JPWO2009017082A1 - Genetically modified non-human mammal exhibiting abnormal behavior and method for producing the same - Google Patents

Abstract

A non-human mammal that exhibits abnormal behavior and has a modified Spsb4 gene in its genome.

Description

  The present invention relates to a genetically modified non-human mammal exhibiting abnormal behavior and a method for producing the same. More specifically, the present invention relates to a genetic modification showing abnormal behavior, useful for elucidating the cause and preventing and treating schizophrenia in human attention deficit / hyperactivity disorder (ADHD) and schizophrenia. The present invention relates to a non-human mammal, a production method thereof, and a screening method for an anti-ADHD drug and a schizophrenia therapeutic drug.

  Attention Deficit / Hyperactivity Disorder (ADHD) is a mental disorder with inattention, hyperactivity and impulsivity as core symptoms, which is observed mainly in children from early childhood to school age. Although the increase in ADHD children has become a major social problem in Europe and the United States, it has recently been clarified that ADHD continues not only for children but also for adults.

  The cause of the onset of ADHD is unknown, and therefore effective preventive and therapeutic methods have not been established. An animal model of this disease is indispensable for elucidating the cause of ADHD, establishing a preventive method thereof, and establishing a therapeutic method and a therapeutic drug. However, a useful model animal has not been sufficiently developed.

  As model animals expressing hyperactivity disorder similar to human ADHD, hepatitis, liver cancer, human Wilson's disease model LEC rats, (a) continuous head vibration during walking, (b) sudden turning motion, And (c) a hyperactivity disorder model rat that exhibits backward head extension movement is disclosed (see Patent Document 1). In this hyperactive model rat, individuals with behavioral characteristics different from normal rats were found in the maintenance process of the original strain of hepatitis / liver cancer / human Wilson disease model LEC rats. It has been suggested that behavioral traits adopt an autosomal single recessive inheritance pattern.

On the other hand, various genetically modified animals have been prepared and used for elucidating gene functions. For example, if a gene involved in the development of ADHD can be identified and a model animal can be obtained by altering the causative gene of ADHD, it will lead to the knowledge of new aspects of the mechanism of ADHD development. This is extremely useful in developing new drugs and the like. Therefore, development of such a model animal is desired.
JP 2001-186828 A

  An object of the present invention is to provide a genetically modified non-human mammal exhibiting abnormal behavior, which is a model animal useful for the development of treatment methods and therapeutic agents for ADHD and schizophrenia. The present invention also provides a method for producing a genetically modified non-human mammal exhibiting such abnormal behavior, and a method for evaluating a substance useful for the prevention and / or treatment of ADHD and schizophrenia using the non-human mammal. The purpose is to provide.

The present inventors have developed a technique that can generate a genetically modified mouse by only mating mice without using mouse embryonic stem (ES) cells, and reported previously (for example, special table) 2002-013602 and Horie et al., Molecular and Cellular Biology, vol. 23, No 24, p. 9189-9207 (2003)). This is a technique capable of efficiently introducing a gene into a chromosome by utilizing the transposition of a Sleeping Beauty (hereinafter also referred to as SB) transposon. Using this mouse transposon system, gene-modified mice were comprehensively produced. As a result, mice having the following abnormal behaviors (1) to (3) were found among the produced gene-modified mice.
(1) Compared with normal (wild-type) mice, no abnormalities are observed after birth to breastfeeding, but from the weaning period (3 weeks of age), it shows irregular right or left rotation when walking Becomes (rotational motion),
(2) Like the normal mouse, it is grouped at the corner of the cage when it is quiet, but when it enters the activity mode, it moves calmly in the cage (hyperactivity), and
(3) In the long-term (more than half a day) self-issued movement measurement using an infrared sensor type measuring instrument, an increase (hyperactivity) of the self-issued movement in the dark period (active period) is observed compared to normal mice. On the other hand, there is no difference in the circadian rhythm of self-issued movements seen in the light-dark cycle.
Further, when a further detailed behavioral analysis was performed on the genetically modified mice exhibiting abnormal behaviors (1) to (3), these mice showed a decrease in anxiety symptoms and a decline in cognitive function. It has been found that the behavior of the patient is very similar to the symptoms of attention defect hyperactivity disorder (ADHD), which is said to be one of developmental disorders. In addition, the hyperactivity of (2) and (3) was found to be sensitive to therapeutic drugs for human schizophrenia.

  Further, when the gene modification site was examined by gene analysis, it was found that in this mouse, the transposon was inserted into the Spsb4 (splA / ryanodine receptor domain and SOCS box containing 4) gene sequence. That is, only the Spsb4 gene was destroyed by the transposon. This result strongly suggests that an abnormality in the Spsb4 gene is associated with ADHD and schizophrenia. So far, there has been no report on studies predicting abnormal behavior of animals from the Spsb4 gene sequence or amino acid sequence, and there has been no report relating Spsb4 gene to ADHD and schizophrenia. Therefore, the non-human mammal of the present invention exhibiting abnormal behavior due to such a single gene abnormality is useful as a model animal for human ADHD and human schizophrenia both in the analysis of basic matters and in clinical application. Is. Furthermore, when human ADHD therapeutic agents or schizophrenia therapeutic agents were administered to Spsb4 gene-modified mice, it was found that abnormal behavior was improved, and abnormal behavior of such animals was associated with pathological conditions related to these diseases. Reflecting this, the inventors have conceived that such genetically modified non-human mammals are very useful for the development of treatments and therapeutic agents for ADHD and schizophrenia, thereby completing the present invention.

That is, the present invention is (1) a non-human mammal that exhibits abnormal behavior and is characterized in that the Spsb4 gene in the genome is modified,
(2) Abnormal behavior is (a) an increase in the amount of spontaneous movement during the active period compared to normal non-human mammals, while there is no difference in the circadian rhythm of the spontaneous movement observed in the light-dark cycle. A non-human mammal according to (1) above,
(3) The non-human mammal according to (1) above, wherein the abnormal behavior is (b) behavior showing irregular right or left rotation during walking,
(4) The non-human mammal according to (1) above, wherein the abnormal behavior is a behavior that moves in a cage in a relaxed manner when (c) enters the activity mode,
(5) Furthermore, (d) the non-human mammal according to any one of (1) to (4), wherein a decrease in anxiety symptoms and a decrease in cognitive function are observed,
(6) The non-human mammal according to any one of (1) to (4) above, which is a homozygous genetically modified animal in which both Spsb4 genes of a pair of chromosomes are modified,
(7) The non-human mammal according to any one of (1) to (4) above, wherein the non-human mammal is a mouse or a rat,
(8) The non-human mammal according to (7) above, wherein the non-human mammal is a mouse,
(9) The non-human mammal according to any one of (1) to (4) above, which is a model animal of human attention deficit / hyperactivity disorder, and
(10) The non-human mammal according to any one of (1) to (4) above, which is a model animal for human schizophrenia,
About.

The present invention also provides
(11) introducing a gene into an embryonic stem (ES) cell using a targeting vector to modify the Spsb4 gene, producing a heterogeneous gene-modified non-human mammal derived from the embryonic stem cell; and The method comprises the step of obtaining a homozygous genetically modified animal in which both Spsb4 genes of a pair of chromosomes are modified by mating the heterogeneous genetically modified non-human mammal for one generation or more. A method for producing a human mammal,
About.

The present invention also provides
(12) (I) a step of administering a candidate substance to a non-human mammal exhibiting abnormal behavior according to any one of (1) to (4), (II) hyperactivity of the non-human mammal And (III) a method for screening an anti-ADHD drug, comprising a step of selecting a candidate substance that has reduced hyperactivity compared to the case of not administering a candidate substance,
About.

The present invention also provides
(13) (I) a step of administering a candidate substance to a non-human mammal exhibiting abnormal behavior according to any one of (1) to (4), (II) hyperactivity of the non-human mammal And (III) a screening method for a therapeutic drug for schizophrenia, comprising: (III) selecting a candidate substance whose hyperactivity is reduced as compared with a case where no candidate substance is administered;
About.

  The genetically modified non-human mammal exhibiting abnormal behavior of the present invention is a model animal for human ADHD and human schizophrenia, and is very useful for elucidating the causes of ADHD and schizophrenia and developing preventive methods, therapeutic methods, and therapeutic drugs It is useful for.

