JP2006075156A - 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 follistatin 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 follistatin 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 follistatin gene is overexpressed.

  Follistatin is a protein that binds to activin and specifically inhibits the function of activin. 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. . Although there are many parts that have not been elucidated about the difference in these functions, etc., activin is a dimer composed of two of these β subunits. For example, in the brain, it consists mainly of a combination of βA / βA. It is known that activin is present. 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).

  The first discovery of follistatin was as a monomeric protein with a sugar chain that suppresses the synthesis and secretion of pituitary follicle-stimulating hormone (see, for example, Non-Patent Documents 8 to 11). Follistatin is localized extracellularly and binds 1: 1 to the secreted activin β subunit to inhibit activin function.

  In order to examine the action of follistatin on the activin signal system having various functions in vivo as described above, mutant mice that have knocked out or overexpressed follistatin have been prepared so far.

  For example, as a report of a transgenic mouse in which follistatin is overexpressed at least in the brain, a transgenic mouse in which follistatin is expressed under the control of metallothionein-1 promoter (see, for example, Non-Patent Document 12) There is. This mouse overexpressed follistatin in the brain, but normal passage was not possible because there were individuals showing abnormalities in the reproductive organs. Also, transgenic mice that are expressed under the control of keratin promoter (skin tissue-specific promoter) (see, for example, Non-Patent Document 13), and those that are expressed under the control of myosin light chain promoter (skeletal muscle-specific promoter). There are transgenic mice (see, for example, Non-patent Document 14), but none of follistatin is overexpressed in the brain, and the activity of activin or follistatin in the cranial nervous system cannot be analyzed. It was.

  Furthermore, in order to analyze the function of follistatin in the whole body including the brain, follistatin knockout mice have been produced. This knockout mouse grows poorly, loses muscles, shines terribly with the skin in tension, shows abnormal rib formation, and abnormal tooth formation. As a result, since breathing cannot be performed normally and the patient died within a few hours after birth, the effect of follistatin on the cranial nervous system could not be analyzed (for example, see Non-Patent Document 15).

  Thus, to date, there have been provided animals that are suitable for elucidating the effects of the activin signal system or follistatin on the cranial nervous system, particularly those overexpressing follistatin in the brain and that can be passaged. There wasn't. In addition, there was no animal that could be used as a model animal for diseases involving learning / memory and other follistatins.

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 Science, 247,836-838,1990 Endocrinology, 128,3313-3315,1991 Mol. Cell Endocrinol. 91,1-11,1993 Endocrinology, 128,1434-1440,1991 Molecular Endocrinology 12, pp96-106, 1998 The EMBO Journal, vol 20, pp5361-5372, 2001 PNAS, vol 98, pp9306-9311,2001 Nature, vol 374, pp360-363,1995

  The present invention has been made to provide a non-human animal in which the follistatin gene is overexpressed. More specifically, the follistatin gene is at least overexpressed in the brain, and has at least a phenotype of any of a decrease in activity, emotional abnormality, information processing mechanism abnormality, and learning memory abnormality. It was made in order to provide a non-human animal that can be substituted, or its offspring. Further, the present invention was made to provide a non-human animal, or a progeny thereof, characterized in that a follistatin gene placed under the control of at least a brain-activated promoter is introduced and can be passaged. Is. Furthermore, the present invention provides a method for producing the animal, a method for screening a therapeutic and / or prophylactic agent for mental illness or memory disorder involving follistatin, and the like.

  As a result of diligent studies to achieve the above-mentioned problems, the present inventors introduced at least a follistatin gene under the control of a promoter activated in the brain, into a non-human animal, It has been found that a follistatin gene can be overexpressed at least in the brain and a non-human animal that can be stably passaged can be produced. In addition, when this animal was analyzed, it was found that it has phenotypes such as decreased activity, abnormal emotionality, abnormal information processing, and abnormal learning and memory. The present invention has been accomplished based on these findings.

