JP2009201501A - alpha2,3-SIALIC ACID TRANSFERASE (ST3GalIV) DEFECTIVE NON-HUMAN ANIMAL AND SCREENING PROCESS USING THE SAME - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an α2,3-sialic acid transferase (ST3GalIV) defective non-human animal and screening process using the same. <P>SOLUTION: An ST3GalIV knockout mouse is prepared to disclose the fact that the mouse relates to physiological phenomenon, such as anxiety, sleep, memory, outgrowth, reproduction, or biological rhythm so that the screening process is constructed for therapeutic agent relating to the physiological phenomenon. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

The present invention relates to a non-human animal in which the gene function of α2,3-sialyltransferase (hereinafter ST3GalIV) is deficient on the chromosome, and preferably to a mouse in which the gene function of ST3GalIV is deficient on the chromosome. In addition, the present invention provides a method for screening a compound useful for the treatment of anxiety disorder or sleep disorder using a non-human animal in which ST3GalIV gene function is deficient on the chromosome, or an in vivo (hereinafter referred to as in vivo) ST3GalIV inhibitor. ) It relates to a method for measuring medicinal effects.

  Sialic acid is a monosaccharide added to the non-reducing end of complex carbohydrates such as glycoproteins and glycolipids. Since this sialic acid has a COOH group, it has a negative charge. Due to this property, sialic acid affects various biological processes such as cell adhesion, differentiation, nerve fiber development and receptor recognition (see Non-Patent Document 1 and Non-Patent Document 2). In addition, as the sugar chain structure is examined in the brain, 40% of N-glycans are sialylated, and a quarter of them are polysialylated (see Non-Patent Document 3). It has been reported that the content of ganglioside, which is a glycolipid containing acid, is about 5 times higher in the brain than in other tissues, and that the ganglioside composition in the brain is conserved among mammals (Non-Patent Literature). 4). These reports suggest that the importance of sialic acid in the brain is high, but the physiological action of sialic acid in the brain, particularly the relationship with the pathological condition, has not yet been fully examined.

Sialyltransferases catalyze the transfer of sialic acid from CMP-sialic acid to the non-reducing end of the acceptor substrate, and 20 types have been reported in mammals. These enzyme groups are divided into α2,3-sialyltransferase (ST3GAII-VI), α2,6-sialyltransferase (ST6GalI, II), α2,8-sial depending on the sugar chain structure synthesized by each enzyme. It is classified into four families: acid transferase (ST8SiaI to VI) and GalNAcα2,6-sialyltransferase (ST6GalNAcI to VI). These sugar chain structures are biosynthesized in the Golgi (see Non-Patent Document 5 and Non-Patent Document 6). Among these α2,3-sialyltransferases, ST3GalIV, which shows substrate specificity for Galβ1-3GlcNAc, Galβ1-4GlcNAc, and Galβ1-3GalNAc, has been confirmed to be expressed in organs throughout the body, and is expressed in normal mice. High in genital organs such as ovary and testis, salivary gland, liver and the like (see Non-Patent Document 7 and Non-Patent Document 8). Although the expression in the brain is low, it is certain that ST3GalIV is thought to perform some physiological function in the brain. In fact, it has been reported that its expression is enhanced in epilepsy model mice (see Non-Patent Document 9). However, sufficient studies have not yet been made on the physiological effects of ST3GalIV, particularly the relationship with pathological conditions.
Rutishauser U .: Polysialic acid at the cell surface: biophysics in service of cell interactions and tissue plasticity., J. Cell Biochem., 70 (3), 304-12, 1998 Muller D., et al .: PSA-NCAM is required for activity-induced synaptic plasticity., Neuron, 17 (3), 413-422, 1996 Zamze S., et al.:Sialylated N-glycans in adult rat brain tissue., Eur. J. Biochem., 258 (1), 243-270, 1998 Iwamori M., et al .: Comparative study on ganglioside compositions of various rabbit tissues.Tissue-specificity in ganglioside molecular species of rabbit thymus., Biochem. Biophys. Acta., 665 (2), 214-20, 1981 Tsuji S .: Molecular cloning and functional analysis of sialyltransferases .: J. Biochem (Tokyo), 120 (1), 1-13, 1996 Takashima S., et al .: Molecular cloning and expression of a sixth type of alpha 2,8-sialyltransferase (ST8Sia VI) that sialylates O-glycans., J. Biol. Chem., 277 (27), 24030-8, 2002 Kono M., et al .: Mouse beta-galactoside alpha 2,3-sialyltransferases: cmparison of in vitro substrate specificities and tissue specific expression., Glycobiology, 7 (4), 469-79, 1997 Ellies LG., Et al .: Sialyltransferase specificity in selectin ligand formation, Blood, 100, 3618-25, 2002 Matsuhashi H., et al .: Region-specific and epileptogenic-dependent expression of six subtypes of α-sialyltransferase in the adult mouse brain, J. Neurochem., 84, 53-66, 2003

  An object of the present invention is to provide a non-human animal deficient in ST3GalIV gene function, such as ST3GalIV knockout mice, to elucidate the relationship between ST3GalIV and a disease state, and further to a method for screening a therapeutic agent for the disease state and an ST3GalIV inhibitor A screening method is provided.

  As a result of diligent research to solve the above problems, the present inventors have produced ST3GalIV knockout mice and found that the mice exhibit physiological phenomena related to anxiety, sleep, growth, reproduction, biological rhythm, and the like. The present invention has been completed.

That is, the present invention relates to non-human animals including rodents such as mice and rats, anxiety disorder model animals, sleep disorder model animals, growth disorder model animals, and reproductive disorder model animals in which the gene function of ST3GalIV is deficient on the chromosome. And a biological rhythm disorder model animal.
The present invention also provides a method for screening a compound useful for the treatment of anxiety disorders using non-human animals including rodents such as mice and rats, in which the gene function of ST3GalIV is deficient on the chromosome, When the compound is administered to the non-human animal, an open field test is performed, and the residence time in the four corners is reduced compared to the non-human animal not administered the compound, the compound is useful for treating anxiety disorders. A method comprising selecting as a compound; a method for screening a compound useful for the treatment of anxiety disorder using the non-human animal, administering the compound to the non-human animal, conducting a fear conditioning test, and administering the compound Selecting the compound as a useful compound for the treatment of anxiety disorders when freezing is reduced compared to the non-human animal A method for screening a compound useful for the treatment of sleep disorders using the non-human animal, wherein the compound is administered to the non-human animal, and an electroencephalogram and / or electromyogram is measured and / or behavior is observed A method comprising selecting the compound as a useful compound for the treatment of sleep disorders when sleep patterns are improved as compared to the non-human animal not administered the compound; the non-human animal is used. A method of screening for compounds useful for the treatment of growth disorders comprising administering a compound to said non-human animal, measuring body weight and / or length, and / or growth hormone (Gh) and / or IGF-1 in blood When the body weight and / or length is increased compared to the non-human animal not administered the compound and / or growth in blood A method comprising selecting the compound as a useful compound for the treatment of growth disorders when the concentration of rumon (Gh) and / or IGF-1 is increased; useful for the treatment of reproductive disorders using the non-human animal A method for screening a compound, comprising administering a compound to the non-human animal, observing the presence of abnormalities in the sexual cycle and / or labor, and comparing the sexual cycle and the non-human animal not administered with the compound. A method comprising selecting the compound as a compound useful for the treatment of a reproductive disorder when abnormalities in labor are improved; a method for screening a compound useful for the treatment of a biological rhythm disorder using the non-human animal The compound is administered to the non-human animal, the sexual cycle is observed, the electroencephalogram and / or electromyogram is measured and / or the behavior is observed, and the compound is administered. Selecting the compound as a useful compound for the treatment of a biological rhythm disorder when the rhythm of the sexual cycle is improved and / or sleep pattern is improved as compared to the non-human animal And a method for measuring the in vivo efficacy of an ST3GalIV inhibitor using said non-human animal, comprising administering a test substance to said non-human animal and wild-type non-human animal, i) open field test, ii) fear conditioning test Iii) measurement of electroencephalogram and / or electromyogram, and / or behavioral observation, iv) measurement of body weight and / or length, v) measurement of concentration of growth hormone (Gh) or IGF-1 in the blood, or Vi) Observing the abnormalities of the sexual cycle and / or parturition, the non-human animal has no effect, and the wild-type non-human animal presents the phenotype of the non-human animal. When: a method for including selecting the subject substance as ST3GalIV inhibitors.

  By carrying out the screening method exemplified above using non-human animals such as rodents including mice, in which the gene function of ST3GalIV in the present invention is deficient on the chromosome, anxiety disorder, sleep disorder, Compounds useful for the treatment of growth disorders, reproductive disorders, biological rhythm disorders, etc. can be found. Moreover, the in vivo efficacy of ST3GalIV inhibitors is evaluated by using non-human animals such as rodents including mice and wild-type non-human animals in which the gene function of ST3GalIV according to the present invention is deficient on the chromosome. can do.

(1) Non-human animal deficient in ST3GalIV gene function on the chromosome The present invention provides a non-human animal deficient in ST3GalIV gene function on the chromosome. The non-human animal in which the gene function of ST3GalIV in the present invention is deficient on the chromosome means that the endogenous gene of the non-human animal encoding ST3GalIV is inactivated by destruction, deletion, substitution, etc., and has substantial enzyme activity. A non-human animal that has lost the function of expressing ST3GalIV. Non-human animals can specifically include rodents such as mice and rats, but are not limited thereto. When the non-human animal is a mouse, the non-human animal is a knockout mouse. More preferably, the non-human animal in which the gene function of ST3GalIV is deleted on the chromosome is a non-human conditional knockout animal in which the gene function of ST3GalIV is inactivated in a time-specific and / or tissue-specific manner. Is a conditional knockout mouse.