It is a figure which shows the result of having measured the self-issued movement and the circadian rhythm of the normal type | mold mouse | mouth and the homo-type gene modification mouse | mouth which showed the abnormal behavior produced in Example 1. FIG. White circles (wt / wt) and black circles (Tp / Tp) represent normal mice and homozygous genetically modified mice, respectively. It is a figure which shows the insertion site | part of the transposon to a Spsb4 gene. It is a figure which shows the identification method of the Spsb4 mutant mouse | mouth by PCR method. p1, p2 and p3 represent primers used for PCR. It is a figure which shows the test result of the anxiety test. A shows the stay time in an open arm. B shows the stay time in a closed arm. C indicates head dips (number of times of peeping into the open arm). * Means P <0.05 for normal mice and ** means P <0.01 for normal mice. wt / wt represents a normal mouse, and Tp / Tp represents a homozygous gene-modified mouse. It is a figure which shows the test result of an open field test. A shows the measurement result of the inner circle walking amount. B shows the measurement result of the amount of walking on the outer circle. wt / wt, Tp / wt and Tp / Tp represent a normal mouse, a heterogeneous genetically modified mouse and a homozygous genetically modified mouse, respectively. It is a figure which shows the calculation result of search preference in the test of cognitive function. The left figure shows the search preference in the training trial, and the right figure shows the search preference in the holding trial. wt / wt, Tp / wt and Tp / Tp represent a normal mouse, a heterogeneous genetically modified mouse and a homogenous genetically modified mouse, respectively. It is a figure which shows the test result of an aggression test. A shows the measurement result of the creeping behavior, and B shows the measurement result of the total contact time. wt / wt represents a normal mouse, and Tp / Tp represents a homozygous gene-modified mouse. It is a figure which shows the result of the medicine administration test to a mouse | mouth. A shows the results of a drug administration test on normal mice. B shows the results of a drug administration test to homozygous genetically modified mice. In the figure, None (circle) is a mouse to which no drug is administered, MPD (triangle) is a mouse to which methylphenidate hydrochloride is administered, and CLZ (square) is a mouse to which clozapine is administered. It is a figure which shows the integrated value of the self-issue amount of movement of a medicine administration group and a non-administration group. In the figure, None is a mouse to which no drug is administered, MPD is a mouse to which methylphenidate hydrochloride is administered, and CLZ is a mouse to which clozapine is administered. wt / wt represents a normal mouse, and Tp / Tp represents a homozygous gene-modified mouse. ** means P <0.01 with respect to normal mice, and ++ means P <0.01 with respect to the drug non-administered group. It is a figure which shows the procedure of the mutation introduction | transduction to the Spsb4 gene by the gene targeting method. It is a figure which shows the result of having confirmed the variation | mutation of Spsb4 gene by Southern blotting. A is the result of analyzing ES cell genomic DNA cleaved with BstZI and KpnI with probeA and B in FIG. 10, and B is the result of analyzing ES cell genomic DNA cleaved with BstZI and KpnI in FIG. This is the result of analysis. It is the schematic which shows the preparation flow of a Spsb4 mutant mouse. It is a figure which shows the result of having investigated the movement distance in the open field test. A and C show the transition of the movement distance of the mouse every 10 minutes in the open field. B and D show the total movement distance of the mouse in the open field for 60 minutes. wt / wt represents a normal mouse. Tp / Tp and Tp / wt represent the homogenous gene-modified mouse and heterogeneous gene-modified mouse prepared in Example 1, respectively. Tp / Del represents a genetically modified mouse in which one Spsb4 gene of a pair of chromosomes is modified by transposon transfer and the other Spsb4 gene is deleted by the gene targeting method. * Means P <0.05 for normal mice, ** means P <0.01 for normal mice, *** means P <0. It means 001. It is a figure which shows the result of having investigated the movement locus | trajectory in the open field test. A and C show the movement trajectory of a normal mouse (wt / wt) in the open field, and B, D, and E show the movement trajectory of the genetically modified mouse, respectively. Tp / Tp and Tp / wt represent the homogenous gene-modified mouse and heterogeneous gene-modified mouse prepared in Example 1, respectively. Tp / Del represents a genetically modified mouse in which one Spsb4 gene of a pair of chromosomes is modified by transposon transfer and the other Spsb4 gene is deleted by the gene targeting method.

  In the present specification, “abnormal behavior” is defined as a statistical difference after normal behavioral analysis as compared to a normal (wild-type) animal that is a control group. That is, the genetically modified non-human mammal of the present invention exhibits rotational movement and / or hyperactivity abnormal behavior during walking under visual observation, and these behaviors are objective in analysis such as medicinal evaluation. Because it is considered difficult to make an index, the behavior of non-human mammals (self-issued behavior, anxiety behavior, environmental habituation, exploration behavior, social behavior, etc.) was measured experimentally, and wild-type animals In the comparison between the measured values of the animals, animals having a significance level of 5% or less (P <0.05) are defined as abnormal behavior animals. Preferably, the experimental measurement value of the abnormal behavior of the non-human mammal is an animal having a significance level of 1% or less (P <0.01) in comparison with the measurement value of the wild-type animal group.

(I) Abnormal behavior Although the non-human mammal of the present invention exhibits abnormal behavior, the abnormal behavior includes (a) an increase in the amount of spontaneous activity during the active period compared to normal non-human mammal. On the other hand, there is no difference in the circadian rhythm of the self-issued movement seen in the light-dark cycle, (b) behavior showing irregular right or left rotation when walking, (c) once entering active mode by external stimulus And the behavior of moving calmly in the cage. Preferably, it is a non-human mammal showing the hyperactivity of (a) or (c) and the rotational movement of (b). More preferably, it is a non-human mammal showing hyperactivity of (a) and (c) and rotational movement of (b).

  Specifically, the non-human mammal of the present invention exhibits the behavior of (a) in the long-term (half day or more) self-issued amount of movement measurement. For example, if the non-human mammal is a mouse, the mouse is nocturnal, and in normal (wild-type) mice, the amount of spontaneous activity increases during the active period (nighttime: dark period), and the quiet period (daytime: light) The self-issued volume will decrease during the period. As shown in FIG. 1, the non-human mammal of the present invention increases the spontaneous movement amount in the active period (dark period) and decreases the spontaneous movement amount in the quiet period (light period), as in normal mice. The daily rhythm of self-issued movement is maintained. However, the amount of spontaneous activity during the active period is significantly increased compared to normal mice.

  In the non-human mammal of the present invention, no abnormality is observed after birth to lactation as compared to a normal non-human mammal. However, the non-human mammal of the present invention exhibits an irregular right or left rotation during walking (b) from the weaning period (3 weeks of age for mice). That is, it frequently shows the behavior of turning to the right or left irregularly during walking.

  Regarding the behavior of (c), the non-human mammal of the present invention has a small amount of behavior and forms a group at the cage corner in the quiet period as in the normal non-human mammal. When entering the activity mode, it moves more calmly in the cage than normal animals.

For hyperactivity of (a) and (c), for example, a non-human mammal is placed in a cage, and the self-issued amount of movement is detected by an infrared sensor type measuring instrument (NS-AS01; Neuroscience, Tokyo) installed at the top. The detection signal can be evaluated by quantifying it using a short-term data acquisition and analysis system (DAS-008; Neuroscience, Tokyo). It can also be evaluated by an open field test.
Regarding the behavior of (b), normal non-human mammals do not exhibit such rotational behavior, and therefore, the difference from normal non-human mammals can be confirmed by visual observation.

  It is preferable that the non-human mammal of the present invention further has (d) a decrease in anxiety symptoms and a decrease in cognitive function. Since the behavior of non-human mammals exhibiting abnormal behaviors (a) to (c) and in which (d) is observed is very similar to the symptoms of ADHD patients, such animals are model animals of ADHD. As very useful.

  About the anxiety symptom of (d), when a non-human mammal is a mouse | mouth, for example, it can be evaluated by the elevated plus maze test and Open field test which are described in an Example. The cognitive function can be evaluated by, for example, a novel object search test described in the examples.

(II) Modification of Spsb4 gene In the non-human mammal exhibiting abnormal behavior of the present invention, the Spsb4 gene in the genome is modified. The Spsb4 gene is a gene widely present in mammals such as humans, dogs, rats, and mice, and is known to encode an ECS complex of a Cullin type E3 ubiquitin ligase. For example, the mouse Spsb4 gene is registered in Mouse Genome Informatics under the ID number MGI: 2183445. Detailed information is described in the following web site.
http://www.informatics.jax.org/javawi2/servlet/WIFetch?
page = markerDetail & key = 82615

  “Gene is altered” means that part or all of the DNA region of the Spsb4 gene is altered. A part or all of the DNA region of the Spsb4 gene is altered means that a part of the genomic DNA of the Spsb4 gene has been changed, added, deleted or replaced, or inverted to a specific region. This means that it is generated. The Spsb4 gene may be modified as long as the Spsb4 gene is inactivated. In order to inactivate the Spsb4 gene, two or more methods of alteration, addition, deletion or substitution may be used for the Spsb4 gene. “Inactivation” means that the altered expression product of the Spsb4 gene does not function or does not exist.