That is, according to the present invention,
(1) The follistatin gene is overexpressed at least in the brain, and can be passaged having at least one of a phenotype of decreased activity, emotional abnormality, information processing mechanism, and learning memory abnormality Non-human animals, or their descendants,
(2) a non-human animal, or a progeny thereof, wherein a follistatin gene placed under the control of at least a brain-activated promoter 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 a decrease in activity amount, abnormal emotionality, decreased information processing mechanism, and abnormal learning memory An animal according to any of the
(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 at least a follistatin gene under the control of a promoter activated in the brain into a fertilized egg of a non-human animal,
(Ii) generating the fertilized egg, analyzing the obtained non-human animal, and selecting an individual into which the follistatin 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 a follistatin 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 follistatin gene is overexpressed,
(Iii) Confirm that the selected individual has at least a phenotype of activity reduction, emotional abnormality, information processing mechanism decline, and learning memory abnormality and can be passaged To
(10) A test substance is administered to the animal according to (1) or (7) above, and the influence of the test substance on the animal is expressed by a phenotype induced by overexpression of follistatin in the animal. A method for screening a therapeutic and / or prophylactic agent for mental disorders or memory disorders involving follistatin, characterized by detecting the change as an index,
(11) Follistatin is characterized in that a test substance is added to a follistatin-expressing cell and the influence of the test substance on the cell is detected using a change in follistatin expression level as an index. A method for screening a therapeutic and / or prophylactic agent for mental illness or memory disorder;
(12) In the above (10) or (11), the mental disease involving follistatin is depression, depression, depression, hyperactivity disorder, autism, schizophrenia, or post-traumatic stress disorder Described method,
(13) Memory disorder involving follistatin 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 according to the above (10) or (11),
(14) Depressant, depression, depression, hyperactivity disorder, autism, integration characterized by formulating a substance selected by the method described in (12) or (13) above 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.

  According to the present invention, a non-human animal in which a follistatin gene is overexpressed at least in the brain is provided. The animal is useful for the screening of therapeutic and / or preventive drugs for mental illness or memory impairment involving the follistatin gene.

  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 relating 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” (published by Cold Spring Harbor Laboratory Press (2003)). It can carry out according to the method of it or it.

1. Non-human animal of the present invention and production method thereof The non-human animal of the present invention has a follistatin gene overexpressed at least in the brain, and at least decreased activity, affective abnormalities, decreased information processing mechanism, and learning The first feature is that it has any phenotype of memory abnormality and can be passaged. In particular, the relationship between overexpression of follistatin, decreased activity, abnormal emotionality, abnormal information processing mechanism, etc. has not been known so far, and the non-human animal of the present invention has been produced. It was revealed for the first time.

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

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

  Here, the follistatin gene is a nucleic acid carrying genetic information of follistatin, which is a protein, and means a gene encoding follistatin. Examples of the gene encoding follistatin include human follistatin cDNA (SEQ ID NO: 2, or GenBank accession number: CR541813).

  In the non-human animal of the present invention, the tissue in which the follistatin gene is overexpressed may be any tissue as long as it includes the brain and the animal has the behavioral abnormality described above. Preferably, overexpression in the forebrain is particularly preferable. When using an artificially overexpressed follistatin gene as the animal of the present invention, a DNA in which the follistatin gene is under the control of an appropriate promoter activated in the above tissue is prepared and introduced. By doing so, the animal can be produced. 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 when a follistatin gene is introduced, any animal can be used. 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 behavior 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 reduced compared to the control individual. Emotionality, information processing mechanism, and learning memory are decreasing or increasing. Preferably, both are lowered. 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 Behavioral Function Test (Biochemistry, published by Medical School, Voi. 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 (Object recognition tes) t) etc. It is preferable to use some of these in combination.