  The wild-type non-human animal in the present invention means an animal of the same type and strain that has normal and healthy ST3GalIV gene function, and is the same as the non-human animal deficient in ST3GalIV gene function on the chromosome. Animals are preferred, and animals of the same sex and age are also preferred. For example, when an ST3GalIV knockout mouse, which is the above-mentioned non-human animal, is produced from a C57BL / 6J mouse, a normal and healthy C57BL / 6J mouse can be adopted as the wild-type non-human animal. Hereinafter, as an example, the production method will be described when the non-human animal is a mouse, that is, a knockout mouse.

Method for preparing ST3GalIV knockout mouse As a method for preparing ST3GalIV knockout mouse, any method can be used as long as it can generate a knockout mouse that has lost the function of ST3GalIV, but a known gene targeting method (for example, Edited by Masaaki Muramatsu and Masaru Yamamoto, “Experimental Medicine Separate Volume, New Handbook of Genetic Engineering, 3rd Edition” (1999, published by Yodosha); Separate Volume, Experimental Medicine, The Protocol Series “Latest Technology of Gene Turding” (2000) , Yodosha) Conditional targeting method p.115-120; Biomanual Series 8 “Gene targeting” —Generation of mutant mice using ES cells (1995, Yodosha) p.71-77); Sambrook et al. , Molecular Clon ing: A LABORATORY MANUAL, 3rd edition, COLD SPRING HARBOR LABORATORY PRESS, 2001, 4.82-4.85, etc.).
For example, (i) a mouse genomic DNA library is screened using a cDNA encoding ST3GalIV as a probe, the ST3GalIV gene of the genomic DNA is isolated, and (ii) a portion of this gene containing an appropriate exon is converted into an appropriate foreign A target vector is prepared by substituting a gene (for example, a marker gene such as a neomycin resistance gene). (Iii) The prepared target vector is derived from an ES cell (for example, MS12 ES cell, 129 / SvJ-derived by electroporation method or the like). Eiv ES cell, TT2 cell line, AB-1 cell line, J1 cell line, R1 cell line, etc.) (iv) ES cells that have undergone homologous recombination are selected and regenerated using this ES cell line Obtained by producing a series of chimeric mice and mating with (v) wild-type mice ST3GalIV knockout mice by mating the heterozygous mice with each other that can be produced wild-type mice littermates.
Alternatively, ST3GalIV knockout mice can be selected from, for example, (i) screening a mouse genomic DNA library using a cDNA encoding ST3GalIV as a probe, isolating the ST3GalIV gene of genomic DNA, and (ii) the genomic DNA region of the ST3GalIV gene. Producing a targeting vector comprising a sequence partially or entirely sandwiched by a recombination target sequence, and homologous recombination of part or all of the genomic DNA region of the ST3GalIV gene with the sequence, (iii) embryo Transducing the targeting vector into a sex stem cell, causing homologous recombination of part or all of the genomic DNA region of the ST3GalIV gene, and (iv) using the embryonic stem cell resulting from the homologous recombination, including Generating a genetically modified mouse having a modified ST3GalIV gene, and (v) expressing the recombinant mouse with a recombinant protein (preferably expressing a recombinant protein in a time-specific and / or tissue-specific manner) ST3GalIV knockout mice in which expression of ST3GalIV protein is suppressed (preferably time-specific and / or tissue-specifically suppressed) can be produced by mating with a recombinant expression gene-modified mouse (JP 2002-2002). -369639 and 2006-141283).

The present invention also provides a non-human animal in which the gene function of ST3GalIV is deficient on the chromosome, which is an anxiety disorder model animal. An anxiety disorder model animal is an animal, preferably an animal that reflects the pathology of anxiety disorder in humans. Using this animal, elucidation of the mechanism of anxiety disorder, treatment method for anxiety disorder, search for prevention method, therapeutic drug It is possible to evaluate and search for preventive drugs.
In the anxiety disorder model animal provided by the present invention, anxiety is caused by the lack of the gene function of ST3GalIV. Since ST3GalIV is a glycosyltransferase, it may be involved in various physiological phenomena, and it is speculated that there are various mechanisms leading to anxiety from ST3GalIV deficiency. On the other hand, in an anxiety disorder model animal, for example, an anxiety disorder model animal prepared by administration of a drug such as a benzodiazepine receptor inverse agonist, anxiety is induced by a single mechanism via a benzodiazepine receptor. Since the mechanism of clinical anxiety is complicated and diverse, the animal model of anxiety disorder provided by the present invention is recognized clinically compared to the animal model of anxiety disorder produced by conventional drug administration. It may be a comprehensive animal model of anxiety disorder that more reflects the anxiety of various mechanisms, and is considered useful for elucidating the mechanism of anxiety disorder.
In addition, since anxiety caused by drug administration involves an artificial operation called drug administration, there is often a variation in the degree of anxiety caused between treated animals. On the other hand, since the anxiety disorder model animal provided by the present invention causes anxiety due to the genetic background of ST3GalIV gene deficiency, the degree of anxiety compared to the anxiety disorder model animal prepared by conventional drug administration There is no variation. This is very advantageous in the evaluation of anxiety disorder therapeutics and preventives.
The anxiety disorder model animal provided by the present invention is preferably a rodent, and more preferably a mouse.

Anxiety disorders are diseases with strong anxiety and symptoms that cause behavioral and psychological disorders, including generalized anxiety disorder, social anxiety disorder (social phobia), panic disorder, PTSD, obsessive-compulsive disorder, etc. , DSM-IV (Diagnostic and Statistical Manual of Mental Disorder) published by the American Psychiatric Association.
Generalized anxiety disorder, social anxiety disorder, panic disorder, PTSD, obsessive compulsive disorder, etc. are also diagnosed with reference to DSM-IV, and generalized anxiety disorder is not limited to a special situation. A panic disorder is a disease characterized by a panic attack that suddenly attacks autonomic symptoms such as palpitation and a strong sense of anxiety. Yes, PTSD is a disease characterized by a strong sense of anxiety caused by trauma experienced in the past. Obsessive-compulsive disorder is an obsession with meaningless behavior (compulsive behavior). It is a disease characterized by

  The present invention further provides a non-human animal in which the gene function of ST3GalIV is deficient on the chromosome, which is a sleep disorder model animal. The sleep disorder model animal is an animal, preferably an animal reflecting the pathology of sleep disorder in humans, and using the animal, elucidation of the mechanism of sleep disorder, treatment method for sleep disorder, search for prevention method, Evaluation and search of therapeutic and prophylactic drugs are possible. One of the features of the sleep disorder model animal provided by the present invention is that the time of REM sleep is short. The sleep disorder model animal provided by the present invention is preferably a rodent, and more preferably a mouse.

  The sleep disorder is a disease accompanied by some abnormality in falling asleep and sleeping. In the present invention, the sleep disorder includes a REM sleep disorder, and further includes, for example, an unnatural sleep state in which the sleep state falls into a sleep state for a short time and returns to the awake state again. A disorder of REM sleep is a state of REM sleep that is not natural, and includes a state in which the time of REM sleep is short.

  The present invention further provides a non-human animal in which the gene function of ST3GalIV, which is a growth disorder model animal, is deficient on the chromosome. A growth disorder model animal is an animal, preferably an animal that reflects the pathology of human growth disorder, and using this animal, elucidation of the mechanism of growth disorder, treatment method for growth disorder, search for prevention method, Evaluation and search of therapeutic and prophylactic drugs are possible. The growth disorder model animal provided by the present invention is not one in which the function of the pituitary gland is significantly impaired unlike the conventional hypophysectomized animal. In addition, the growth disorder model animal provided by the present invention has a slower increase in body weight and length than that of a wild-type non-human animal, and the concentrations of growth hormone (Gh) and IGF-1 in plasma are also reduced. . The growth disorder model animal provided by the present invention is preferably a rodent, and more preferably a mouse.

  A growth disorder is a disease with some abnormality in growth, for example, an abnormality in height and / or weight gain. In the present invention, the growth disorder is dwarfism (including pituitary dwarfism), and short stature, preferably epiphyseal line in Turner syndrome, chronic renal failure, Praderwillie syndrome and adult growth hormone secretion deficiency Dwarfism without closure (including pituitary dwarfism without epiphyseal closure) and short stature in the following diseases without epiphyseal closure: Turner syndrome, chronic renal failure, Praderwilly syndrome And adult growth hormone secretion deficiency. The body length corresponds to the height, for example, in humans.

  The present invention further provides a non-human animal in which the gene function of ST3GalIV is deficient on the chromosome, which is a reproductive disorder model animal. A reproductive disorder model animal is an animal, preferably an animal reflecting the pathology of a reproductive disorder in humans, and using this animal, elucidation of the mechanism of reproductive disorder, treatment method for reproductive disorder, search for prevention method, Evaluation and search of therapeutic and prophylactic drugs are possible. The reproductive disorder model animal provided by the present invention has an irregular female sexual cycle. In addition, in the reproductive disorder model animal provided by the present invention, abnormal labor is observed and the baby is still born without being delivered, and this tendency is observed by older animals. The reproductive disorder model animal provided by the present invention is preferably a rodent, and more preferably a mouse.

  A reproductive disorder is a disease in which there is some abnormality in fertility regardless of male and female and normal fetal birth is not achieved, for example, a disease with abnormalities in the sexual cycle, pregnancy, and / or parturition. In the present invention, the sexual cycle means, for example, in the case of humans, the menstrual cycle from the day when menstruation starts until the day before the next menstruation starts, in the case of mice, the cycle from the estrus period to the next estrus period ( Including estrus → estrus → late estrus → rest period), abnormalities of the sexual cycle include irregular sexual cycle, disturbed rhythm of sexual cycle, shortened cycle of sexual cycle Abnormalities in labor include miscarriages, premature births, term labor and stillbirths.