  In the present invention, “change” of genomic DNA refers to introducing one or more base substitutions into the genomic DNA sequence of the Spsb4 gene so that the altered Spsb4 gene expression product does not function or does not exist. That means. In the present invention, “deletion” of genomic DNA means that the expression product of Spsb4 gene does not function or does not exist by deleting part or all of the genome of Spsb4 gene. In the present invention, “insertion” with respect to genomic DNA means that an expression product of the Spsb4 gene in which DNA other than the Spsb4 gene is inserted by inserting DNA having a sequence other than the Spsb4 gene into the Spsb4 gene. It means not to exist or not to exist. In the present invention, “replacement” of genomic DNA means that a part or all of the genome of the Spsb4 gene is replaced by a separate sequence not related to the Spsb4 gene so that the expression product of the Spsb4 gene does not function or does not exist. To do.

  In addition, when the modified position, that is, the portion into which the mutation is introduced is on the 3 ′ side of the exon, the N-terminal side of the protein encoded by the Spsb4 gene is expressed when the 5 ′ side of the exon is normally transcribed and translated. there is a possibility. Therefore, the modified position of the Spsb4 gene is preferably between exon 1 and exon 2 upstream of the gene in the case of an intron. When the modified position of the Spsb4 gene is an exon, it is preferable to delete exon 2 in which most of the protein is encoded.

  The non-human mammal of the present invention may be a hetero-type Spsb4 gene-modified animal in which only one Spsb4 gene of a pair of chromosomes is modified, or a homo-type gene in which both Spsb4 genes of a pair of chromosomes are modified. It may be a modified animal. Preferably, it is a homozygous genetically modified animal. As described later, homozygous genetically modified animals can be obtained by crossing one or more generations of heterozygous Spsb4 genetically modified animals.

  The kind of the non-human mammal of the present invention is not particularly limited, and examples thereof include mouse, rat, rabbit, guinea pig, dog, pig, sheep, goat and the like. Among these, a mouse or a rat is preferable because it is easy to handle and easy to reproduce, and a mouse is most preferable.

  As a method for producing the non-human mammal of the present invention, for example, as described in the Examples, (I) a heterogeneous gene-modified non-human mammal having a transposon sequence and having at least one translocation site in cells throughout the body. Step (A) for producing a human mammal, step (B) for selecting an animal in which the Spsb4 gene has been altered from the produced heterogeneous gene-modified non-human mammal, and a heterogeneous gene in which the Spsb4 gene has been altered A method comprising the step (C) of obtaining a homozygous genetically modified animal in which both Spsb4 genes of a pair of chromosomes are modified by crossing two or more generations of the modified animal, or (II) A step (D) of modifying a Spsb4 gene by introducing a gene into a sex stem (ES) cell, a heterogeneous gene-modified non-human mammal derived from the ES cell Step (E) for producing, and Step (F) for obtaining a homogenous genetically modified animal in which both Spsb4 genes of a pair of chromosomes are modified by mating the heterogeneous gene-modified non-human mammal for one generation or more The method containing is mentioned. In order to produce a non-human mammal exhibiting abnormal behavior of the present invention with good reproducibility, the method (II) is preferred. These methods are examples of the method for producing the non-human mammal of the present invention, and the method for producing the non-human mammal of the present invention is not limited to the following method. Moreover, a modification and / or a change can be appropriately added to the method shown below as necessary.

  In the method of (I), a heterogeneous genetically modified non-human mammal having a transposon sequence and having at least one translocation site in cells of the whole body has a gene mutation derived from transposon translocation of the non-human mammal. It is common to most cells. Such a non-human mammal can be obtained, for example, by crossing a non-human mammal having both a transposon sequence and a transposase gene (double transgenic non-human mammal) with a wild-type non-human mammal.

  The transposon sequence in the present invention is a non-transposable DNA segment that can be inserted at various positions in the genome by the action of transposase, and usually has repetitive sequences (reverse repeat structures in both ends) at both ends. It is. However, the repetitive structure may include an incomplete repetitive portion as long as it can be translocated (inserted at another position in the genome) by the action of transposase. Various DNA sequences, for example, a marker gene, a gene expression regulatory sequence, a desired gene, and the like can be inserted into the portion sandwiched between opposite repeat structures at both ends of the transposon sequence. The short nucleotide sequence that functions in transposition of the transposon in the DNA into which the transposon is inserted is called the target sequence, duplication of the target sequence occurs during transposon insertion, and the transposon is sandwiched between two identical copies of the target sequence. It will be in the state. For example, in the Sleeping Beauty (SB) transposon described later, the target sequence is TA, and the transposon translocation site is TA-transposon-TA.

An example of a preferred embodiment of the step (A) in the method (I) is shown below.
First, a transgenic non-human mammal containing a transposase gene and a transgenic non-human mammal containing a transposon sequence are prepared (step a1). In step a1, a transposase gene-containing transgenic non-human mammal or a transposon sequence-containing transgenic non-human mammal is produced by introducing a transposase gene or transposon sequence into a non-human mammal ES cell or fertilized egg. be able to. As a method used for transposon sequence or transposase gene introduction into ES cells and fertilized eggs, known methods can be used. Preferably, for example, a method using a retroviral vector or an electroporation method can be used as a method for introduction into ES cells. As a method for introducing the fertilized egg, a microinjection method and the like can be mentioned.

  The transposase gene is preferably a Sleeping Beauty (SB) transposase gene. Moreover, as a transposon sequence, a Sleeping Beauty transposon (SB) sequence is preferable. For example, when a genetically modified mouse is prepared, a SB transposase gene-containing trans is produced according to the method described in Horie et al., Molecular and Cellular Biology, vol. 23, No 24, p. 9189-9207 (2003). A transgenic mouse and a transgenic mouse containing a transposon sequence can be produced. In the present specification, “SB transposon” or “Sleeping Beauty transposon” refers to a Tc1 / mariner type transposon having transposon activity in a mammal or a cell thereof (Z. Ivics et al .; Cell, 91: 501-510 (1997)).

  A double transgenic non-human mammal is produced by crossing this transposase gene-containing transgenic non-human mammal with a transposon sequence-containing transgenic non-human mammal (step a2). The genotype of the obtained double transgenic non-human mammal is identified by PCR or the like, and a transgenic non-human mammal having both a transposon sequence and a transposase gene (also referred to as a seed non-human mammal) is selected (step a3). . By crossing this seed non-human mammal with a normal (wild-type) non-human mammal, a heterogeneous gene-modified non-human mammal in which one gene of a pair of chromosomes is modified is obtained from the offspring. (Step a4). Next, an animal in which the Spsb4 gene is modified is selected from the heterogeneous gene-modified non-human mammal (step B). A method for selecting an animal in which the Spsb4 gene is modified from the produced heterogeneous gene-modified non-human mammal is not particularly limited. For example, a gene obtained by cutting the tail or the like of the obtained non-human mammal can be selected, and an animal in which the Spsb4 gene is modified can be selected by PCR using a specific primer.

  By crossing two or more generations of heterogeneous genetically modified animals in which the Spsb4 gene has been modified, homozygous genetically modified animals in which both Spsb4 genes of a pair of chromosomes have been modified can be obtained (Step C).

  The method (II) using ES cells includes steps (D), (E) and (F). In step (D), for example, a targeting vector is prepared in vitro using a gene recombination technique. Step (d1) may be included. Further, in the step (D), after performing the step (d2) of introducing a gene into an embryonic stem (ES) cell to modify the Spsb4 gene using a targeting vector, It is preferable to perform the step (d3) of selecting a modified Spsb4 gene. In addition, the step (E) includes a step (e1) of obtaining a chimeric non-human mammal by transferring ES cells having a modified Spsb4 gene into a blastocyst derived from a normal non-human mammal, and the obtained chimeric non-human mammal. It is preferable to include a step (e3) of mating the animal with a normal non-human mammal. In step (E), if necessary, step (e2) may be performed after performing step e1, and further step (e2) may be performed after step e3. The step (e4) of selecting a non-human mammal that has been performed may be performed.

  In one preferred embodiment of the method of (II) using ES cells, for example, first, a targeting vector is prepared in vitro using a gene recombination technique (step d1), and the vector is transferred to the ES cell. The Spsb4 gene is altered (for example, destroyed) by introduction (step d2). Among the ES cells into which the gene has been introduced, those in which the Spsb4 gene has been modified are selected by a method such as Southern blotting (step d3). Next, the ES cell in which the Spsb4 gene is modified is transferred into a blastocyst derived from a normal non-human mammal to produce a chimeric non-human mammal (step e1), and the resulting chimeric non-human mammal is converted into a normal non-human mammal. By mating with a mammal (step e3), an ES cell-derived non-human mammal can be produced. This ES cell-derived non-human mammal is a heterogeneous gene-modified non-human mammal in which one Spsb4 gene of one of a pair of chromosomes is modified. Next, by crossing one or more generations of this heterogeneous gene-modified non-human mammal, a homozygous gene-modified animal in which both Spsb4 genes of a pair of chromosomes have been modified can be obtained (Step F).