  Among the behavioral abnormalities of the non-human animal of the present invention, “decrease in the amount of activity” generally means that the activity level, curiosity, and aggressiveness are lower 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 reduced 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 shorter 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, those in which darkness occupancy time is significantly higher than control and anxiety is increasing in the light and dark test, forced swimming test (Forced swimming test) ) Immobility time is significantly longer than control and is less resistant to despair, or is present in a closed arm with a plastic wall in an elevated plus maze test For example, the time is significantly higher than that of the control, and the fear of height is increasing. “Degradation of information processing mechanism” generally means that there is an abnormality in an action or the like that ignores unnecessary information. As such a phenotype, for example, a prepulse inhibition test (Prepulse inhibition test) in which the degree of inhibition by a prepulse is lower than that of a control can be mentioned.

“Learning / memory abnormality” generally means that learning ability and memory ability decline. Such phenotypes include, for example, lower learning and memory abilities compared to controls in behavioral tests such as Morris water maze, fear conditioning, and object recognition test. And the like.
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 artificially overexpressed follistatin gene is usually crossed with a wild-type animal of the same strain, and repeatedly selecting the animal into which the gene has been introduced from the resulting offspring, The system is 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 follistatin gene may be used, or a follistatin gene overexpressed by mutagenesis or the like may be selected. As a preferred example, a non-human animal into which a follistatin gene placed under the control of at least a brain-activated promoter has been introduced and can be passaged (hereinafter referred to as “follistatin introduced animal”). There is).

  Any promoter can be used as long as it is activated in at least the brain, overexpresses the follistatin gene in the body of the introduced non-human animal, and the introduced animal has the ability to be passaged. obtain. 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 follistatin gene is overexpressed at least in the brain and the function of follistatin 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 follistatin in cranial nerve activity in a 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 follistatin relating to memory, sensation, emotion, etc. can be analyzed in the forebrain.

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

  Here, the follistatin gene is a nucleic acid carrying genetic information of follistatin, which is a protein, and means a gene encoding follistatin. The follistatin gene may be any as long as it can produce follistatin having the ability to bind to activin in the animal and inhibit the function of activin when it is expressed. For example, a region encoding follistatin on the genome may be obtained and used, but in the present invention, cDNA is preferably used. Examples of such follistatin gene include human follistatin cDNA (SEQ ID NO: 2 or GenBank accession number: CR541813) encoding human follistatin. 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 mutation introduced into the gene using a point mutagenesis method or the like may be used as long as the generated follistatin maintains the function as follistatin as described above. it can. 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, the expression “a follistatin gene is expressed” means production of mRNA encoding follistatin or follistatin.

  In addition, “being under control” of the promoter means that the expression of the follistatin gene is induced by activating the promoter in the introduced tissue, specifically, For example, a DNA (hereinafter sometimes referred to as “transgene”) in which the follistatin gene is linked downstream of the promoter and the tag sequence, an expression control region such as a terminator, and the like are appropriately linked is prepared. This condition can be induced by introducing it into a non-human animal by the 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” a follistatin gene means introducing it into any of the genomes of a non-human animal to be introduced. Here, a DNA (transgene) containing the follistatin gene in the genome is preferably introduced as one 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 follistatin-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 non-human animal of the present invention thus obtained has a phenotype of at least a decrease in activity, an emotional abnormality, a deterioration in information processing mechanism, and an abnormality in learning and memory, and can be passaged. However, those having other phenotypes are also included in the follistatin-introduced animal of the present invention.

The follistatin-introduced animal of the present invention can be prepared according to a known method for preparing a foreign gene-introduced animal that is generally used per se, using a promoter and a follistatin gene as described above. Specifically, for example,
(1) introducing at least a follistatin gene under the control of a promoter activated in the brain 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 follistatin gene has been introduced.
(3) Confirm that the selected individual can be passaged.
Can be produced.