  The present invention further provides a non-human animal in which the gene function of ST3GalIV is deficient on the chromosome, which is a model animal of a biological rhythm disorder. The biological rhythm disorder model animal is an animal, preferably an animal that reflects the pathology of a human biological rhythm disorder. Using this animal, elucidation of the mechanism of biological rhythm disorder, treatment method for biological rhythm disorder, prevention It is possible to search for laws, evaluate and search for therapeutic drugs and preventive drugs. The biological rhythm disorder model animal provided by the present invention has an irregular female sexual cycle, and rhythm failure during delivery is observed. The biological rhythm disorder model animal provided by the present invention has irregular REM sleep and non-REM sleep cycles, does not represent a natural sleep rhythm, and has a short REM sleep time. The biological rhythm disorder model animal provided by the present invention is preferably a rodent, and more preferably a mouse.

  The biological rhythm disorder is a disease in which a normal physiological function is impaired due to some abnormality in a biological rhythm such as circadian rhythm, sleep rhythm, sexual cycle, hormone secretion rhythm, and the like.

(2) Screening method for therapeutic agent for anxiety disorder using a non-human animal in which ST3GalIV gene function is deficient on the chromosome using open field test The present invention is a non-human deficient in ST3GalIV gene function on the chromosome Methods of screening for compounds useful for the treatment of anxiety disorders using animals are provided.
For example, the present invention provides a method for screening a compound useful for the treatment of anxiety disorders by administering a compound to a non-human animal deficient in ST3GalIV gene function on the chromosome, conducting an open field test, and administering the compound. Selecting a compound as a useful compound for the treatment of anxiety disorders when the residence time at the four corners is reduced compared to a non-human animal in which the gene function of ST3GalIV is not chromosomally deficient. Preferably, the non-human animal is a rodent such as a mouse or a rat.

An anxiety disorder is as described in (1) above.
The open field test is a test that uses the habit of walking more favorably around the inside of the device in a wide new environment called a square device, and when a benzodiazepine anxiolytic such as diazepam is administered, This is a test method to evaluate the degree of anxiety as an increase in staying time in the central part of the device is an indicator that the degree of anxiety is decreasing because of increased search behavior in the central part of the device (Taku Yamaguchi, Yoshioka) Mitsuhiro, Journal of Japanese Pharmacology, 2007, 130, 105-111). Thus, animals administered compounds useful for the treatment of anxiety disorders have a reduced residence time at the four corners of the device and an increased residence time in the middle in this test system.
As described above, the open field test is a test using the natural habits of animals, and is not a test method for inducing pathological anxiety and evaluating the degree of anxiety. Furthermore, among the clinically effective drugs for anxiety disorders, benzodiazepine anti-anxiety drugs can detect anxiolytic effects in this test system, but are tricyclic antidepressants and first-line drugs for anxiety disorder treatment. The anxiolytic effect of SSRI is not detected, and central stimulants and other drugs have increased spontaneous motor activity and increased exploratory behavior in the center of the device. It is recommended to evaluate in combination with other test systems (see Taku Yamaguchi, Mitsuhiro Yoshioka, Nihon Pharmacou, 2007, 130, 105-111).
Since the screening method for a therapeutic agent for anxiety disorder according to the present invention uses a non-human animal deficient in ST3GalIV gene function on the chromosome, which has been recognized in a fear conditioning test as having high anxiety sensitivity, a healthy wild-type animal is used. It is considered that the evaluation system is superior to the conventional open field test using only The screening method for a therapeutic agent for anxiety disorder of the present invention uses a non-human animal in which the gene function of ST3GalIV is deficient on the chromosome, thereby evaluating the conventional open field test using only healthy wild-type animals. This is a system that has a large window (maximum change amount that can be changed by test compound administration) and can detect the anxiolytic effect more accurately (for example, wild type (+ / +) and ST3GalIV knockout mice (-/ Comparing the difference between each black column and white column in-), the difference in ST3GalIV knockout mice is larger than that in wild-type mice, so that the efficacy of anxiolytic drugs can be detected with a larger change amount).

  The screening method for a therapeutic agent for anxiety disorders of the present invention includes a step of administering a compound to a non-human animal in which the gene function of ST3GalIV is deficient on the chromosome. Administration is not particularly limited as long as it is carried out by an appropriate administration method, and administration methods such as oral administration, subcutaneous administration, intraperitoneal administration, and intravenous administration can be adopted. A person skilled in the art can appropriately set conditions such as a compound to be administered, a medium of the compound, a dose, and a method of administration.

The measurement device for the open field test is not particularly limited, and a device including a square cage and a stay location recording device can be adopted. Preferably, in the case of a mouse, the cage is made of a transparent plastic, the width is 45 cm × 55 cm, and the height is an appropriate height that the mouse cannot easily jump over. The stay location recording device is not limited as long as the stay location is recorded and the stay time at the four corners can be measured, but a video camera with a recording function or AUTOMEX II (manufactured by Tokai Medical Science) or the like can be employed. The cage is divided into appropriate sizes, and the four corners or the central part for measuring the residence time of the animals are preset. The size of the four corners can be appropriately set by those skilled in the art. For example, in the case of a cage having a width of 45 cm × 55 cm, each of the four corners can be set to a size of 11.25 cm × 13.75 cm including the corners of each cage. The size of the central part can also be appropriately set by those skilled in the art. For example, in the case of a cage having a size of 45 cm × 55 cm, a 11.25 cm × 27.27 including a 45 cm × 55 cm square center of gravity (intersection of diagonal lines) that is not in contact with the cage wall. Can be set to a size of 5 cm.
Those skilled in the art can appropriately set the experiment schedule such as the number of trials, the number of animals, and the schedule of the open field test.

As a result of the open field test, a non-human animal in which the gene function of ST3GalIV to which the compound was administered was deficient on the chromosome was not administered to the non-human animal in which the compound was not administered (or only the compound vehicle was administered). When the residence time at the four corners is reduced compared to a human animal, the compound is selected as a useful compound for the treatment of anxiety disorders.
Alternatively, as a result of an open field test, a non-human animal in which the gene function of ST3GalIV administered with the compound is deficient on the chromosome does not administer the compound (or only the compound vehicle is administered), and the gene function of ST3GalIV is deficient on the chromosome When the residence time in the central part is increased compared to the non-human animals, the compound is selected as a compound useful for the treatment of anxiety disorders.
In some cases, the screening method for an anxiety disorder therapeutic agent of the present invention further comprises administering the compound to a wild-type non-human animal, preferably a mouse, to confirm that the compound has no effect on the wild-type animal. You may include the process to do. As a result, it can be selected as a compound useful for the better treatment of anxiety disorders that alleviates the abnormal anxiety only to animals with abnormally increased anxiety sensitivity and has no effect on normal animals.

(3) A method for screening a compound useful for the treatment of anxiety disorder using a non-human animal in which ST3GalIV gene function is deficient on the chromosome using a fear conditioning test. Provided is a method for screening a compound useful for the treatment of anxiety disorder using a deficient non-human animal.
For example, the present invention provides a method for screening a compound useful for the treatment of anxiety disorders by administering a compound to a non-human animal deficient in ST3GalIV gene function on the chromosome, conducting a fear conditioning test, and administering the compound. Selecting a compound as a useful compound for the treatment of anxiety disorders when freezing is reduced compared to a non-human animal in which the gene function of ST3GalIV is not chromosomally defective. Preferably, the non-human animal is a rodent such as a mouse or a rat.

An anxiety disorder is as described in (1) above.
The fear conditioning test, also known as the contextual fear conditioning test or fear conditioning stress test, experienced an electric shock that was loaded from an inevitable electric shock (electrical stimulus) from the floor grid of the experiment box under certain conditions Condition in the experimental box (fear experiences are perceived by tactile sensation on the interior and floor grid in the experimental box, or by sound stimulation presented before electric shock) and present the same conditions to the experimental animal again Assess anxiety by measuring psychological stress responses caused by (in the same experimental box that received the shock shock, or by presenting the same sound stimulus presented before the shock shock) It is a test. As one of the psychological stress reactions, a defensive reaction, that is, “shrinking behavior (freezing)” in which the rat shrugs in a non-moving state other than respiratory motion without giving an electric shock. Show. Freezing is regarded as an index of fear and anxiety, and the more it appears, the more intense it is, and the less it appears, the weaker it is. The expression of freezing depends on the intensity of electric shock, the intensity of sound stimulation, the intensity of illumination in the experiment box, etc. when conditioning, but the experiment environment and experimental conditions including these are appropriately set by those skilled in the art. it can. The determination of freezing can also be made visually, but an automatic analysis system based on image processing using a video camera may be used.
The fear conditioning test is a system that can detect the anxiolytic effects of many drugs such as benzodiazepine anxiolytics, serotonin 5-HT _1A receptor agonists, and SSRIs that are applied to anxiety disorders in clinical practice. It is very useful as a system (see Japanese Journal of Psychosomatic Medicine Vol. 37, No. 3, pp. 223-229, etc.).
In the screening method for a therapeutic agent for anxiety disorder according to the present invention, a non-human animal in which the gene function of ST3GalIV, which is highly susceptible to anxiety, is deficient on the chromosome is used, which is more than a fear conditioning test using only healthy wild-type animals. Since a large evaluation window is obtained, the evaluation system is superior to the conventional fear conditioning test.

  The screening method for a therapeutic agent for anxiety disorders of the present invention includes a step of administering a compound to a non-human animal in which the gene function of ST3GalIV is deficient on the chromosome. Administration is not particularly limited as long as it is carried out by an appropriate administration method, and administration methods such as oral administration, subcutaneous administration, intraperitoneal administration, and intravenous administration can be adopted. A person skilled in the art can appropriately set conditions such as a compound to be administered, a medium of the compound, a dose, and a method of administration.

The measuring device for the fear conditioning test is not particularly limited, and a person skilled in the art can select an appropriate one.
Also, the experiment schedule such as the number of trials, the number of animals, and the schedule can be appropriately set by those skilled in the art.

