation time (sec). It is a graph which shows the result of the light and dark test (Light and dark test) performed about the activin introduction | transduction mouse | mouth of this invention. It is a graph which shows the result of the forced swimming test (Forced swimming test) performed about the activin introduction | transduction mouse | mouth of this invention. It is a graph which shows the result of the elevated plus maze test performed about the activin introduction | transduction mouse | mouth of this invention. It is a graph which shows the result of the prepulse inhibition test (Prepulse inhibition test) performed about the activin introduction | transduction mouse | mouth ACM3 of this invention. It is a graph which shows the result of the fear conditioning learning test done about activin introduction | transduction mouse | mouth ACM4 of this invention.

Claims (16)

A non-passable non-human animal in which the activin gene is overexpressed at least in the brain and has at least a phenotype of increased activity, emotional abnormality, information processing mechanism abnormality, and learning memory abnormality, Or its descendants. A non-human animal or a progeny thereof, wherein an activin gene placed under the control of a promoter activated at least in the brain is introduced and can be passaged. The animal according to claim 2, wherein the promoter is further not activated in the reproductive organs. The animal according to claim 2 or 3, wherein the promoter is activated after the developmental period of the brain neural network. The animal according to any one of claims 2 to 4, wherein the promoter is activated specifically in the forebrain. The animal according to any one of claims 2 to 5, wherein the promoter is a CaMKII promoter. The animal according to any one of claims 2 to 6, wherein the animal has a phenotype of at least an increase in activity, an emotional abnormality, an information processing mechanism abnormality, or a learning memory abnormality. . The method for producing an animal according to any one of claims 2 to 6, comprising the following steps (1) to (3).
(1) An activin gene placed under the control of a promoter activated at least in the brain is introduced into a fertilized egg of a non-human animal.
(2) Generate the fertilized egg, analyze the obtained non-human animal, and select an individual into which the activin gene has been introduced.
(3) Confirm that the selected individual can be passaged. The method for producing an animal according to claim 1 or 7, comprising the following steps (1) to (3).
(1) The activin gene is introduced into a non-human animal fertilized egg.
(2) The fertilized egg is generated, and the obtained non-human animal is analyzed to select an individual in which the activin gene is overexpressed.
(3) Confirm that the selected individual has at least one of the phenotypes of increased activity, emotional abnormality, information processing mechanism abnormality, and learning memory abnormality and can be passaged. To do. A test substance is administered to the animal according to claim 1 or 7, and the influence of the test substance on the animal is detected by using a phenotypic change induced by overexpression of activin in the animal as an index. A screening method for a therapeutic and / or prophylactic agent for a psychiatric disorder or memory disorder involving activin. Activin and test substance, or only test substance are added to cells expressing activin receptor, and the effect of the test substance on the cells is detected using changes in the activin signal system as an indicator A method for screening a therapeutic and / or prophylactic agent for a mental illness or memory disorder involving activin. A test substance is added to an activin-expressing cell, and the effect of the test substance on the cell is detected by using a change in the activin expression level as an index. A screening method for therapeutic and / or prophylactic agents. The method according to any one of claims 10 to 12, wherein the psychiatric disorder involving activin is depression, depression, depression, hyperactivity disorder, autism, schizophrenia, or post-traumatic stress disorder. The memory disorder involving activin is learning memory disorder, memory disorder associated with aging, memory disorder associated with cerebrovascular disorder, memory disorder associated with dementia, memory disorder associated with Parkinson's disease, or memory disorder associated with depression The method in any one of 10-12. Depressant, depression, depression, hyperactivity disorder, autism, schizophrenia, post-traumatic stress, characterized by formulating a substance selected by the method according to claim 13 or 14 Disorders, learning and memory disorders, memory disorders associated with aging, memory disorders associated with cerebrovascular disorders, memory disorders associated with dementia, memory disorders associated with Parkinson's disease, or memory disorders associated with depression Method. The animal according to claim 1 or 7, wherein the follistatin gene is overexpressed, and at least in any of the indicators of activity, emotionality, information processing mechanism, and learning memory Treatment of depression or schizophrenia, comprising administering a test substance to an animal having an opposite phenotype and selecting either of the following substances (1) or (2): / Or screening method for preventive drugs.
(1) In the animal according to claim 1 or 7, the value is at least normal in any of the indicators of activity, emotionality, and information processing mechanism, the follistatin gene is overexpressed, and at least the activity A substance that does not affect the value of the indicator in an animal having a phenotype opposite to that of the animal according to claim 1 or 7 in any of emotion, information processing mechanism, and learning memory.
(2) at least in an animal in which the follistatin gene is overexpressed and has a phenotype opposite to that of the animal of claim 1 or 7 in at least one of activity, emotionality, information processing mechanism, and learning memory The substance which does not affect the value of the said index in the animal of Claim 1 or 7, in which the value becomes close to normal in any of the index of activity amount, emotionality, and information processing mechanism.

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