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

  In the above production method, for example, preparation of a fertilized egg of a non-human animal, introduction of a follistatin gene into the fertilized egg, generation of the fertilized egg, acquisition of a litter derived from the fertilized egg, an individual into which the follistatin gene has been introduced These can be selected by a commonly used method known per se.

  The non-human animal may be a non-human animal that can be expressed by introducing the follistatin gene 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, follistatin cDNA (SEQ ID NO: 2 or GenBank accession No. CR541813) and αCaMKII promoter (Science, vol 274, pp1678-1683, 1996) etc. are introduced into mouse fertilized eggs, and follistatin is introduced. The case where a mouse is produced will be specifically described as an example.

  For example, a follistatin cDNA can be prepared by extracting mRNA from a tissue expressing mouse or human follistatin, further preparing a cDNA library, and screening and preparing it by a conventional method. . 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, by performing a PCR reaction using a primer containing the base sequence of Myc tag protein downstream of follistatin cDNA, a DNA in which these are easily linked can be prepared. 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 follistatin gene-introduced fertilized egg can be transplanted into the oviduct of a female mouse that has been pseudopregnant to generate and give birth.

  An individual into which the follistatin gene has been introduced is 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, and the follistatin gene introduced into the genome using the Southern blot method, PCR method, etc. Presence / absence and number of copies can be analyzed.

  In addition, it is preferable to confirm that the follistatin gene is actually expressed in the individual and follistatin is generated. Furthermore, the expression of follistatin can be indirectly confirmed using an antibody against Myc tag protein expressed together with follistatin. Such follistatin gene expression can be confirmed by, for example, ELISA, Western blotting, or the like. Further, for example, it may be analyzed by the expression level of mRNA encoding follistatin. In addition, the expression of the follistatin gene can be indirectly confirmed using the SV40 poly A sequence constituting the transgene together with the follistatin gene as a probe. Such follistatin 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 follistatin-introduced mouse of the present invention, an individual in which at least the follistatin gene is overexpressed in the brain is preferably selected. Preferably, the expression level of the follistatin gene in the brain of the mouse of the present invention is analyzed, and compared with the expression level of the follistatin gene in the brain of the wild-type mouse, It is preferable to select. However, especially when trying to analyze follistatin itself, the amount of follistatin normally produced in wild-type animals is very small, so follistatin is below the detection limit in the commonly used ELISA method, Western blot method, etc. It may become. On the other hand, when follistatin is detected in the animal of the present invention, it can be said that the non-human animal of the present invention contains more follistatin than the wild-type animal of the same species.

  The follistatin-introduced mouse of the present invention thus obtained is preferably crossed with a syngeneic wild-type mouse to maintain the strain. Each time mating is performed, an individual overexpressing the follistatin 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 follistatin-introduced mouse is used to conduct a behavioral test, and has at least one of a phenotype of activity reduction, emotional abnormality, information processing mechanism decline, and learning memory abnormality Choose one. 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. Screening method for therapeutic and / or prophylactic agent for mental illness or memory disorder involving follistatin 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 follistatin gene is overexpressed, and at least the amount of activity is decreased, emotional abnormality, information processing mechanism is decreased, and learning memory Animals that have any of the abnormal phenotypes and can be passaged are very useful for the screening of drugs for the treatment and / or prevention of mental illness or memory impairment involving follistatin that presents pathologies similar to these behavioral abnormalities Useful. Mental illness or memory impairment involving follistatin also includes mental illness or memory impairment involving activin, which it specifically inhibits. Examples of mental disorders involving follistatin include depression, depression, depression, hyperactivity disorder, autism, hyperactivity disorder, schizophrenia, post-traumatic stress disorder, and the like. In addition, memory disorders involving follistatin include 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, memory disorders associated with depression Etc.

  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. . If the light-occupied time is long in the light and dark test, which is an emotional behavior test, or if the immobility time is short in the forced swimming test, the elevated plus maze test It is also understood that the animal has similar symptoms to human mania and hyperactivity disorder when the presence time in the open arm is long. 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 a decrease in learning ability or retention of memory 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.