As a result of the fear conditioning test, a non-human animal in which the gene function of ST3GalIV administered with the compound is deficient on the chromosome is not administered to the non-human animal in which the compound is not administered (or only the compound vehicle is administered). When freezing is reduced compared to a human animal, the compound is selected as a useful compound for the treatment of anxiety disorders.
In some cases, the screening method for an anxiety disorder therapeutic agent of the present invention further comprises administering the compound to a wild-type non-human animal, preferably a mouse, to confirm that the compound has no effect on the wild-type animal. You may include the process to do. As a result, it can be selected as a compound useful for the better treatment of anxiety disorders that alleviates the abnormal anxiety only to animals with abnormally increased anxiety sensitivity and has no effect on normal animals.

  In addition, a screening method for a therapeutic agent for anxiety disorder using a non-human animal in which the gene function of ST3GalIV is deficient on the chromosome using the open field test described in (2), and the fear conditioning test described in (3) A method for screening compounds useful for the treatment of anxiety disorders using a non-human animal in which the gene function of ST3GalIV is deficient on the chromosome is performed in parallel or continuously in both methods. By selecting a compound selected as a compound useful for the treatment of both anxiety disorders as a compound useful for the treatment of anxiety disorders, screening with higher accuracy becomes possible.

(4) Learning / memory enhancers, cognitive function enhancers, memory maintenance enhancers, or anti-dementia drug fear conditioning tests including ST3GalIV inhibitors can also serve as an evaluation system for learning / memory (Taku Yamaguchi, Mitsuhiro Yoshioka, Nihon Pharmacology) Journal, 2007, 130, 105-111; Kazuaki Takuma et al., Nichi Pharmacology, 2007, 130, 112-116), if a large amount of freezing appears, it can be evaluated that learning / memory is enhanced. If a non-human animal deficient in ST3GalIV's gene function on the chromosome appears in a conditioned conditioning test that requires memory and more freezing occurs than a wild-type nonhuman animal, ST3GalIV deficiency is It suggests the possibility of enhancing cognitive function, or memory maintenance. Therefore, in such a case, the present invention can provide a non-human animal in which the gene function of ST3GalIV is deficient on the chromosome with enhanced learning / memory, cognitive function, or memory maintenance. Preferably, the present invention can provide a rodent such as a mouse or a rat having an ST3GalIV gene function deficient on the chromosome and having enhanced learning / memory, cognitive function, or memory maintenance. More preferably, the present invention can provide a mouse in which ST3GalIV gene function is deficient on the chromosome with enhanced learning / memory, cognitive function, or memory maintenance. Furthermore, when it is suggested that ST3GalIV deficiency may enhance learning / memory, cognitive function, or memory maintenance, the present invention provides a learning / memory enhancing agent, a cognitive function promoting agent, memory containing ST3GalIV inhibitor. Maintenance enhancers or anti-dementia drugs may be provided.
Anti-dementia drugs include drugs for treating and / or preventing dementias such as Alzheimer's disease and cerebrovascular dementia.

Here, the ST3GalIV inhibitor is a substance that inhibits the enzyme activity of ST3GalIV, and is not limited to a low-molecular compound, and includes peptides, proteins, DNA, RNA, and further, ST3GalIV antisense oligonucleotides or siRNAs. Including.
A pharmaceutical composition containing an ST3GalIV inhibitor and its administration method can be appropriately determined by those skilled in the art.
The antisense oligonucleotide and siRNA of ST3GalIV may be incorporated into an appropriate vector, for example, an adenovirus vector so that it can be expressed in neurons.

(5) Method for screening a compound useful for treatment of sleep disorders using a non-human animal deficient in ST3GalIV gene function on the chromosome The present invention uses a non-human animal deficient in ST3GalIV gene function on the chromosome. A method of screening for compounds useful in the treatment of sleep disorders that were present.
For example, the present invention provides a method for screening a compound useful for the treatment of sleep disorders by administering a compound to a non-human animal in which the gene function of ST3GalIV is deficient on the chromosome, and measuring EEG and / or electromyogram and / or Or, when the behavioral observation is made and the sleep pattern is improved compared to a non-human animal in which the gene function of ST3GalIV not administered the compound is deficient on the chromosome, the compound is useful for the treatment of sleep disorders Selecting as a compound. Preferably, the non-human animal is a rodent such as a mouse or a rat.

  The sleep disorder is a disease accompanied by some abnormality in falling asleep and sleeping. In the present invention, the sleep disorder includes a REM sleep disorder, and moreover, for example, an unnatural sleep state such as a wake state that falls into a sleep state for a short time and then returns to a wake state again, wakefulness, non-REM sleep, REM sleep time and cycle Includes sleep states that are different from the natural state of sex. In some cases, sleep disorders may be accompanied by other diseases, such as anxiety disorders. A disorder of REM sleep is a state of REM sleep that is not natural, and includes a state in which the time of REM sleep is short.

The measurement and analysis of electroencephalogram and electromyogram are not particularly limited. For example, electrodes can be implanted in appropriate locations in the brain and subcutaneously, and electroencephalogram and electromyogram can be measured using Vital Recorder (KISSEI COMTEC). The brain wave analysis research program SleepSign (KISSEI COMTEC) was used to determine the ratio of each of the delta, theta, alpha, and spindle wavelengths of the electroencephalogram, and the electromyogram size and mouse behavior were recorded. From the video image, it is possible to identify each state of wakefulness, REM sleep, and sleep and analyze the sleep pattern.
Although observation of behavior is not particularly limited, by taking a picture with a photographing means such as a video camera or a digital video camera over the whole day, recording on a recording medium such as a video or a DVD, and analyzing the recorded video by visual observation. ,It can be carried out. For example, it is possible to determine whether or not an animal is awake by visual observation of a recorded video.
In the screening method for therapeutic agents for sleep disorders of the present invention, non-human animals exhibiting unnatural sleep states and having ST3GalIV gene function deficient on the chromosome are used. Therefore, sleep disorders using only healthy wild-type animals are used. This test system is superior to screening methods for therapeutic drugs. In addition, since the REM sleep time of a non-human animal in which the gene function of ST3GalIV is deficient on the chromosome is significantly reduced compared to that of a wild-type non-human animal, the non-human animal in which the gene function of ST3GalIV is deficient on the chromosome The present invention provides a novel method of screening for compounds useful in the treatment of REM sleep disorders.

  The screening method for a therapeutic agent for sleep disorders of the present invention includes a step of administering a compound to a non-human animal in which the gene function of ST3GalIV is deficient on the chromosome. Administration is not particularly limited as long as it is carried out by an appropriate administration method, and administration methods such as oral administration, subcutaneous administration, intraperitoneal administration, and intravenous administration can be adopted. A person skilled in the art can appropriately set conditions such as a compound to be administered, a medium of the compound, a dose, and a method of administration.

  A person skilled in the art can appropriately set the experimental schedule such as the conditions such as the measurement of electroencephalogram and electromyogram, behavioral observation and analysis thereof, the number of trials, the number of animals, and the schedule.

As a result of measurement of electroencephalogram and / or electromyogram and / or behavioral observation and analysis thereof, a non-human animal in which the gene function of ST3GalIV administered with the compound is deficient on the chromosome does not receive the compound (or When the sleep pattern is improved as compared to a non-human animal in which the gene function of ST3GalIV is deficient on the chromosome (administered only as a compound vehicle), the compound is selected as a compound useful for the treatment of sleep disorders. Here, the improvement of the sleep pattern means that the transition of sleep, REM sleep, and awakening (sleep pattern) approaches a more natural state, and sleep of a non-human animal in which the gene function of ST3GalIV is deficient on the chromosome Improved pattern means that sleep, REM sleep, and awakening cycle and time approach a wild-type non-human animal.
In addition, as a result of measurement of electroencephalogram and / or electromyogram and / or behavioral observation and analysis thereof, non-human animals in which the gene function of ST3GalIV administered with the compound is deficient on the chromosome do not receive the compound A compound that is useful in the treatment of REM sleep disorders when the duration of REM sleep is increased compared to a non-human animal in which the gene function of ST3GalIV is chromosomally deficient (or administered only with the compound vehicle) Selected as.
Optionally, the method of screening for compounds useful for the treatment of sleep disorders in the present invention further comprises administering the compound to a wild type non-human animal, preferably a mouse, wherein the compound exhibits an effect on the wild type animal. You may include the process of confirming that there is no. Thereby, the abnormal sleep can be improved only for animals exhibiting abnormal sleep, and the compound can be selected as a compound useful for better treatment of sleep disorders that does not affect normal animals.

(6) Method for screening compound useful for treatment of growth disorder using non-human animal deficient in ST3GalIV gene function on chromosome The present invention uses a non-human animal deficient in ST3GalIV gene function on chromosome. A method of screening for compounds useful in the treatment of growth disorders that were present is provided.
For example, the present invention provides a method for screening a compound useful for the treatment of growth disorders, comprising administering a compound to a non-human animal in which ST3GalIV gene function is deficient on the chromosome, measuring body weight and / or length, and / or Measurement of the concentration of growth hormone (Gh) and / or IGF-1 in the blood is carried out to compare the body weight and / or relative to a non-human animal in which the gene function of ST3GalIV not administered the compound is deficient on the chromosome When the body length and / or the concentration of growth hormone (Gh) and / or IGF-1 in the blood is increased, the compound is selected as a compound useful for the treatment of growth disorders. Preferably, the non-human animal is a rodent such as a mouse or a rat.

The measurement and analysis of body weight and body length are not particularly limited, and can be performed using, for example, a weight scale, a body length scale, and the like.
The measurement of the concentration of growth hormone (Gh) and IGF-1 in blood is not particularly limited. For example, serum or plasma is obtained from a blood sample collected from an animal, and the concentration of Gh or IGF-1 in the blood sample is determined. , And can be measured using ELISA.
The screening method for a therapeutic agent for growth disorders according to the present invention has less variation, accuracy, and less intervention because of complicated interventions such as surgery compared to a conventionally used system using hypophysectomized animals. Screening results with good reproducibility can be obtained.