The method for producing the abnormal behavior gene-modified non-human mammal by the method (II) is specifically described below.
(1) Preparation of targeting vector (step d1)
First, a DNA fragment containing a site to be mutated in the Spsb4 gene of a mutant animal to be produced is obtained from a DNA library. The DNA fragment is used to construct a targeting vector for homologous recombination of ES cells. This DNA fragment is preferably used by isolating DNA having the same sequence as the ES cell to be produced so that recombination occurs more efficiently when homologous recombination is performed. For example, when a mouse is used as an animal into which a mutation is introduced, a DNA fragment of a mouse that can delete exon 2 of the Spsb4 gene or replace it with another gene is prepared from a genomic DNA library derived from ES cells.

  As a method for modifying the Spsb4 gene, a normal site-specific gene mutation introduction method can be used. For example, the target mutation can be introduced using a method such as BAC recombineering (see Lee, E.C. et al. Genomics, vol. 73, p. 56-65 (2001)).

  A targeting vector is prepared by inserting a DNA fragment containing the modified Spsb4 gene into the vector. The targeting vector preferably contains, as essential elements, a chromosomal DNA fragment into which a mutation has been introduced, a DNA fragment encoding a selection marker, a promoter for controlling transcription thereof, and a selection marker expression unit including a terminator. The chromosomal DNA fragment into which the mutation is introduced is a part necessary for causing homologous recombination in the ES cell, and the chromosomal DNA fragments before and after the part into which the mutation has been introduced are necessary. That is, the targeting vector has a DNA fragment different from the original chromosomal DNA only in the mutated base. The length of the chromosomal DNA fragment before and after the site where the mutation is introduced is preferably about 10 kbp, but in general, some increase or decrease in length is allowed. However, if it is too short, the frequency of homologous recombination in ES cells may decrease.

  As the DNA fragment encoding the selectable marker, a drug resistance gene, a reporter gene and the like are preferable. Examples of drug resistance genes include neomycin phosphotransferase II (nptII) gene and hygromycin phosphotransferase (hpt) gene. Reporter genes include, for example, green fluorescent protein (GFP) gene and β-galactosidase (lacZ). Gene, chloramphenicol acetyltransferase (cat) gene, and the like. As the promoter, CAG promoter, phosphoglycerate kinase-1 (PGK-1) promoter, elongation factor 2 (EF-2) promoter, MC-1 promoter and the like can be used.

  The vector serving as the basic skeleton of the targeting vector into which the DNA fragment containing the modified Spsb4 gene is introduced is not particularly limited as long as it is capable of self-replication in a cell to be transformed (for example, E. coli). For example, commercially available pBluescript (manufactured by Stratagene), pZErO 1.1 (Invitrogen), pGEM-1 (Promega) and the like can be used.

(2) Introduction of vector into ES cells (step d2) and selection of ES cells into which genes have been introduced (step d3)
After introducing the prepared targeting vector into ES cells, the ES cells are cultured and colonies appearing are collected. As the ES cell, any of already established cell lines and newly established cell lines can be used. For gene introduction into ES cells, methods such as calcium phosphate coprecipitation method, electroporation method, lipofection method, retrovirus infection method, aggregation method, microinjection method, and particle gun method can be adopted. The electroporation method is preferable in that the cells can be treated.

  ES cells into which the transgene has been integrated are those in which the Spsb4 gene has been disrupted by screening chromosomal DNA isolated from colonies obtained by culturing single cells on feeder cells by Southern hybridization or PCR. Can be selected. Alternatively, selection may be performed by using a vector containing a drug resistance gene or a reporter gene as described above. When the ES cells after introduction of the targeting vector are cultured in the presence of a selection marker, the ES cells that survive and form colonies may have undergone homologous recombination. A PCR method is generally used as a method for examining cells that have undergone homologous recombination among the ES cells that have formed these colonies. However, DNA fragments, RNA fragments, synthetic oligodeoxynucleotides that can be used as probes, or It is also possible to use an antibody or the like.

(3) Production of transgenic non-human mammal (Step E)
By transferring the ES cell in which the disruption of the Spsb4 gene has been confirmed into a blastocyst derived from the same kind of non-human mammal, the ES cell is incorporated into the cell mass of the host embryo to form a chimeric embryo. A chimeric transgenic animal is obtained by transplanting this chimeric embryo to a foster parent and allowing it to develop and grow (step e1). For example, when a mouse is used, a blastocyst is separately taken out from a pregnant mouse, and the selected ES cell is transferred, and then introduced into the uterus of a female mouse subjected to pseudopregnancy. Then, a chimeric animal is selected from the offspring born from the temporary parents (step e2). A chimeric animal can be selected by hair color, for example, when ES cells are derived from 129 mice. The chimeric animal is mated with a normal animal (step e3), genomic DNA is extracted from the tail of the born pup and the like, and a mutation introduced by the PCR method is selected (step e4). Modified heterozygous animals can be obtained.

As a specific selection method, a part of the tail of the resulting pup is collected and chromosomal DNA is extracted. Using the extracted chromosomal DNA as a substrate, PCR is performed using two oligodeoxynucleotides having primers designed to sandwich the mutation site of the Spsb4 gene as primers. Perform agarose gel electrophoresis of the PCR product, confirm the band containing mutations by hybridization using oligodeoxynucleotides with the presence or absence of bands, mobility of the bands on the gel, and base sequences containing mutation sites, etc. It can be examined whether or not the extracted Spsb4 gene is contained in the extracted chromosomal DNA. The nucleotide sequence of the oligonucleotide used for the PCR primer may be any as long as it can detect the modified Spsb4 gene. By selecting an animal having a modified Spsb4 gene based on the PCR result, an animal having a modified Spsb4 gene heterozygously can be obtained.
By crossing one or more generations of animals having a modified Spsb4 gene heterozygously, a homozygous genetically modified animal in which both Spsb4 genes of a pair of chromosomes are modified can be obtained (Step F).

  The abnormal behavior of the non-human mammal of the present invention is sensitive to human ADHD and schizophrenia drugs. That is, when an ADHD therapeutic agent or a schizophrenia therapeutic agent is administered to the non-human mammal of the present invention, hyperactivity is improved. Therefore, the abnormal behavior of the non-human mammal of the present invention is considered to reflect the pathological condition associated with these diseases. Although it does not specifically limit as a therapeutic agent for ADHD and a therapeutic agent for schizophrenia, for example, methylphenidate hydrochloride (chemical name: methyl α-phenyl-2-piperidine acetate hydrochloride), clozapine (chemical name: 8-chloro-11- (4-methylpiperazin-1-yl) -5H-dibenzo [b, e] [1,4] diazepine).

  The non-human mammal of the present invention has (1) the behavior of the animal is similar to the symptoms of ADHD patients (surface validity), and (2) the drug that acts on ADHD is an abnormal behavior of the animal such as hyperactivity. It satisfies the improvement of sex (predictive validity) and is very useful as a model animal for human ADHD. Therefore, various test substances are administered to the non-human mammal of the present invention, and the change in abnormal behavior such as hyperactivity in animals in the non-administration group or control substance-administered animals is compared. Alternatively, substances useful for treatment can be evaluated. As a representative example of evaluation, screening of candidate substances can be mentioned, and test items include symptoms, pathological findings, pharmacological tests and the like. Such a step of administering a candidate substance to a non-human mammal exhibiting abnormal behavior of the present invention, (II) a step of assaying a change in abnormal behavior of the non-human mammal, and (III) no candidate substance being administered A screening method for an anti-ADHD drug including a step of selecting a candidate substance that decreases abnormal behavior compared to the case is also one aspect of the present invention. For example, when a candidate substance is administered, if a result that confirms that abnormal behavior such as hyperactivity has been improved is obtained, the used candidate substance can be selected as an anti-ADHD drug. Hyperactivity is suitable as the abnormal behavior to be tested in the screening method.