  Therefore, the test substance is administered to the non-human animal of the present invention, and the effect of the test substance on the animal is detected using the phenotypic change of the animal as an index. Screening for therapeutic and / or prophylactic agents for psychiatric or memory disorders involving statins can be performed.

  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 a reduced amount of activity and emotionality compared to a wild-type animal. A decrease in activity or emotionality means that the animal has symptoms similar to human depression and autism. Thus, for example, when a non-human animal of the present invention administered with a certain test substance is subjected to an activity test or emotional behavior test and an increase in activity or emotional activity is confirmed, the test substance may be depressed or It can be seen that it can be used as a therapeutic and / or prophylactic agent for autism. On the other hand, if a further decrease 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 common diseases and hyperactivity disorders. For example, the information processing mechanism of the non-human animal of the present invention is preferably lower than that of 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 follistatin The analysis of behavioral abnormalities in the follistatin-introduced animal of the present invention revealed the relationship between follistatin overexpression and mental illness or memory impairment. By controlling, it is possible to treat and / or prevent these diseases. That is, screening for therapeutic and / or prophylactic drugs for mental illness or memory impairment involving follistatin uses cells expressing follistatin (hereinafter, sometimes referred to as “follistatin expressing cells”). Can also be done. Specifically, a test substance can be added to a follistatin-expressing cell, and the influence of the test substance on the cell can be detected using the change in follistatin expression level as an index.

  If the amount of follistatin increases, the activin signal system is inhibited. For example, a substance that increases follistatin production by participating in the follistatin gene enhances inhibition of the activin signal system by follistatin. It is. If follistatin-expressing cells are used, a substance that affects follistatin expression can be screened.

  The follistatin-expressing cell may be any cell that can express follistatin, for example, a brain neuron expressing follistatin prepared from the non-human animal of the present invention described in detail in 1 above. Primary cultured cells and the like. 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 follistatin 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 expression level of follistatin in the cells to which the test substance has been 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 expression level of follistatin is decreased, it can be determined that the test substance is useful as a therapeutic and / or prophylactic agent for depression or autism. On the contrary, when the expression level is increased, it can be determined that the test substance is useful as a therapeutic and / or prophylactic agent for common disease or hyperactivity disorder. Furthermore, by using an animal having a phenotype opposite to that of the non-human animal of the present invention, such as an activin-introduced animal, it is possible to screen for therapeutic and / or preventive drugs for depression and the like.

2-3. Method for Formulating Substance Obtained by Screening Method of the Present Invention Thus, the substance selected by the screening method of the present invention detailed in 2-1 and 2 is formulated by a commonly used method known per se, and the substance is prepared. Depression, depression, depression, hyperactivity disorder, mental disorders such as autism, schizophrenia, or learning memory impairment, memory impairment associated with aging, memory impairment associated with cerebrovascular disorder A therapeutic and / or prophylactic agent for memory impairment such as memory impairment associated with dementia, memory impairment associated with Parkinson's disease, and memory impairment 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, sterile pharmaceutical compositions used as injections 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, memory disorder associated with depression, and / 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.

  EXAMPLES Hereinafter, although an Example demonstrates this invention, this invention is not limited at all by these Examples.

Preparation of follistatin forebrain-specific overexpression mice The animal of the present invention does not cause abnormalities in the early stages of development in order to analyze the function of follistatin in the brain. A transgene (see Fig. 1) is prepared so that human follistatin is expressed under the control of the αCaMKII promoter (Science, vol 274, pp1678-1683, 1996), which is expressed approximately after 4 weeks of age). Statin-introduced mice were prepared. The mouse strain used is C57BL.