  The screening method for a therapeutic agent for growth disorders of the present invention comprises a step of administering a compound to a non-human animal in which the gene function of ST3GalIV is deficient on the chromosome. Administration is not particularly limited as long as it is carried out by an appropriate administration method, and administration methods such as oral administration, subcutaneous administration, intraperitoneal administration, and intravenous administration can be adopted. A person skilled in the art can appropriately set conditions such as a compound to be administered, a medium of the compound, a dose, and a method of administration.

  A person skilled in the art is responsible for measuring the body weight and length, measuring the concentration of growth hormone (Gh) and IGF-1 in the blood, and analyzing the conditions, the number of trials, the number of animals, the schedule of the experiment, etc. It can be set appropriately.

As a result of measurement of body weight and / or length, and / or concentration of growth hormone (Gh) and / or IGF-1 in blood, and analysis thereof, the gene function of ST3GalIV administered with the compound was observed on the chromosome. The deficient non-human animal does not receive the compound (or administers only the compound vehicle), and the body weight and / or body length and / or blood compared to the non-human animal deficient in the gene function of ST3GalIV on the chromosome When the concentration of growth hormone (Gh) and / or IGF-1 is increased, the compound is selected as a compound useful for the treatment of growth disorders.
Optionally, the method of screening for compounds useful for the treatment of growth disorders in the present invention further comprises administering the compound to a wild type non-human animal, preferably a mouse, wherein the compound exhibits an effect on the wild type animal. You may include the process of confirming that there is no. Thereby, the abnormal growth can be improved only for an animal exhibiting abnormal growth, and the compound can be selected as a compound useful for the treatment of a superior growth disorder that does not affect normal animals.

(7) Method for screening compound useful for treatment of reproductive disorder using non-human animal deficient in ST3GalIV gene function on chromosome The present invention uses a non-human animal deficient in ST3GalIV gene function on chromosome Methods of screening for compounds useful in the treatment of reproductive disorders were provided.
For example, the present invention provides a method for screening a compound useful for the treatment of reproductive disorders, comprising administering a compound to a non-human animal in which ST3GalIV gene function is deficient on the chromosome, and confirming whether or not there is an abnormality in the sexual cycle and / or labor. An observation is made that when the sexual cycle and / or delivery abnormalities are improved compared to non-human animals in which the gene function of ST3GalIV not administered the compound is chromosomally deficient, the compound is Including selecting as a therapeutically useful compound. Preferably, the non-human animal is a rodent such as a mouse or a rat.

Observation of the presence or absence of abnormal sexual cycles is not particularly limited, and can be performed using, for example, vaginal plaque.
Observation of the presence or absence of abnormalities in labor is not particularly limited, and can be performed, for example, by measuring the number of days of pregnancy and, optionally, observing the life or death of the fetus through an incision.
The method for screening a therapeutic drug for reproductive disorders of the present invention is compared to a conventional system using an animal in which the sexual cycle is abnormal by treating a normal animal with a drug or the like. Since the intervention of treatment is small, screening results with less variation and good accuracy and reproducibility can be obtained.

  The screening method for a therapeutic agent for reproductive disorders of the present invention includes a step of administering a compound to a non-human animal in which the gene function of ST3GalIV is deficient on the chromosome. Administration is not particularly limited as long as it is carried out by an appropriate administration method, and administration methods such as oral administration, subcutaneous administration, intraperitoneal administration, and intravenous administration can be adopted. A person skilled in the art can appropriately set conditions such as a compound to be administered, a medium of the compound, a dose, and a method of administration.

  A person skilled in the art can appropriately set the experimental schedule such as observation of the presence or absence of abnormalities in the estrous cycle and labor and analysis thereof, the number of trials, the number of animals, and the schedule.

As a result of observing the presence or absence of abnormalities in the estrous cycle and / or labor and analyzing them, a non-human animal in which the gene function of ST3GalIV to which the compound was administered is deficient on the chromosome does not administer the compound (or the compound vehicle) The compound is selected as a useful compound for the treatment of reproductive disorders when abnormalities in the sexual cycle and / or parturition are improved as compared to non-human animals in which the gene function of ST3GalIV is chromosomally deficient .
Optionally, the method of screening for compounds useful in the treatment of reproductive disorders in the present invention further comprises administering the compound to a wild-type non-human animal, preferably a mouse, wherein the compound exhibits an effect on the wild-type animal. You may include the process of confirming that there is no. Thereby, the abnormal reproduction is improved only for animals exhibiting abnormal reproduction, and the compound can be selected as a compound useful for better treatment of reproductive disorders that does not affect normal animals.

(8) Method for screening compound useful for treatment of biological rhythm disorder using non-human animal deficient in ST3GalIV gene function on chromosome The present invention relates to a nonhuman animal deficient in ST3GalIV gene function on chromosome. Provided is a method for screening a compound useful for treatment of a biological rhythm disorder used.
For example, the present invention provides a method for screening a compound useful for treatment of a biological rhythm disorder by administering a compound to a non-human animal in which the gene function of ST3GalIV is deficient on the chromosome, and observing the sexual cycle and / or electroencephalogram and / or Alternatively, the rhythm and / or sleep pattern of the sexual cycle is improved as compared with non-human animals in which the gene function of ST3GalIV not administered with the compound is measured and electromyogram measurement and / or behavioral observation is performed. When selected, the compound is selected as a compound useful for the treatment of a biological rhythm disorder. Preferably, the non-human animal is a rodent such as a mouse or a rat.

The observation of the estrous cycle is not particularly limited, and can be performed using, for example, vaginal plaque.
Observation of the presence or absence of abnormalities in labor is not particularly limited, and can be performed, for example, by measuring the number of days of pregnancy and observing the life and death of the fetus through incision.
The measurement and analysis of electroencephalogram and electromyogram are not particularly limited. For example, electrodes can be implanted in appropriate locations in the brain and subcutaneously, and electroencephalogram and electromyogram can be measured using Vital Recorder (KISSEI COMTEC). The brain wave analysis research program SleepSign (KISSEI COMTEC) was used to determine the ratio of each of the delta, theta, alpha, and spindle wavelengths of the electroencephalogram, and the electromyogram size and mouse behavior were recorded. From the video image, it is possible to identify each state of wakefulness, REM sleep, and sleep and analyze the sleep pattern.
Although observation of behavior is not particularly limited, by taking a picture with a photographing means such as a video camera or a digital video camera over the whole day, recording on a recording medium such as a video or a DVD, and analyzing the recorded video by visual observation. ,It can be carried out. For example, it is possible to determine whether or not an animal is awake by visual observation of a recorded video.
The method for screening a therapeutic agent for biological rhythm disorders according to the present invention is compared to a conventional system using an animal in which the sexual cycle is abnormal by treating a normal animal with a drug or the like. Since there are few interventions in the treatment, screening results with less variation and better accuracy and reproducibility can be obtained.

  The screening method for a therapeutic agent for a biological rhythm disorder of the present invention includes a step of administering a compound to a non-human animal in which the gene function of ST3GalIV is deficient on the chromosome. Administration is not particularly limited as long as it is carried out by an appropriate administration method, and administration methods such as oral administration, subcutaneous administration, intraperitoneal administration, and intravenous administration can be adopted. A person skilled in the art can appropriately set conditions such as a compound to be administered, a medium of the compound, a dose, and a method of administration.

  A person skilled in the art is in charge of the experimental schedule such as the observation of the estrous cycle and / or the measurement of the electroencephalogram and / or electromyogram and / or the observation of the behavior and the analysis thereof, the number of trials, the number of animals, the schedule, etc. It can be set appropriately.

As a result of observation of the estrous cycle and / or measurement of electroencephalogram and / or electromyogram and / or observation of behavior and analysis thereof, a non-human animal in which the gene function of ST3GalIV administered with the compound is deficient on the chromosome, When the rhythm and / or sleep pattern of the sexual cycle is improved compared to a non-human animal in which the gene function of ST3GalIV is not administered on the compound (or only the vehicle of the compound) is deleted on the chromosome Selected as a compound useful for the treatment of biological rhythm disorders. The rhythm of the sexual cycle is a concept including the regularity of the sexual cycle. For example, elements constituting the cycle (for example, follicular phase, ovulation phase, luteal phase, menstrual phase, etc. for human women) In the case of a mouse, it means that the estrus first period, the estrus period, the estrus late stage, the estrus rest period, etc.) are repeated periodically in a constant and normal period. Therefore, “the rhythm of the sexual cycle has been improved” includes normalization of the sexual cycle, for example, normalization of the periodicity of the elements constituting the cycle.
In some cases, the method for screening a compound useful for treatment of a biological rhythm disorder in the present invention further comprises administering the compound to a wild-type non-human animal, preferably a mouse, so that the compound has an effect on the wild-type animal. A step of confirming that it is not shown may be included. Thereby, the abnormal biological rhythm can be improved only for an animal exhibiting an abnormal biological rhythm, and the compound can be selected as a compound useful for better biological rhythm disorder treatment that does not affect normal animals.