  The non-human mammal of the present invention also has (1) that the hyperactivity exhibited by the animal is similar to the symptoms of patients with schizophrenia (surface validity), and (2) drugs that are effective against schizophrenia. It satisfies the improvement of hyperactivity (predictive validity) and is very useful as a model animal for human schizophrenia. Therefore, prevention and / or treatment of schizophrenia is achieved by administering various test substances to the non-human mammal of the present invention and comparing the hyperactivity changes in the animals of the non-administration group or the control substance-administered animals. Useful substances can be evaluated. As a representative example of evaluation, screening of candidate substances can be mentioned, and test items include symptoms, pathological findings, pharmacological tests and the like as described above. The step of administering a candidate substance to such a non-human mammal exhibiting abnormal behavior of the present invention, (II) the step of assaying the hyperactivity change of the non-human mammal, and (III) the candidate substance being administered A screening method for a therapeutic drug for schizophrenia, which includes a step of selecting a candidate substance with reduced hyperactivity as compared with the case where it is not, is also one aspect of the present invention. For example, when a candidate substance is administered, if a result that confirms that hyperactivity has been improved is obtained, the used candidate substance can be selected as a therapeutic drug for schizophrenia.

  In the screening method of the present invention, the method for examining changes in abnormal behavior such as hyperactivity is not particularly limited. For example, for one or more of the abnormal behaviors (a) to (c) and the symptom (d), before and after administration of the candidate substance, by performing the above-described evaluation methods or the tests and methods described in the following examples. It is possible to evaluate behavioral changes in Specifically, the change in hyperactivity is detected, for example, by an infrared sensor type measuring instrument (NS-AS01; Neuroscience, Tokyo), and the detected signal is converted into a short-term data acquisition and analysis system (DAS-008; It can be evaluated by a numerical method using Neuroscience, Tokyo), an Open field test, or the like.

  Candidate substances include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, cell culture supernatants, plant extracts, mammals (eg, mice, rats, pigs, cows, sheep) , Monkeys, humans, etc.) tissue extracts, plasma and the like. The candidate substance may be a novel substance or a known substance. These candidate substances may form salts, and as salts of candidate substances, salts with physiologically acceptable acids and bases are used. Such salts include salts with inorganic acids such as hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid; acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid, Preference is given to salts with organic acids such as succinic acid, benzoic acid, methanesulfonic acid and benzenesulfonic acid.

  Examples of the method of contacting the test animal with the candidate substance include oral administration, intravenous injection, application, subcutaneous administration, intradermal administration, and intraperitoneal administration. The method is appropriately selected according to the symptom of the test animal and the nature of the candidate substance. You can choose. The dosage of the candidate substance can be appropriately selected according to the administration method, the nature of the candidate substance, and the like.

  EXAMPLES Next, although an Example demonstrates this invention concretely, this invention is not limited to these.

Example 1
1. Generation of genetically modified mice using transposon (I) Generation of Sleeping Beauty (SB) transposase gene-containing transgenic mice (SB transgenic mice) Japanese Patent Publication No. 2002-013602 and Horie et al .; Proc. Natl. Acad. Sci SB transgenic mice were prepared by the method described in USA 98: 9191-9196 (2001) (hereinafter also referred to as Reference A). A specific method is shown below.

(I) Preparation of pCX-SB construct for preparation of SB transgenic mouse After cutting SB transposase gene from pSB10 plasmid (Z. Ivics et al .; Cell, 91: 501-510 (1997)) with restriction enzyme SacII , Smoothed. The SB-DNA fragment was inserted into the blunted EcoRI site of the pCX-EGFP plasmid (M.Okabe et al .; FEBS Letter, 407: 313-319 (1997)) in the state of replacing the EGFP fragment, and PCX-SB After obtaining the construct, the orientation of the SB transposase gene in the construct was confirmed.
(Ii) Preparation of SB transgenic mouse from pCX-SB construct A unit DNA fragment containing a promoter / SB / PolyA addition signal was cleaved from pCX-SB construct with restriction enzyme SalI-BamHI and purified by electrophoresis. About 5 fg of this DNA fragment was transferred to a fertilized egg of B6C3F2 mouse (fertilized egg obtained by crossing a C57BL / 6 female and a C3H male) and then transplanted to the oviduct of a pseudopregnant mouse. Thus, SB transgenic mice were produced around 20 days thereafter.

(II) Production of transgenic mouse containing transposon sequence a) Construction of trap vector
A trap vector was prepared by the method described in Horie et al., Molecular and Cellular Biology, vol. 23, No 24, p. 9189-9207 (2003). A specific method is shown below.

(I) The cloning site of pBluescript II (Stratagene) is used as a primer.
5'-GCCG CTCG AGGG CGCG CCAG ATTT AAAT CAGC TTTT GTTC CCTT TAGT GAG-3 '(SEQ ID NO: 1)
as well as
5'-CGCA GCGG CCGC ATTT AAAT GAGG CGCG CCGC TCCA ATTC GCCC TATA GTG-3 '(SEQ ID NO: 2)
The AscI, XhoI, NotI and SwaI sites were replaced by PCR amplification of pBluescript II using The PCR product 2.9 kb XhoI-NotI fragment was compared to the 0.8 kb XhoI-NotI fragment of IR / DR (R, L) from pBS-IR / DR (R, L) (see Reference A). Ligation yielded pBS-IR / DR-AS containing AscI and SwaI sites flanked by inverted repeats and direct repeats.

(Ii) oligonucleotides
5'-GTAC GGCG CGCC GGTA CCAT TTAA AT-3 '(SEQ ID NO: 3)
as well as
5'-GTAC ATTT AAAT GGTA CCGG CGCG CC-3 '(SEQ ID NO: 4)
And oligonucleotide
5'-CGTT TAAA CTTA ATTA AGAG CT-3 '(SEQ ID NO: 5)
as well as
5'-CTTA ATTA AGTT TAAA CGAG CT-3 '(SEQ ID NO: 6)
Was annealed to prepare linker DNA containing AscI-KpnI-SwaI site and PmeI-PacI site. After removing the TransCX-GFP fragment, each linker was inserted into the peculiar KpnI and SacI sites of pTransCX-GFP: Neo (see Reference A) to obtain pAKS: Neo: PP.

(Iii) SalI-BamHI fragment of pCX-EGFP-PigA (see Reference A) containing CAG-EGFP and Neo cassette blunted with BamHI (Ishida and Leder, Nucleic Acids Res., Vol. 27: e35 (1999) )) 256 bp XhoI fragment was inserted into the NotI site blunted with pBluescript II SalI to obtain pCAG-GFP-SD. The Neo cassette is composed of a splice donor (SD) sequence derived from the mouse hprt gene exon 8 / intron 8 region, and an mRNA destabilization signal derived from the 3 'untranslated region of human granulocyte macrophage colony stimulating factor cDNA.

(Iv) Isolation of a lacZ gene SacII-NotI fragment containing a HindIII fragment smoothed with XbaI of the rabbit β-globin poly (A) addition signal and a nuclear localization signal from pRTonZ (Koji Horie et al., Unpublished data) PBluescript II was inserted into the XbaI and SmaI sites of pBluescript II, and the SacII and NotI sites, respectively, to obtain pLacZ-BS. After insertion of LacZ-poly (A), the HindIII site was removed by smoothing and self-ligated. The SalI-XhoI fragment of CAG-GFP-SD derived from pCAG-GFP-SD was inserted into the XhoI site of pLacZ-BS to obtain pLacZ-CAG-GFP-SD. The human bcl-2 intron 2 / exon 3 splice acceptor (SA) sequence was amplified using the RET vector (Ishida et al .: Nucleic Acids Res. 27: e35 (1999)) as a template and the following primers.
5'-CGGC AAGC TTCT CGAG CTGT ATCT CTAA GATG GCTG G-3 '(SEQ ID NO: 7)
5'-GCCA CGGT CGAC GCCT GCAT ATTA TTTC TACT GC-3 '(SEQ ID NO: 8)
The in-sequence ribosome entry site (IRES) was amplified again using the RET vector as a template and the following primers.
5'-GGAG CGTC GACT ACGT AAAT TCCG CCCC TCTC CCTC-3 '(SEQ ID NO: 9)
5'-GGAG CGTC GACT ACGT AAAT TCTC CCTC CCC-3 '(SEQ ID NO: 10)
The HindIII-SalI SA-containing fragment and SalI-BamHI IRES-containing fragment were simultaneously cloned into the HindIII and BamHI sites of pLacZ-CAG-GFP-SD to obtain pSA-IRESLacZ-CAG-GFP-SD.