  Follistatin cDNA (SEQ ID NO: 2) was obtained by extracting mRNA from an ovary expressing human follistatin and amplifying it by RT-PCR. A known base sequence (SEQ ID NO: 1) 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 the follistatin 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 resulting follistatin gene-introduced fertilized egg was transplanted to the oviduct of a female mouse pseudo-pregnant to generate and give birth.

  As a result, follistatin-introduced mice (Follistatin-Myc transgenic mice, hereinafter referred to as “FSM”) were prepared as one line (hereinafter referred to as “FSM4”). There was). 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 the FSM compared with the wild type mouse.

  Next, in order to confirm that the follistatin gene was actually expressed and follistatin was produced in the obtained individuals, analysis by ELISA was performed. The analysis was performed using a follistatin assay kit “AN'ALYZA Follistatin Immunoassay” (Catalog No. 8085: manufactured by GT (Minneapolis, MN, USA)). 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, follistatin expression could not be confirmed in wild type mice in the hippocampus, but the presence of follistatin was confirmed in FSM (FIG. 2). In the figure, “Wild” indicates a control wild-type mouse, and FSM indicates the result of a sample obtained from the follistatin transgenic mouse of the present invention. “N.D.” means not detected (Not detectable). The vertical axis of the graph represents the amount of follistatin (ng) per hippocampus obtained from one side of one mouse brain.

A significant difference in the amount of follistatin was observed between wild-type mice and FSM in the hippocampus, which clearly indicates that follistatin is overproduced specifically in the forebrain under the control of the αCaMKII promoter. It was. In addition, since there is very little endogenous follistatin, it was suggested that it is difficult to analyze endogenous follistatin in wild-type mice using this ELISA kit.
FSM4 grew normally in all individuals, and there was no abnormality in 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 FSM was measured by an open field test. In the exploratory behavior test, the mouse's behavioral distance, behavioral time, and number of rises in a normal state are left in a 30cm x 30cm square (Muromachi Science Co., Ltd.) surrounded by a high wall for 30 minutes. This is a test to measure the rise time.
When this experiment was performed on FSM (100-150 days old, male), the action time and action distance were significantly decreased in FSM4 compared to control individuals (ANOVA, p <0.0001). There was a significant decrease (ANOVA, p <0.0001) (Figure 3).
FIG. 3 is a graph showing the results of an open field test performed on the follistatin-introduced mouse of the present invention. In the figure, the values indicated by squares (□) are the results of FSM, and the values indicated by circles (◯) are the results of control wild-type mice. 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) every 5 minutes. It is said that a lot of standing up action shows a high degree of curiosity.

  These results indicate that FSM has significantly reduced activity, curiosity and aggressiveness compared to 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 FSM, it was found that the occupation time of the dark part was significantly longer than that of the control individual (ANOVA, p <0.0001) (FIG. 4). This indicates that the degree of anxiety is increasing in FSM. In FIG. 4, the values indicated by squares (□) are the results of FSM, and the values indicated by circles (◯) are the results of control wild type mice. In addition, the horizontal axis of the graph is the experiment time (minutes), and the vertical axis is the dark space occupation time (%).

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 FSM, the immobility time on the second day was significantly increased compared to the control individuals (ANOVA, p = 0.01) (FIG. 5). This indicates that FSM has a strong tendency to despair. The horizontal axis of the graph of FIG. 5 is the experiment time (minutes), and the vertical axis is the immobility time (sec) for each one minute. In the figure, the values indicated by squares (□) are the results of FSM, and the values indicated by circles (◯) are the results of control wild-type mice.

Prepulse inhibition test
The prepulse inhibition test (hereinafter sometimes referred to as “PPI test”) is a behavioral test used for screening of schizophrenia 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 listening to a loud sound is reduced (this phenomenon is called “prepulse inhibition” ( Hereinafter, this is sometimes referred to as “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). When this behavioral test was performed on FSM, the degree of suppression in PPI was significantly reduced compared to the wild type (ANOVA, p = 0.013) (FIG. 6).