(9) Method for Measuring In Vivo Drug Efficacy of ST3GalIV Inhibitor The present invention provides a method for measuring the in vivo drug efficacy of ST3GalIV inhibitor.
For example, in the method for measuring the in vivo efficacy of an ST3GalIV inhibitor provided by the present invention, a test substance is administered to a non-human animal and a wild-type non-human animal in which the gene function of ST3GalIV is deficient on the chromosome,
i) open field test ii) fear conditioning test,
iii) EEG and / or electromyogram measurements and / or behavioral observations iv) Body weight and / or body length measurements,
v) measurement of the concentration of growth hormone (Gh) or IGF-1 in the blood, or
vi) Observing abnormalities of the sexual cycle and / or parturition, ST3GalIV gene function is not affected on non-human animals deficient on the chromosome, wild type non-human animals are deficient on ST3GalIV gene function on the chromosome Selecting the test substance as an ST3GalIV inhibitor when presenting the phenotype of the non-human animal. Preferably, the non-human animal is a rodent such as a mouse or a rat.
The in vivo drug effect is a pharmacological action detected in an in vivo test. The in vivo drug effect of ST3GalIV inhibitor includes anxiety-inducing action, learning / memory enhancing action, cognitive function promoting action, memory maintenance enhancing action, REM sleep time. Examples include sleep pattern disorder effects including a decrease effect, body weight and / or body length increase suppression effect, blood growth hormone (Gh) or IGF-1 concentration suppression effect, sexual cycle and / or labor abnormalities, and the like.

  The ST3GalIV inhibitor is a substance that inhibits the enzyme activity of ST3GalIV, and is not particularly limited, and includes low molecular weight compounds, peptides, proteins, DNA, and RNA, and further includes ST3GalIV antisense oligonucleotides or siRNAs.

  Open field test, fear conditioning test, electroencephalogram and electromyogram measurement, behavioral observation, body weight and length measurement, blood growth hormone (Gh) or IGF-1 concentration measurement, sexual cycle and / or parturition The observation of the abnormality can be appropriately performed by those skilled in the art as described in the above (2) to (8).

The method for measuring the in vivo efficacy of the ST3GalIV inhibitor of the present invention comprises the step of administering a test substance to a non-human animal and a wild-type non-human animal in which the gene function of ST3GalIV is deficient on the chromosome. The method of administering the test substance can be appropriately set by those skilled in the art, and is not particularly limited, but administration methods such as oral administration, subcutaneous administration, intraperitoneal administration, and intravenous administration can be adopted. A person skilled in the art can appropriately set conditions such as a substance to be administered, a medium of a test substance, and a dosage.
The test substance is a substance that may inhibit the enzyme activity of ST3GalIV, and is not particularly limited, and includes low molecular weight compounds, peptides, proteins, DNA, RNA, and further, ST3GalIV antisense oligonucleotides, or siRNA including.
The antisense oligonucleotide and siRNA of ST3GalIV may be administered by being incorporated into an appropriate vector, for example, an adenovirus vector so that it can be expressed in nerve cells.

Open field test, fear conditioning test, or electroencephalogram and / or electromyogram measurement, behavioral observation, body weight and length measurement, blood growth hormone (Gh) or IGF-1 concentration measurement, and / or As a result of observation of abnormalities in the estrous cycle and / or parturition, non-human animals in which ST3GalIV gene function is deficient on the chromosome have no effect, and wild-type nonhuman animals have non-human genes in which ST3GalIV gene function is deficient on the chromosome When presenting the animal phenotype, the test substance is selected as an ST3GalIV inhibitor.
Here, the phenotype includes a trait that is recognized in behavioral pharmacology, and a phenotype of a non-human animal in which the gene function of ST3GalIV is deficient on the chromosome indicates that the gene function of ST3GalIV is on the chromosome. As with non-human animals deficient in the above, anxiety-inducing action, learning / memory-enhancing action, cognitive function promoting action, memory maintenance-enhancing action, sleep pattern disorder action including REM sleep time-decreasing action, body weight and / or body length increase suppression It expresses an action, a blood growth hormone (Gh) or IGF-1 concentration inhibitory action, an abnormal sexual cycle and / or parturition.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited by these.

ターゲッティング配列を有するES細胞を、標準的な手法により、C57BL/6Jblastcysts（MS12）に注入した。キメラをC57BL/6Jマウスと交配させた。ターゲットした遺伝子座を伝達する生殖系列を有する子孫を内部繁殖させた（targetedマウス）。そして、targetedマウスから得た精子をC57 Bl/6Jマウスの卵にインビトロで受精させ、ＣＡＧ−プロモーター−Ｃｒｅ−ポリプラスミドを、受精卵に注入し、そして２細胞期を子宮に移植した。ＳＴ３ＧａｌＩＶ遺伝子座を欠損するマウスを、また、C57BL/6Jマウスと交配させた。さらに、プロモーター−frep-ポリＡ注入プラスミドを受精卵に注入し、そして、freptaseがターゲット遺伝子座において、floxで挟まれたneo遺伝子を取り除くように、２細胞期を子宮に移植した。組織特異的ＳＴ３ＧａｌＩＶ欠損マウスが、Ｃｒｅ−ｌｏｘｐ系により得られた。 Example 1: Construction of knockout mice deficient in ST3GalIV throughout the body (I) Construction of targeting vector To create ST3GalIV deficient mice, genomic fragments were isolated from the C57BL / 6J library (BAC library, BAC Resources PAC). did. As shown in FIG. 1, a targeting vector was constructed.
The 10.5 kb FspI-SnaBI fragment was first isolated and ligated into pBluscript SK + (A32). Then, the SpeI-XhoI fragment was isolated and ligated to pBluscript SK + (C12). C12 was modified so that a HindIII site was inserted into the intron region between exons 2 and 3 and an EcoRV site was inserted into the intron region between exons 3 and 4 (DC3). A loxp (39mer primer pair) was inserted into the HindIII site of the DC3 plasmid, and a 2.0 kb Not I-Xho I fragment (loxP-frt-pgkneo) having an SsPI site isolated from the loxp-frp-pgkneo plasmid was added to the DC3 plasmid. Was inserted into the EcoRV site (DC311_F7). The 5.0 kb Bcl I-Xho I fragment in the DC311_F7 plasmid was inserted into the Bcl I-Xho I site of the A32 plasmid (GD6). Finally, a 1.2 kb Not I fragment of pMC1DTpA (Transgenic Res 1999, 8: 215-21, Yanagawa et al) was inserted into the NotI site of the GD6 plasmid (HH5; 13.6 kb fragment).
(II) Creation of ST3GalIV-/-mice
HH5 targeting vector was introduced into MS12ES cells (derived from C57BL / 6J) by electroporation. G418 resistant ES clones were screened from accurate ST3GalIV locus targeting by Southern blotting using three probes. Probes were 235bp fragment (3 'side), 220bp fragment (inside), 299bp fragment (5' side) by PCR, Df77 / Dr45 (probe N), Df90 / Dr91 (probe R), and Df71 / Dr72, respectively. Amplification was performed using a (probe F) primer pair (forward primer / reverse primer).
Df77: 5'-GTTAGTTAGCTGTTGCCCGGTT-3 '(SEQ ID NO: 1)
Dr45: 5'-CTTGTTGGAGGCATCTGGATAG-3 '(SEQ ID NO: 2)
Df90: 5'-GGAGACAGCCATGCAGGAGAAG-3 '(SEQ ID NO: 3)
Dr91: 5'-AACTTGAATTCCTCCTCCTTCT-3 '(SEQ ID NO: 4)
Df71: 5'-AGAGTACACTGTAACTGGTCTG-3 '(SEQ ID NO: 5)
Dr72: 5'-ACTCAGTGCCAGCTCTGTAGAT-3 '(SEQ ID NO: 6)

ES cells with targeting sequences were injected into C57BL / 6Jblastcysts (MS12) by standard techniques. Chimeras were bred with C57BL / 6J mice. Offspring with germline carrying the targeted locus were bred internally (targeted mice). Sperm from targeted mice were then fertilized in vitro in C57 Bl / 6J mouse eggs, CAG-promoter-Cre-polyplasmid was injected into fertilized eggs, and the 2-cell stage was transplanted into the uterus. Mice lacking the ST3GalIV locus were also bred with C57BL / 6J mice. Furthermore, the promoter-frep-poly A injection plasmid was injected into fertilized eggs, and the 2-cell stage was transplanted into the uterus so that freptase removes the neo gene sandwiched between flox at the target locus. Tissue specific ST3GalIV deficient mice were obtained with the Cre-loxp system.

Example 2: Histochemical analysis of ST3GalIV knockout mice 13-15-week-old ST3GalIV homo-deficient, hetero-deficient, and wild-type mice were perfused and fixed, followed by paraffin embedding in the brain to prepare coronal sections. Hematoxylin-eosin staining, anti-GAFP antibody staining, and TUNEL staining were performed.
As a result, growth of astrocytes in the hippocampal dentate gyrus, neuronal atrophy in the cerebral cortical zonal gyrus, and abnormal cerebellar layer structure were observed (FIG. 3).

Example 3: Behavioral analysis of ST3GalIV knockout mice (I) Open field test
Using AUTOMEXII (manufactured by Tokai Medical Science Co., Ltd.), trials were performed on 8-9 week old ST3GalIV homo-deficient, hetero-deficient, and wild-type mice. The AUTOMEX floor (45cmX55cm) was divided into 12 squares (A to L squares) as shown in FIG. The mouse was placed at the position shown in FIG. 4, and the position where the mouse stayed and the number of staying seconds were recorded during the observation for 5 minutes. The state where the mouse body (head side) entered the square was recorded as the staying position. After observing for 5 minutes, they went out and rested for 5 minutes. The trial of the above 5 minute observation → 5 minute break was performed 3 times (FIG. 4). The observation results for the third time for 5 minutes are shown in FIG. In this trial, mice not accustomed to open fields were used. The recorded seconds are divided into two groups of four corners (A, D, I, L) and the other (B, C, E, F, G, H, J, K), and the average number of seconds (four corners: (A + D + I + L) / 4X12; otherwise: (B + C + E + F + G + H + J + K) / 8X12) was calculated. Statistical analysis was conducted by one-way ANOVA followed by Turkey's multiple comparison test.
As a result, compared to the wild type, the homozygous and heterozygous animals have a greater difference in the number of staying seconds at the four corners and the other, and there is a strong tendency to move to the four corners (FIG. 5), and ST3GalIV knockout mice It was suggested that anxiety tends to be provoked.
This demonstrates that ST3GalIV knockout mice are useful as model animals for anxiety disorders, and that the in vivo efficacy of ST3GalIV inhibitors can be evaluated by an open field test using ST3GalIV knockout mice and wild type mice. Indicated.
Moreover, it was suggested that the open field test using ST3GalIV knockout mice can be used for a screening method for compounds useful for anxiety disorders, preferably a screening method for compounds useful for anxiety disorders caused by ST3GalIV deficiency.