(V) Smoothing the XhoI fragment of pSA-IRESLacZ-CAG-GFP-SD containing SA, IRES, lacZ, poly (A) and CAG-GFP-SD, and both EcoRI and BamHI sites of pBS-IR / DR-AS Was smoothed and inserted into these sites to obtain pTrans-SA-IRESLacZ-CAG-GFP-SD. The AscI-SwaI fragment of pTrans-SA-IRESLacZ-CAG-GFP-SD was inserted into the AscI and SwaI sites of pAKS: Neo: PP to obtain pTrans-SA-IRESLacZ-CAG-GFP-SD: Neo.

b) pTrans-SA-IRESLacZ-CAG-GFP-SD: Production of Transposon Transgenic Mice from Neo The obtained pTrans-SA-IRESLacZ-CAG-GFP-SD: Neo was linearized using Pac I, and this DNA The fragment was injected into a fertilized egg (BDF1 mouse × BDF1 mouse). By transplanting this fertilized egg into the oviduct of a pseudopregnant mouse, a transgenic mouse containing a transposon sequence was produced around 20 days thereafter.

(III) Production of Double Transgenic Mouse The SB transgenic mouse produced in (I) and the transposon sequence-containing transgenic mouse produced in (II) are mated to produce a double transgenic mouse (referred to as a seed mouse). did. The genotype was identified using primers specific to the above-mentioned GFP and SB genes (see Reference A). All animal experiments were performed according to Osaka University guidelines.

(IV) Screening of transgenic mice (i) Selection of transgenic mice DNA obtained from the tail of the obtained seed mice immediately after birth was analyzed by PCR, and pTrans-SA-IRESLacZ-CAG-GFP-SD: Neo And transgenic mice carrying both pCX-SB were selected. pTrans-SA-IRESLacZ-CAG-GFP-SD: Transgenic mice with both Neo and pCX-SB did not fluoresce green.
(Ii) A mouse in which the transposon sequence is transposed by transposase and emits green fluorescence. Transgenic mouse having both pTrans-SA-IRESLacZ-CAG-GFP-SD: Neo and pCX-SB of (a) above, and female And wild-type ICR mice were mated to obtain genetically modified mice from the offspring. The tail of the obtained mouse immediately after birth was excised, and the fluorescence was measured using a fluorescence stereomicroscope (WILD M10, manufactured by Leica) having a GFP-specific filter. In order to avoid a decrease in detection sensitivity, screening was performed before the appearance of hair color. Individuals expressing GFP and shining in green fluorescence were selected as mice (transgenic mice) in which the transposon sequence was rearranged by SB transposase. Among these genetically modified mice, mice exhibiting abnormal behavior were isolated and subjected to genetic analysis described below. Mice that were found to have altered Spsb4 gene by genetic analysis (heterogeneous gene-modified mouse: genotype Tp / wt) were crossed for two or more generations, and Spsb4 gene was altered in both genes of a pair of chromosomes. A mouse (homotype-modified mouse: genotype Tp / Tp) was obtained.

2. Gene analysis Next, the transposon insertion site was determined by ligation-mediated PCR (LM-PCR) using the tail DNA of heterogeneous genetically modified mice (Horie et al., Molecular and Cellular Biology, vol. 23, No 24). , p. 9189-9207 (2003)).
FIG. 2 schematically shows a transposon insertion site into the Spsb4 gene. The Sleeping Beauty transposon is inserted into the TA sequence. The upper diagram of FIG. 2 shows a partial sequence of a normal (wild-type) Spsb4 gene, and the lower diagram shows a partial sequence of the Spsb4 gene into which an SB transposon has been inserted. After insertion, TA sequences overlap on both sides of the SB transposon. As a result of gene analysis, the SB transposon was inserted at the site shown in FIG. 2 in the intron portion between exon 1 and exon 2 of the Spsb4 gene. Since mutations at other sites were not confirmed, this Spsb4 gene modification is considered to be the cause of abnormal behavior in mice.

Based on the sequence at the insertion site, a system for genotype identification was established by PCR using the following primer set.
FIG. 3 is a schematic diagram showing a method for identifying Spsb4 mutant mice by PCR. The upper diagram of FIG. 3 shows the Spsb4 gene of a normal (wild type) mouse, and the lower diagram shows a mutant gene into which an SB transposon has been inserted. In FIG. 3, P1, P2 and P3 represent the primers shown below.
P1: 5'-AACAAATCAGACAGACAGCATCTGAGGAAC-3 '(SEQ ID NO: 11)
P2: 5'-TAGTCTGTGTTGTCTTATCTTCGAGGCATG-3 '(SEQ ID NO: 12)
P3: 5'-CTTGTGTCATGCACAAAGTAGATGTCC-3 '(SEQ ID NO: 13)
Primers P1 and P2 are located outside pTrans-SA-IRESLacZ-CAG-GFP-SD: Neo, respectively. PCR was performed using the HotStarTaq system (Qiagen) under the following conditions: 95 ° C. for 15 minutes, 35 cycles (94 ° C. for 30 seconds, 55 ° C. for 30 seconds, 72 ° C. for 30 seconds), then 72 ° C. for the final step. One minute cycle.

  The obtained PCR product was 334 bp when P1 and P2 were used, and 439 bp when P3 and P2 were used.

3. Behavioral analysis of abnormal behavior mice Using the obtained homozygous gene-modified mice, heterogeneous gene-modified mice and normal mice, the test described below was conducted. The mice were raised as follows.
Breeding conditions for mice Both genetically modified mice and normal mice were bred in exactly the same way under standard conditions as breeding conditions for experimental animals. That is, under a light / dark cycle of temperature 23 ± 1 ° C., humidity 50 ± 10%, 12 hours (light period: 8:45 to 20:45), after weaning, 5-6 animals / cage of the same sex only, And water freely. In the experiment, only males were used. The homozygous genetically modified mice frequently showed a rotational motion that randomly turned to the right or left during walking as compared to normal mice during breeding.

(I) Circadian rhythm
(I) Test method A mouse is placed in a polycarbonate cage (35 x 30 x 17 cm), and short-term data acquisition analysis of signals detected by an infrared detection type self-issued dynamic sensor (NS-AS01; Neuroscience, Tokyo) installed at the top Quantification was performed using a system (DAS-008; Neuroscience, Tokyo). The measurement was carried out continuously for 7 days for each animal, and the count number per hour was calculated.
(Ii) Evaluation method The amount of behavior of a mouse usually increases during the dark period and decreases during the light period. Differences in circadian rhythms were determined by comparing the changes in the amount of spontaneous movement per hour between the homozygous genetically modified mouse and the wild-type mouse (time zone showing the maximum spontaneous movement). About the difference of the self-issue amount of movement between 2 groups, it determined by using p value calculated by repeated measurement ANOVA as a parameter | index.
(Iii) Results Regarding the homozygous gene-modified mice, an increase in the amount of spontaneous movement was recognized immediately after extinction, as in the normal mice, and it lasted for 5 to 6 hours. The result is shown in FIG. No difference was found between homozygous genetically modified mice and normal mice in the diurnal rhythm of spontaneous movement observed in 7 days of continuous measurement. However, the self-issued amount of homozygous gene-modified mice was markedly increased compared to normal mice [Repeated measurements ANOVA: genotype, F _(1,7) = 7.908, p = 0.0261 (p <0.05); time, F _{(167, 1169)} = 11.172, p <0.0001; time × genotype, F _{(167, 1169)} = 3.664, p <0.0001].
(Iv) Evaluation It was evaluated that homogenous genetically modified mice maintained a circadian rhythm during the active period but showed a marked increase in spontaneous behavior compared to normal animals.

(II) Anxiety test (i) Test method Anxiety behavior was evaluated by an elevated plus maze test. Mouse in the middle of an elevated plus maze (50 cm high, arm width 10 cm, wall height 30 cm) with a structure where two arms without walls (open arms) and two arms with side walls (closed arms) intersect Set the head of the arm in the direction of the open arm, 5 minutes stay in the open arm, number of times to enter the open arm, time to stay in the closed arm, number of times to enter the closed arm and head dips (open arm The number of peeping) was recorded.
(Ii) Evaluation method Mice usually have the property of preferring closed and dark places. Therefore, in this study, it is observed that normal mice stay longer in closed closed arms than in open arms. In addition, it is known that as the anxiety of the mouse increases, entry and stay in the open arm decrease. Therefore, an inter-group test (Student's t-test) is performed on the basis of the measurement values of normal mice for each measurement item, and in particular, when a significant difference is observed in the stay time and the number of times of entry to the open arm, The mice were evaluated as having changed anxiety behavior.
(Iii) Results Compared with normal mice, an increase in the residence time in the open arm (FIG. 4A) and a corresponding decrease in the residence time in the closed arm (FIG. 4B) were observed in the homozygous genetically modified mice. In addition, the number of head dips (FIG. 4C) increased in homozygous genetically modified mice (Student's t-test: * P <0.05, ** P <0.01, relative to normal mice) . On the other hand, the number of times of entry into the open arm (normal mouse: 4.00 ± 0.80, homozygous gene-modified mouse: 7.13 ± 1.37, p = 0.0689) and the number of times of entry into the closed arm (normal mouse) : 11.9 ± 1.60, homozygous genetically modified mice: 12.9 ± 1.49, p = 0.6544), no difference was observed between the two groups.
(Iv) Evaluation In homozygous genetically modified mice, it was evaluated that anxiety feeling was reduced or impulsiveness was increased as compared with normal mice.