  FIG. 6 shows the results of measurement of startle response to 120 dB sound when the prepulse (PP) is 68, 71, and 77 dB, respectively. The vertical axis represents the pre-pulse suppression rate. From the above results, the follistatin-introduced mice prepared by the present inventors have a reduced activity, weak curiosity, strong anxiety, and a lack of information processing mechanisms compared to wild-type mice. It was. From these results, it was shown that this follistatin-introduced mouse becomes a model mouse with negative symptoms such as depression, autism, and schizophrenia.

Spatial learning ability test (1) Morris water maze test
In order to know the FSM's ability to learn space, a Morris water maze test was conducted. The control group used FSM littermate wild. The Morris water maze test is a test in which a 1.5m diameter pool is filled with opaque water and a mouse is placed in it. There is an evacuation site called a platform in the pool. Mice usually don't like the water, so they swim to the platform to escape from the water. Since the position of the platform cannot be visually recognized by the mouse, its own spatial position information in the pool is determined from the positions of various objects outside the pool, and the spatial information is stored.

(2) Spatial learning acquisition ability test The platform position was fixed, and 6 trials per day were conducted for 4 consecutive days (Fig. 7) to learn the procedure and learn the platform position. Three trials are shown as a block. The vertical axis represents the time to reach the platform, and is the average of 3 trials.
As a result, both Wild and FSM learned that the arrival time decreased as the day progressed, and the location of the platform was learned. However, the rate of arrival time reduction in the FSM group was significantly slower than in the wild group, indicating that the FSM learning speed was slower than in the wild group.

(3) Transfer test 24 hours after the completion of the test in (2), a test (transfer test) was performed to see if the platform position was remembered. In this test, the place where the mouse swims intensively with the platform removed from the pool is determined. If the mouse remembers the platform position, it will swim intensively around where the platform was. The assay method divides the pool into four and measures the time spent swimming in each section at a fixed time (FIG. 8). When the time in the section where the platform exists is higher than that in the other sections, it is determined that the user remembers.
As a result, there was no significant difference between the FSM group and the Wild group, and both memorized the platform position to the same extent.

(4) Working memory test Working memory (working memory) is memory that lasts for several seconds to several minutes, which is shorter than short-term memory. It is called a working memory because it is a memory required to perform a certain type of work. In the water maze test, a test for changing the platform position every day was performed for 5 consecutive days to measure the working memory (FIG. 9). The platform position on each day was fixed and 6 trials were performed at 1 minute intervals per day. The platform position for each day is indicated by a black circle at the top of the graph. The vertical axis represents the platform arrival time.
As a result, it was found that the FSM group was significantly higher than the wild group in the arrival time of the final block of each day (two trials were grouped together). This result shows that FSM cannot cope with the new environment where the platform position has changed, and that FSM has a failure in working memory.

When a mouse is placed in the fear conditioning learning test box and a fear condition (electric shock) is applied, the mouse learns that there is a relationship 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.

(1) Fear-conditioned learning test with weak stimulation Conditioned stimulation was performed by applying electric shock (training) at 0.5mA-1sec three times at 1-minute intervals 2 minutes after placing the mouse in the box, and finishing the stimulation for 2 minutes It was later removed from the box and returned to the home cage (the mouse that normally keeps mice). On the next day, the mouse was again placed in the box (test 1), and the freezing reaction was analyzed (FIG. 10). At this time, no electric shock is given.
As a result, the wild group showed a freezing reaction in test 1, but the FSM group had a significantly lower freezing value than the wild group.