(II) Fear conditioning test 8-9 weeks old ST3GalIV homo-deficient or wild type mice were placed in a chamber with an electric grid, and after 60 seconds, 120 dB, 10 seconds of sound were presented, and immediately after that, 0 .5V, 1 second electrical stimulation was applied. Furthermore, after 20 seconds (that is, 90 seconds after being placed in the cage), a sound of 120 dB and 10 seconds was presented again, and immediately after that, 0.5 V, 1 second of electrical stimulation was applied. Two days after conditioning, mice were placed in cages, 120 dB, 10 seconds sound was exhibited after 60 seconds and 90 seconds, and the percentage of mice that exhibited freezing in 180 seconds after being placed in the cage was measured. Freezing is defined as a state in which skeletal muscles and whiskers do not move except for movement related to breathing. Statistical analysis was performed by two-way factorial ANOVA.
As a result, it was recognized that freezing was significantly increased in the homozygote compared with the wild type (FIG. 6), and ST3GalIV knockout mice were more anxious than the wild type. Thus, it was shown that the in vivo efficacy of the ST3GalIV inhibitor can be evaluated by a fear conditioning test using ST3GalIV knockout mice and wild type mice. In addition, ST3GalIV knockout mice showed an increase in freezing in a fear conditioning test conditioned by sound and no increase in freezing in a fear conditioning test conditioned by contextual memory. It was shown to be involved in emotional memory.
In addition, SSRI, which is effective in the treatment of anxiety disorders and depression, including generalized anxiety disorder, panic disorder, PTSD, obsessive compulsive disorder and social anxiety disorder in clinical practice, significantly improves freezing in the fear conditioning test similar to this study. ST3GalIV knockout mice are useful because suppression and fear conditioning tests are useful as evaluation systems for therapeutic agents for anxiety disorders (see Japanese Journal of Psychosomatic Medicine Vol. 37, No. 3, pp. 223-229, etc.) It was shown to be useful as a model animal for anxiety disorders including generalized anxiety disorder, panic disorder, PTSD, obsessive-compulsive disorder and social anxiety disorder. Furthermore, a fear conditioning test using ST3GalIV knockout mice is a screening method for compounds useful for the treatment of anxiety disorders including generalized anxiety disorder, panic disorder, PTSD, obsessive compulsive disorder and social anxiety disorder, preferably ST3GalIV deficiency It has been shown that the present invention can be used in a screening method for compounds useful for the treatment of anxiety disorders including generalized anxiety disorder, panic disorder, PTSD, obsessive compulsive disorder, social anxiety disorder and the like.

(III) Electroencephalogram analysis 1) Preparation of electroencephalogram measurement electrode Prepare two stainless steel wires (COONER WIRE Inc) cut into 15 mm for electroencephalogram electrodes, and peel off the coating on one end of the stainless steel wire 0.5-1 mm. After applying phosphoric acid, it was left for 10 minutes. Subsequently, solder was applied to the stainless steel wire (hereinafter, preliminary solder), and then phosphoric acid was applied again and left for 10 minutes. An electroencephalogram screw electrode (hereinafter referred to as “bis”) (manufactured by Biotex Kyoto) was also allowed to stand for 10 minutes after applying phosphoric acid. After soldering the stainless steel wire and screw coated with phosphoric acid, peel off the coating on the tip of the opposite stainless steel wire that was soldered, leave it for 10 minutes after applying phosphoric acid, perform preliminary soldering, apply phosphoric acid again and apply 10 Left for a minute. At the same time, pre-soldering was also performed on the mouse connector (Biotex Kyoto), and the stainless wire and the connector were soldered. Subsequently, two stainless steel wires cut to 20 mm were prepared for electromyogram measurement, and stainless steel wires were prepared in the same manner as for the electroencephalogram electrodes, and soldered to the connector. After all the soldering is completed, a two-component mixed epoxy resin is mixed, drawn with a vacuum pump to eliminate bubbles in the resin, and then solidified so as to hide the soldered portion, and dried and fixed at room temperature for one day. .

2) Surgery to install electrodes for electroencephalogram measurement After anesthetizing the mouse with an influrane anesthesia machine (medical agent) for small animals, use a dental drill (manufactured by MINITOR), 1mm from bregma, 1.5mm right side and lambda 1mm forward and 1.5mm on the right, two holes were drilled in the skull, and the screw was embedded in one and a half turns. Thereafter, two stainless steel wires for electromyogram measurement were inserted as far as possible from side to side in the subcutaneous part of the back of the neck. Thereafter, the connector was temporarily fixed to the skull with Aron Alpha (registered trademark) and completely fixed with dental cement (manufactured by GC Corporation). One week after the operation, the electroencephalogram and electromyogram were measured for 24 hours using a Vital Recorder (manufactured by KISSEI COMTEC).

3) Analysis using electroencephalogram analysis software The measured electroencephalogram was analyzed using an electroencephalogram analysis research program SleepSign (KISSEI COMTEC). SleepSign classifies brain waves into 4 patterns: δ waves of 0.5-4Hz, θ waves of 4-9Hz, α waves of 9-13Hz, spindles of 13Hz or higher, ratio of each wavelength, electromyogram size and mouse From the video images recording the behaviors of KO mice and W (wake state), R (REM sleep state), and S (sleep state), three patterns were analyzed, and the tendency of KO mice and wild type sleep was analyzed.
As a result, ST3GalIV knockout mice have disrupted sleep and have less long-time sleep than the wild type, and the waveform of the θ wave that is characteristic of REM sleep is not seen. It was confirmed that there were few compared (FIGS. 7 and 9). Moreover, it was confirmed that the situation of falling from awake state to a sleep state for a short time and returning to the awake state again, and the time and periodicity of awakening, REM sleep, and non-REM sleep are more disturbed than the wild type. Therefore, it was recognized that ST3GalIV knockout mice exhibit sleep disorders.
This demonstrates that ST3GalIV knockout mice are useful as model animals for sleep disorders including REM sleep disorders, and that the in vivo efficacy of ST3GalIV inhibitors is the analysis of sleep patterns in ST3GalIV knockout mice and wild-type mice. It was shown that it can be evaluated.
In addition, analysis of sleep patterns of ST3GalIV knockout mice is useful for screening methods for sleep-improving agents, for example, REM sleep-improving agents, and preferably for screening methods for improving sleep disorders caused by ST3GalIV-deficiency, for example, REM sleep-disorders It was shown that.

Example 4: Measurement of length and body weight of ST3GalIV knockout mice DNA was extracted from the tail of 7-day-old mice, and homo, hetero or wild type genotypes were confirmed using PCR. At 9 days of age, the number of litters fed with litters was unified to 6 and the body weight and body length were measured once every 2 days from 9 days to 10 weeks of age to create a growth curve. As a result, both male and female homozygous ST3GalIV knockout mice showed an effect of suppressing body weight and length as compared to wild type and heterozygous mice, and particularly significant suppression of body weight and length was observed at 25-57 days of age. (FIGS. 10 and 11). Statistical processing was performed by two-way factorial ANOVA, and p <0.0001 was considered significant.

Example 5: Measurement of Gh and IGF-1 concentrations in plasma of ST3GalIV knockout mice Male homozygous ST3GalIV knockout mice and male wild type mice (C57BL / 6J mice) with marked growth inhibition at 3, 4, 6 and 8 weeks of age Blood was collected from the orbital vein and centrifuged at 2500 rpm for 10 minutes. The concentrations of growth hormone (Gh) (SPI-BIO A05104-96 wells) and insulin-like growth factor (IGF-1) in the obtained plasma were measured using ELISA (R & D systems Quantikine No. MG100). As a result, the homo type showed a tendency to decrease the Gh secretion amount compared with the wild type, and the IGF-1 secretion amount significantly decreased (FIG. 12). Statistical processing was performed by two-way factorial ANOVA, and p <0.0001 was considered significant.

Example 6: Examination of fertility of ST3GalIV knockout mice (I) Examination of fertility of male ST3GalIV knockout mice 12-week-old male homozygous ST3GalIV knockout mice and wild-type mice were treated with 10-week-old female wild-type mice (C57BL). / 6j) and each. As a result, normal birth was confirmed both in the case of using male homozygous ST3GalIV knockout mice and male wild-type mice. Therefore, no effect on fertility was observed in males due to STSGalIV deficiency.

(II) Examination of reproductive ability of female ST3GalIV knockout mice Reproductive ability of female ST3GalIV knockout mice 1) Observation of sexual cycle by vaginal plaque examination 2) Confirmation of mating by confirmation of vaginal plug 3) Confirmation of abdominal dilation It was examined by confirmation of conception and 4) confirmation of labor or fetal death.
1) Observation of sexual cycle by vaginal plaque test A 26-33 week-old female homozygous ST3GalIV knockout mouse and a wild type mouse were subjected to a vaginal plaque test for one month. In the vaginal plaque test, the inside of the vagina was smeared with filter paper according to a conventional method, and the obtained vaginal reservoir was observed with a microscope to determine the early estrus, estrus, late estrus, or estrus rest period. And the number of days was measured from the late estrus to the next late estrus.
As a result, compared to wild-type mice, homozygous ST3GalIV knockout mice have irregular sexual cycles composed of the early estrus, estrus, late estrus, and estrus rest periods (FIG. 13). . In addition, the number of days from the late stage of estrus to the next late stage of estrus in the homozygous ST3GalIV knockout mouse was significantly smaller than that in the wild type mouse (FIG. 13). In addition, statistical processing used Mann-Whitney U-test, and was considered significant at p <0.05. Therefore, abnormalities of the sexual cycle were observed in homozygous ST3GalIV knockout mice.
2) Confirmation of mating by confirming vaginal plugs The sexual cycle was judged by vaginal plaque examination, and a female found to be in the early estrus coexisted with a wild-type male at 7 pm and mating was confirmed at 7 am the next morning by the presence or absence of vaginal plug . As a result, in both 8-12 week old mice and 31-38 week old mice, homozygous ST3GalIV knockout mice tended to have higher mating probabilities compared to wild type mice. There wasn't. Therefore, in homozygous ST3GalIV knockout mice, mating was normal as in wild type mice.