(III) Open field test
(I) Test method A mouse is placed in the center of a gray acrylic circular open field (diameter 60 cm, height 35 cm), and the outer circle walking amount, inner circle walking amount, and number of times of entering the inner circle after 5 minutes and 10 minutes. The stay time in the inner circle, the number of rearing, the number of climbing, the grinding time, and the movement latency (time until the start of walking) were measured.
(Ii) Evaluation method As described above, a mouse usually has a property of preferring a closed and dark place. Therefore, in this test, it is observed that normal mice stay longer in the periphery than in the open center. Moreover, in this test, it is known that the approach and stay to the center increase as the anxiety of the mouse, including habituation to the device, decreases. Furthermore, the movement between the compartments described in this field (inner circle walking amount and outer circle walking amount) is considered as an index of the self-issued movement amount of the mouse. Therefore, one-way analysis of variance and post hoc test (Bonferroni / Dunn test) were performed for each measurement item, and evaluation was made as a change in anxiety or self-issue of the mouse depending on the item in which a significant difference was observed.
(Iii) Results Compared with normal mice and heterogeneous gene-modified mice, the amount of inner circle walking in homozygous gene-modified mice (FIG. 5A) [ANOVA: F _{(2, 11)} = 7.234, p = 0.0099] And the amount of walking in the outer circle (FIG. 5B) [ANOVA: F _{(2, 11)} = 10.593, p = 0.527] (post-hoc Bonferroni / Dunn test: * P <0.05, ** P <0.01, relative to normal mice). On the other hand, there was no difference between groups in other measurement items. The results are shown in Table 1.

(Iv) Evaluation The amount of behavior in the inner circle compartment of homozygous gene-modified mice was increased compared to normal mice, and was evaluated as reflecting a decrease in anxiety feeling or an increase in behavior.

(IV) Cognitive function (i) Test method Cognitive function was evaluated by a novel object search test. It calculated based on the search preference with respect to two substances (two substances selected from a cylinder block, a golf ball, and an adapter of an outlet) installed in an acrylic open field (30 × 30 × 35 cm). First, an animal was acclimatized for 3 days (10 minutes / day) in an experimental apparatus in which an object was not placed, and then allowed to freely search for 10 minutes in an apparatus in which two different substances were placed (training trial). After 24 hours, they were allowed to search freely for 5 minutes in a device in which one of the two was replaced with a novel object (holding trial). In the training trial and the retention trial, the search time for each of the two objects was measured. In the training trial, the ratio (%) of the search time for any object to the total search time, and in the holding trial, the ratio (%) of the search time for the novel object to the total search time. (Exploratory preference: EP). Assuming that the time for searching for a novel substance is A and the time for searching for the same substance as in the training trial is B, search preference (%) = A / (A + B) × 100 is calculated.
(Ii) Evaluation method Mice usually show curiosity for novel substances. Therefore, normal mice show a similar search behavior for the two substances in the training trial, while showing a stronger search preference for the novel substance in the holding trial. That is, the EP will be significantly higher than about 50% for training trials and 50% for holding trials. On the other hand, animals with reduced cognitive function do not remember the substance at the time of the training trial, so the search behavior for a novel substance in the holding trial decreases. In this study, in the one-way analysis of variance and post hoc test (Bonferroni / Dunn test), when a significant decrease in EP in the retention trial was observed, it was evaluated that the visual cognitive function of the mouse was reduced.
(Iii) Results In the training trial, there was no difference in EP, exploratory action time and action amount between the group of normal mice, heterogeneous genetically modified mice and homozygous genetically modified mice [EP, ANOVA: F _{(2, 11)} = 0.639, p = 0.5463], but in the retention trial, the EP of the homozygous gene-modified mouse was lower than that of the normal mouse (FIG. 6) [ANOVA: F _{(2, 11)} = 5.124, p = 0.0268; post-hoc Bonferroni / Dunn test: ** P <0.01, for normal mice]. In addition, about the search action time and action amount at the time of a holding trial, a difference was not observable between three groups. Table 2 shows the results.

(Iv) Evaluation It was evaluated that cognitive function and attention were reduced in homozygous genetically modified mice compared to normal mice.

(V) Aggressive (aggressive behaviors)
(I) Test method Each mouse was transferred to a cage made of polycarbonate (20 × 12 × 10 cm) smaller than the breeding environment, acclimated for 10 minutes, and then the same-sex same-age C57BL / 6J mouse (Japan SLC) More) was introduced as a novel invasion mouse, and the subsequent behavior was analyzed. The analysis was carried out for 10 minutes, and the number of times and the time were measured by taking the social behavior (intrusion, dive), attack and hostile behavior (biting, tail tremor, hair repair) as attacking behavior.
(Ii) Evaluation Method Mice usually show vigilance and hostility against newly invading animals. However, normal mice rarely show aggressive behavior against animals of the same strain. In this test, a test between groups (t-test) is performed for each measurement item based on the measurement value of normal mice, and the change is evaluated as a change in aggression or social behavior of the mouse depending on the item for which a significant difference is observed. did.
(Iii) Results Compared with normal mice, homozygous genetically modified mice showed a decrease in creeping behavior (FIG. 7A) and total contact time (FIG. 7B) with respect to the invading mice (Student's t-test: * P <0.05 against normal mice). On the other hand, there was no difference between the two groups in other measurement items. The results are shown in Table 3.

(Iv) Evaluation Although homogenous genetically modified mice showed a decrease in social behavior (contact) compared to normal mice, it was evaluated that no aggressiveness was expressed.

4). Drug administration test to mice (i) Test method Methylphenidate hydrochloride (chemical name: methyl α-phenyl-2-piperidine acetate hydrochloride) is dissolved in physiological saline and clozapine (chemical name: 8-chloro-11). -(4-Methylpiperazin-1-yl) -5H-dibenzo [b, e] [1,4] diazepine) is suspended in 0.5% carboxymethylcellulose and 0% per body weight 30 minutes before the start of behavioral analysis. Orally administered at doses of 5 mg / kg and 3 mg / kg. As in the circadian rhythm test, mice were placed in a polycarbonate cage (35 × 30 × 17 cm), and the amount of spontaneous exercise was measured from 20:00 to 8:00 the next day. In the methylphenidate administration test, mice obtained by crossing homozygous gene-modified mice with C57BL / 6J mice three times were used.
(Ii) Evaluation method An integrated value of the spontaneous exercise amount from 21: 00 to 3:00 of each mouse in the drug administration group and the non-administration group is produced, and is calculated in a one-way analysis of variance and a post-test (Tukey-Kramer test). P value was determined as an index.
(Iii) Results The doses of methylphenidate hydrochloride and clozapine hydrochloride (FIGS. 8A and 9) that do not affect the spontaneous behavior in the dark period of normal mice are suppressed against the increase in the spontaneous activity of homozygous genetically modified mice. Showed effect and improved to normal mouse level (FIG. 8B and FIG. 9) [FIG. 9, ANOVA: F ₍ 5,21 ₎ = 8.674, p = 0.0001; post-hoc Tukey-Kramer test: ** P <0.01 for normal mice; ++ P <0.01 for non-drug group].
(Iv) Evaluation The hyperactivity of homozygous gene-modified mice was sensitive to ADHD drugs and schizophrenia drugs, and this behavior was evaluated to reflect the pathological conditions associated with these diseases.

Example 2
Mutation introduction and phenotype analysis to Spsb4 gene by gene targeting method In order to confirm that the causative gene of the phenotype of the mutant mouse prepared using transposon is Spsb4 gene, gene targeting method in ES cells was used. Mice in which mutations were introduced into the Spsb4 gene were prepared and phenotypic analysis was performed. The specific procedure is shown below.

1. Mutation Introduction into ES Cells A bacterial artificial chromosome (BAC) containing the Spsb4 gene fragment (SEQ ID NO: 14) was obtained from BACPAC Resources Center at Children's Hospital Oakland Research Institute, Oakland, California, USA. Into the BAC containing this Spsb4 gene fragment (SEQ ID NO: 14), the BAC recombineering method (Genome Research, vol. 13, no. 3, p. 476-484, 2003) by Liu et al. Two loxP sequences shown in the upper row and the neo gene were inserted. Furthermore, the region from the first intron to the second intron was cloned into a plasmid vector to obtain a targeting vector. This vector was introduced into mouse-derived ES cells by electroporation, and a cell line in which mutations were introduced into the genome of ES cells by homologous recombination shown in Step I of FIG. 10 was obtained from neo-resistant colonies. . As shown in FIG. 10 and FIGS. 11A and B, the mutation introduced into the ES cell genome is confirmed by Southern blotting using a probe in the Spsb4 gene region and a probe in the neo gene after digesting the genomic DNA with BstZ17I or KpnI. This was done by law. FIG. 11A shows the result of analysis using probe A and B in FIG. 10 in Southern blotting, and FIG. 11B shows the result of analysis using probe neo in FIG. As shown in FIGS. 11A and 11B, since a DNA band that was not detected in the wild type (wt / wt) was detected, it was found that the loxP sequence and the neo gene were inserted into the Spsb4 gene.