(2) Fear-conditioning learning test that gave a strong stimulus Next, the stimulus condition was strengthened. Two minutes after placing the mouse in the box, electric shock was applied 5 times at 1 minute intervals at 0.5 mA-1 sec, and the mouse was taken out of the box 2 minutes after the completion of stimulation and returned to the home cage. On the next day, mice were placed in the box and the freezing reaction was observed (FIG. 11).
As a result, there was no significant difference between the wild group and the FSM group. Considering together with the result of (1), FSM shows that fear learning is not established under weak fear conditions, but fear learning is established under strong fear conditions. These results indicate that FSM has poor learning ability, which is the same tendency as the results of spatial learning.

  The non-human animal of the present invention can be used for psychosis or learning memory impairment, aging, such as depression, depression, depression, hyperactivity disorder, autism, schizophrenia, posttraumatic stress disorder, etc. Is used as a model animal for memory impairment such as memory impairment associated with cerebrovascular disorder, memory impairment associated with cerebrovascular disorder, memory impairment associated with dementia, memory impairment associated with Parkinson's disease, memory impairment associated with depression, etc. Can be screened for therapeutic and / or prophylactic agents for mental disorders or memory disorders.

It is the figure which showed the structure of the transgene used for preparation of the follistatin introduction | transduction mouse | mouth of this invention. It is the result of having analyzed the production amount of follistatin in the hippocampus by ELISA method about the follistatin introduction mouse | mouth of this invention. It is a graph which shows the result of the search action test (open field test) performed about the follistatin introduction | transduction mouse | mouth of this invention. It is a graph which shows the result of the light and dark test (Light and dark test) performed about the follistatin introduction | transduction mouse | mouth of this invention. It is a graph which shows the result of the forced swimming test (Forcedswimming test) performed about the follistatin 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 follistatin introduction | transduction mouse | mouth of this invention. It is a graph which shows the result of the Morris type water maze test (test of the ability to acquire spatial learning with a fixed platform position) performed on the follistatin-introduced mouse of the present invention. It is a graph which shows the result of the Morris type water maze test (the transfer test except a platform) performed about the follistatin introduction | transduction mouse | mouth of this invention. It is a graph which shows the result of the Morris type water maze test (working memory test which changed the position of the platform) performed about the follistatin introduction mouse of the present invention. It is a graph which shows the result of the fear conditioning learning test which gave the weak irritation | stimulation performed about the follistatin introduction | transduction mouse | mouth of this invention. It is a graph which shows the result of the fear conditioning learning test which gave the strong irritation | stimulation performed about the follistatin introduction | transduction mouse | mouth of this invention.

Claims (14)

A passable non-human animal in which the follistatin gene is overexpressed at least in the brain, and has at least a phenotype of reduced activity, emotional abnormality, information processing mechanism, and learning memory abnormality Or its descendants. A non-human animal, or a progeny thereof, wherein a follistatin 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 a decrease in activity, an emotional abnormality, a deterioration in information processing mechanism, and an abnormality in learning and memory. . The method for producing an animal according to any one of claims 2 to 6, comprising the following steps (1) to (3).
(1) A follistatin 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 follistatin 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 follistatin 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 follistatin gene is overexpressed.
(3) Confirm that the selected individual has at least one of the following phenotypes: active mass reduction, emotional abnormality, information processing mechanism decline, 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 effect of the test substance on the animal is detected by using a phenotypic change induced by overexpression of follistatin in the animal as an index. A method for screening a therapeutic and / or prophylactic agent for a mental disorder or memory disorder involving follistatin. A test substance is added to a follistatin-expressing cell, and the influence of the test substance on the cell is detected using a change in follistatin expression as an index, A screening method for a therapeutic and / or preventive drug for memory impairment. 12. The method according to claim 10 or 11, wherein the mental illness involving follistatin is depression, depression, depression, hyperactivity disorder, autism, schizophrenia, or post-traumatic stress disorder. The memory disorder involving follistatin 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 Item 12. The method according to Item 10 or 11. Depressant, depression, depression, hyperactivity disorder, autism, schizophrenia, post-traumatic stress, characterized by formulating a substance selected by the method according to claim 12 or 13 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.

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