3) Confirmation of conception by confirming abdominal expansion Conception was confirmed by abdominal expansion of a mouse with a confirmed vaginal plug. As a result, both the 8-12 week old mouse and the 31-38 week old mouse were found to have a lower conception probability in homozygous ST3GalIV knockout mice than in the wild type mice. There was no (Figure 14). Thus, in homozygous ST3GalIV knockout mice, conception was normal as in wild type mice.
4) Confirmation of childbirth or fetal death Confirmation of the birth of a confirmed mouse or a mouse that could not confirm the birth, incised the abdomen, and confirmed the life and death of the fetus and the state of the uterus (Fig. 15). As a result, three wild-type mice confirmed to be pregnant delivered as normal after a gestation period of 18-20 days. On the other hand, although 5 homozygous ST3GalIV knockout mice became pregnant, there were cases in which they did not finally deliver but died in the womb. In particular, in the elderly (31-38 weeks old) homozygous ST3GalIV knockout mice, the gestation period was prolonged, and one of the elderly homozygous ST3GalIV knockout mice was 3 on the 21st day of pregnancy and on the 22nd day of pregnancy. One animal was born dead on the 23rd day of pregnancy, 2 animals on the 25th day of pregnancy, and 2 animals were born on the 25th day of pregnancy. When dissected on the 27th day of pregnancy, 2 fetuses died in the uterus. In addition, one of the elderly homozygous ST3GalIV knockout mice gave birth to 4 mice on the 21st day of pregnancy (survived, but 1 mouse died the next day), and 2 dead animals on the 22nd day of pregnancy (with a placenta) Was delivered). One of the causes of such a prolonged pregnancy period was considered to be an abnormality in labor, for example, an abnormality in labor rhythm as a biological rhythm. The results are listed in Table 1.

Fig. 2 shows a targeting vector constructed for the production of ST3GalIV knockout mice of the present invention. To generate ST3GalIV knockout mice, genomic fragments were isolated from the C57BL / 6J library (the BAC library in pBACe3.6). A targeting vector was constructed as represented in FIG. The linearized targeting vector was introduced into MS12 embryonic stem cells by electroporation to obtain G418-resistant ES cells. Chimeric mice were prepared from ES cells (Targeted) into which a targeting vector was introduced. The CAG-promoter-Cre plasmid was introduced into fertilized eggs derived from mouse sperm into which a targeting vector had been introduced to obtain ST3GalIV-deficient mice. The result of Southern blot hybridization is shown. w / w is a wild type, w / targeted is a mouse having a targeting vector heterozygously, w / floxed is a mouse having a gene excluding the neo gene from the targeting vector heterozygous, w /-is a heterozygous ST3GalIV-deficient mouse, /-Represents a homozygous ST3GalIV-deficient mouse. The results of histochemical examination of the cingulate and hippocampus are shown. In the fusiform layer of the zonal gyrus, there are many atrophied cells instead of granule cells (arrows). In the dentate gyrus polymorphic cell layer of ST3GalIV-deficient mice (+/− and − / −), proliferation of GFAP positive astrocytes was observed. Shows the open field test equipment and test schedule. The result of the open field test of ST3GalIV knockout mouse is shown. ST3GalIV-deficient mice (+/− and − / −) showed a tendency to move to the four corners compared to the wild type (+ / +), and showed a strong sensitivity to stress. The result of the fear conditioning test of ST3GalIV knockout mice is shown. The sleep pattern obtained by the electroencephalogram analysis etc. of ST3GalIV knockout mouse | mouth is shown. Each column represents sleep (light brown (brown indicated by $)), REM sleep (dark brown (brown indicated by #)), and arousal (black) from the top. ST3GalIV knockout mice were shown to have shorter REM sleep (#) times than wild type mice. An example of the electromyogram analysis of ST3GalIV knockout mouse is shown. R represents a REM sleep state, W represents an awake state, and S represents a sleep state. The comparison of the REM sleep time of ST3GalIV knockout mouse | mouth and a wild type mouse | mouth is shown. The changes in body weight and length of female homozygous ST3GalIV knockout mice, female heterozygous ST3GalIV knockout mice, and female wild type mice are shown. The changes in body weight and length of male homozygous ST3GalIV knockout mice, male heterozygous ST3GalIV knockout mice, and male wild type mice are shown. A comparison of plasma Gh and IGF-1 concentrations in ST3GalIV knockout mice and wild type mice is shown. The number of days from the late stage of estrus to the next late stage of estrus of wild type mice and female homozygous ST3GalIV knockout mice are shown (left figure). Early estrus ("front" in the figure), estrus ("middle" in the figure), late estrus ("after" in the figure), estrus rest period ("rest" in the figure) of wild-type and female homozygous ST3GalIV knockout mice ) Is shown respectively (right figure). The fertility probabilities of 8-12 week old wild type mice and female homozygous ST3GalIV knockout mice are shown (left figure). The conception probabilities of 31-38 week-old wild type mice and female homozygous ST3GalIV knockout mice are shown (right diagram). A deadborn baby (left photo) and uterus (right photo) taken from the abdomen of a female homozygous ST3GalIV knockout mouse, and a uterus (middle photo) of a wild type mouse are shown.

Claims (14)

  A non-human animal in which the gene function of ST3GalIV is deficient on the chromosome.   2. The non-human animal according to claim 1, which is an anxiety disorder model animal, a sleep disorder model animal, a growth disorder model animal, a reproductive disorder model animal, or a biological rhythm disorder model animal.   A method for screening a compound useful for the treatment of an anxiety disorder, comprising administering the compound to a non-human animal according to claim 1, performing an open field test, and not administering the compound. Selecting the compound as a useful compound for the treatment of anxiety disorders when the residence time in the four corners is reduced compared to the non-human animal.   A method for screening a compound useful for the treatment of anxiety disorder, comprising administering the compound to a non-human animal according to claim 1, performing a fear conditioning test, and not administering the compound. Selecting the compound as a compound useful in the treatment of anxiety disorders when freezing is reduced compared to a non-human animal.   A method for screening a compound useful for the treatment of an anxiety disorder, wherein the method according to claim 4 and 5 is carried out, and the compound selected as a compound useful for the treatment of an anxiety disorder by both methods is used for anxiety. Selecting a compound useful in the treatment of the disorder.   A method for screening a compound useful for the treatment of sleep disorders, comprising administering a compound to the non-human animal according to claim 1, measuring an electroencephalogram and / or an electromyogram and / or observing a behavior, 2. A method comprising selecting the compound as a compound useful for the treatment of sleep disorders when sleep pattern is improved as compared to the non-human animal of claim 1 wherein the compound is not administered.   A method for screening a compound useful for treatment of a disorder of REM sleep, wherein the compound is administered to a non-human animal according to claim 1, and an electroencephalogram and / or electromyogram is measured and / or behavior is observed. And when the REM sleep time is increased compared to the non-human animal according to claim 1 wherein the compound is not administered, the compound is selected as a compound useful for the treatment of REM sleep disorder. Including methods.   A method for screening a compound useful for the treatment of growth disorders, comprising administering a compound to the non-human animal according to claim 1, measuring body weight and / or length, and / or growth hormone (Gh) in blood. And / or when the concentration of IGF-1 is measured to increase body weight and / or body length and / or in blood compared to the non-human animal of claim 1 not administered the compound A method comprising selecting a compound as a useful compound for the treatment of growth disorders when the concentration of growth hormone (Gh) and / or IGF-1 is increased.   A method for screening a compound useful for the treatment of reproductive disorders, comprising administering the compound to the non-human animal according to claim 1 and observing whether there is an abnormality in the sexual cycle and / or labor, and 2. A method comprising selecting the compound as a useful compound for the treatment of a reproductive disorder when the sexual cycle and / or delivery abnormalities are improved compared to the non-human animal of claim 1 not administered .   A method for screening a compound useful for treatment of a biological rhythm disorder, comprising administering the compound to a non-human animal according to claim 1, observing the sexual cycle, measuring an electroencephalogram and / or electromyogram, and / or acting And wherein the compound is regenerated when the rhythm of the sexual cycle is improved and / or sleep pattern is improved as compared to the non-human animal of claim 1 wherein the compound is not administered. Selecting a compound useful in the treatment of the disorder.   The method according to any one of claims 3 to 10, further comprising the step of administering the compound to a wild-type non-human animal to confirm that the compound has no effect on the wild-type animal. A method for measuring the in vivo efficacy of an ST3GalIV inhibitor, comprising administering a test substance to the non-human animal and the wild-type non-human animal according to claim 1,
i) Open field test ii) Fear conditioning test iii) EEG and / or electromyogram measurement and / or behavioral observation iv) Weight and / or body length measurement,
v) measurement of the concentration of growth hormone (Gh) or IGF-1 in the blood, or
vi) Observing abnormalities in the sexual cycle and / or parturition, the non-human animal according to claim 1 has no effect, and the wild-type non-human animal presents the phenotype of the non-human animal according to claim 1 A method comprising selecting the test substance as an ST3GalIV inhibitor.   The non-human animal according to claim 1 or 2, wherein the non-human animal is a mouse.   The method according to claim 3, wherein the non-human animal is a mouse.

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