2. Production of genetically modified mice ES cells modified with the Spsb4 gene produced above were injected into mouse blastocysts and further transplanted into the uterus of pseudopregnant mice to obtain male chimeric mice (Neo / wt) . On the other hand, the hetero-type Spsb4 mutant mouse (Tp / wt) prepared in Example 1 was transformed into a CAG-Cre mouse (Sakai K. and Miyazaki J, Biochemical and Biophysical Research Communications, vol 237, no. 2, p. 318-324, 1997) and a female mouse having the Spsb4 gene heterozygous (Tp / wt) and having a CAG-Cre transgene was prepared (Tp / wt, CAG-Cre). In general, in female mice having a CAG-Cre transgene, Cre protein accumulates in eggs, so when mated with male mice having a loxP sequence, recombination between loxPs occurs frequently immediately after fertilization. It is known. Therefore, as shown in FIG. 12, when female Tp / wt, CAG-Cre mice are mated with male Neo / wt mice, the allele (2) exon is deleted by recombination shown in Step II of FIG. Del allele) was obtained. Since most of the translational region of the Spsb4 gene is present in exon 2, this exon deletion is thought to completely inactivate gene function. Among the offspring obtained by the crossing in FIG. 12, whether or not an individual having the Spsb4 gene Tp / Del genotype shows the same phenotype as Tp / Tp was analyzed by the method described in the next section.

The sequence of the PCR primer (FIG. 10) used to examine recombination between loxPs is shown below.
Primer 1 5′-TAATGAGGGGTTTGGAACAGTGCTGTCTGG-3 ′ (SEQ ID NO: 15)
Primer2 5′-GTCTTAAAATGTAACCAGGCTTGGGAAACA-3 ′ (SEQ ID NO: 16)
Primer3 3 5'-TTAGCAACTGCTGGGACAGTTCACACAATC-3 '(SEQ ID NO: 17)
PCR was performed using HotStarTaq system (Qiagen) under the following conditions: 95 ° C for 15 minutes for 1 cycle, (94 ° C for 30 seconds, 55 ° C for 30 seconds, 72 ° C for 30 seconds) for 35 cycles, One cycle of 7 minutes at 72 ° C.

3. Phenotype analysis The phenotypes of Tp / Del mice and Tp / Tp mice were analyzed using the distance and locus of movement in the open field test as indices.

(I) Breeding conditions for mice Wild-type mice (wt / wt mice) and genetically modified mice (Tp / Del mice and Tp / Tp mice) were respectively treated in the same manner under standard conditions as breeding conditions for experimental animals. Raised. That is, under the light-dark cycle of temperature 23 ± 1 ° C, humidity 50 ± 10%, 12 hours (light period: 8:45 to 20:45), the animals are reared only after being weaned, and freely ingested with food and water. I let you.

(Ii) Test method A mouse is placed in the center of a gray acrylic circular open field (diameter 60 cm, height 35 cm), and the travel distance every 10 minutes is analyzed using the Etho Vision tracking system (Brain Science, Osaka). The total travel distance for a total of 60 minutes was calculated. In addition, the behavior trajectories for the first 150 seconds after the mouse was placed in the open field were compared. P is calculated by performing repeated measures analysis of variance for comparison of movement distance every 10 minutes, and one-way analysis of variance and Bonferroni's test for comparison of total movement distance of 60 minutes. The value was determined as an index.

(Iii) Results As shown in FIGS. 13A and B, the transition of the travel distance every 10 minutes (FIG. 13A) and the total travel distance for 60 minutes (FIG. 13B) in the Tp / Tp mice compared to the wt / wt mice. Significant increases were observed [Repeated measurements ANOVA: genotype, F _(1,18) = 17.865, p = 0.0005 (p <0.001); time, F _(5,90) = 0 868, p = 0.05062; genotype × time; F _(5,90) = 2.359, p = 0.0463 (p <0.05): Student's t-test; *** p <0 .001 vs. wt / wt].

Similarly, in Tp / Del, it was recognized that the transition of the movement distance every 10 minutes (FIG. 13C) and the total movement distance for 60 minutes (FIG. 13D) increased significantly compared to wt / wt [Repeated measurements]. ANOVA: genotype, F _(2,27) = 4.981, p = 0.144 (p <0.05); time, F _(5,135) = 11.838, p <0.0001; genotype × time , F _(5,135) = 1.54, p = 0.1319: post hoc Bonferroni's test; * p <0.05 vs. wt / wt: ANOVA: F _(2,27) = 4.981, p = 0.0144: post-hoc Bonferroni's test; * p <0.05 vs. wt / wt. ].
Further, as shown in FIG. 14, at the beginning of the open field test (the first 150 seconds from the start of the test), wt / wt mice (FIGS. 14A and C) mainly walk along the wall around the open field. In comparison, Tp / Tp mice (FIG. 14B) and Tp / Del mice (FIG. 14E) showed exploratory behavior in the middle of the open field.

(Iv) Evaluation Tp / Del mice were evaluated to exhibit a phenotype (hyperactivity and reduced anxiety) similar to the Tp / Tp mice (homotype gene mice) prepared in Example 1.

  From Examples 1 and 2, it was found that the Spsb4 gene abnormality was associated with ADHD and schizophrenia. Therefore, it was found that the non-human mammal of the present invention in which the Spsb4 gene was modified is useful as a model animal for human ADHD and human schizophrenia.

  The genetically modified non-human mammal of the present invention is very useful as a model animal for human ADHD and schizophrenia, for elucidating the causes of ADHD and schizophrenia and for developing preventive methods, therapeutic methods and therapeutic agents. is there.

Claims (13)

  A non-human mammal that exhibits abnormal behavior and has a modified Spsb4 gene in its genome.   The abnormal behavior is a behavior in which (a) an increase in the amount of spontaneous movement during the active period is recognized as compared with a normal non-human mammal, but no difference is observed in the circadian rhythm of the spontaneous movement observed in the light-dark cycle. The non-human mammal according to 1.   The non-human mammal according to claim 1, wherein the abnormal behavior is (b) behavior that exhibits irregular right or left rotation during walking.   The non-human mammal according to claim 1, wherein the abnormal behavior is (c) behavior that moves in a cage in a calm manner when entering the activity mode.   Further, (d) the non-human mammal according to any one of claims 1 to 4, wherein a decrease in anxiety symptoms and a decrease in cognitive function are observed.   The non-human mammal according to any one of claims 1 to 4, which is a homogenous gene-modified animal in which both Spsb4 genes of a pair of chromosomes are modified.   The non-human mammal according to any one of claims 1 to 4, wherein the non-human mammal is a mouse or a rat.   The non-human mammal according to claim 7, wherein the non-human mammal is a mouse.   The non-human mammal according to any one of claims 1 to 4, which is a model animal of human attention deficit / hyperactivity disorder.   The non-human mammal according to any one of claims 1 to 4, which is a model animal for human schizophrenia.   A step of introducing a gene into an embryonic stem cell using a targeting vector to modify the Spsb4 gene, a step of producing a heterogeneous gene-modified non-human mammal derived from the embryonic stem cell, and the heterogeneous gene-modified non-human A method for producing a non-human mammal exhibiting abnormal behavior, comprising a step of obtaining a homozygous gene-modified animal in which both Spsb4 genes of a pair of chromosomes are modified by crossing mammals for one generation or more.   (I) a step of administering a candidate substance to the non-human mammal exhibiting abnormal behavior according to any one of claims 1 to 4, (II) a step of assaying a change in hyperactivity of the non-human mammal, And (III) a method for screening an anti-ADHD drug, comprising a step of selecting a candidate substance having reduced hyperactivity compared to a case where no candidate substance is administered.   (I) a step of administering a candidate substance to the non-human mammal exhibiting abnormal behavior according to any one of claims 1 to 4, (II) a step of assaying a change in hyperactivity of the non-human mammal, And (III) a screening method for a therapeutic drug for schizophrenia, comprising a step of selecting a candidate substance having reduced hyperactivity compared to a case where no candidate substance is administered.