Application of exosc9 gene mutant animals in construction of anxiety model and screening of anti-anxiety drugs

Abstract

The application belongs to the technical field of biological medicine, and specifically discloses a kind of Exosc9 The application discloses application of a gene mutation animal in construction of an anxiety model and in screening of an anti-anxiety drug. Exosc9 The gene mutation animal is a known model, but the application finds that the animal shows behavior phenotypes related to anxiety through systematic behavior experiments, including a decrease in central zone activity in an open field experiment, a decrease in open arm staying time in a high cross maze experiment, and a decrease in exploration behavior. Through behavior observation and quantitative analysis of the animal, the application can be used to study behavior characteristics of anxiety, compare phenotype differences of animals of different genotypes, genders and ages, and analyze influences of genetic and environmental factors on anxiety behavior. The application also constructs a method for screening of an anti-anxiety candidate drug and evaluation of drug efficacy based on the model, which can greatly shorten an early screening period of drug research and development, and has a wide application prospect in the field of neuropsychiatric disease research and drug research and development.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more particularly to a... Exosc9 Application of genetically mutant animals in constructing anxiety models and screening anti-anxiety drugs. Background Technology

[0002] Anxiety disorder is the most common mental disorder worldwide, with main clinical symptoms including anxiety, worry, fear, dread, tension, and autonomic nervous system dysfunction. In recent years, the global prevalence of anxiety disorder has been on the rise, and its prevalence is positively correlated with the level of social development, severely impairing individual physical and mental health and social functioning. However, in clinical treatment, psychotherapy has limited effectiveness, and drug treatment suffers from slow onset of action, drug tolerance, and addiction. Therefore, in-depth research into the pathogenesis of anxiety disorder is of significant practical importance.

[0003] Exosc9 It is the core subunit of the RNA exonuclease complex (EXOSC1-EXOSC9), which is a highly conserved 3'-5' ribonuclease in eukaryotes. In the nucleus and cytoplasm, it participates in RNA processing, quality control and turnover by binding catalytic subunits and cofactors, maintaining the stability of RNA metabolism and the fine regulation of gene expression.

[0004] Exosc9 With over 90% homology in humans, mammals, mice, and zebrafish, studies have shown that gene mutations in this species lead to abnormal development of midbrain neurons, structural defects in the hindbrain and cerebellum, developmental defects of neural crest cells, and developmental and migration disorders of motor neurons. It is also accompanied by a neurological syndrome caused by the destruction of RNA exosomes, resulting in cerebellar atrophy and spinal motor neuron lesions.

[0005] Currently on Exosc9 Gene research has focused on epigenetics and neuronal changes, and currently there is very little... Exosc9 Research on the influence of genes on anxiety-like behaviors. Summary of the Invention

[0006] The purpose of this invention is to address the aforementioned shortcomings of the prior art by providing a [specific solution / method]. Exosc9 The application of genetically mutant animals in constructing anxiety models and screening anti-anxiety drugs has been clarified for the first time. Exosc9 The key application value of gene knockout animal models in the study of anxiety-related mental disorders provides new technical tools and research ideas for the construction of models, the exploration of pathogenesis, and the development of treatment plans for such diseases.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: The first aspect of the present invention is to provide Exosc9The application of genetically modified animals in constructing anxiety models or screening anti-anxiety drugs, the Exosc9 The genetically mutated animals are rodents or zebrafish.

[0008] Furthermore, the aforementioned Exosc9 Gene mutations include Exosc9 Gene knockout or Exosc9 Point mutations in genes.

[0009] Furthermore, the rodent is a mouse.

[0010] Furthermore, the anxiety disorder is selected from generalized anxiety disorder, social anxiety disorder, panic disorder, acute stress disorder, and stress-related pathological avoidance behavior.

[0011] A second aspect of the present invention provides a method for screening anti-anxiety drugs, comprising the following steps: Provided Exosc9 Animals with genetic mutations; To the Exosc9 Gene mutations can be used to apply candidate drugs for testing. The evaluation of the Exosc9 Behavioral and physiological indicators related to anxiety symptoms caused by gene mutations; If the candidate drug can improve the behavioral indicators related to the anxiety symptoms, it is identified as an anti-anxiety candidate drug.

[0012] Furthermore, the behavioral indicators include open field experiments, light and dark box experiments, diving experiments, or elevated cross maze experiments, and the physiological indicators include head length and body length measurements.

[0013] A third aspect of the present invention is to provide a method for evaluating the efficacy of candidate anti-anxiety drugs, utilizing the aforementioned Exosc9 Using non-human mammals with gene mutations as test subjects, the study observed the degree of improvement in anxiety-like behavioral phenotypes caused by drugs.

[0014] Furthermore, the aforementioned Exosc9 Gene mutations include Exosc9 Gene knockout or Exosc9 Point mutations in genes.

[0015] Furthermore, the aforementioned Exosc9 Gene mutations lead to mice.

[0016] Furthermore, the anxiety disorder is selected from generalized anxiety disorder, social anxiety disorder, panic disorder, acute stress disorder, and stress-related pathological avoidance behavior.

[0017] Compared with the prior art, the beneficial effects of the present invention are: (1) This invention is the first to demonstrate that Exosc9 There is a significant causal relationship between gene mutations and the pathological phenotype of anxiety disorders. This has been demonstrated through behavioral experiments. Exosc9 Gene deletions or mutations can lead to stable anxiety-like behaviors in non-human mammals, providing new genetic evidence for the pathogenesis of anxiety disorders.

[0018] (2) Compared with traditional anxiety models constructed using drug-induced or environmental stressors (such as foot shocks or chronic unpredictable stress), the anxiety model described in this invention... Exosc9 Gene mutation models exhibit better phenotypic stability and individual consistency. This model is unaffected by external manipulation and can stably simulate the pathological state of anxiety disorders over the long term, reducing experimental error.

[0019] (3) This invention has explored known Exosc9 New uses for genetically mutant animals, due to Exosc9 Gene mutations directly point to specific molecular deletions, making this model highly effective for screening targeted drugs. This not only saves the cost of developing entirely new gene-mutant animals but also provides an ideal, multifunctional research platform for studying the comorbidity mechanisms of anxiety disorders and other complications, significantly shortening the early screening cycle in drug development.

[0020] (4) The behavioral data on anxiety disorder provided by this invention can be used to study the effects of genetic factors, environmental stress, and their interactions on the phenotype of anxiety disorder, providing an important reference for research on the pathogenesis and neurobehavioral basis of anxiety disorder. The behavioral assessment system and animal phenotypic data established by this invention can provide basic support for the experimental design, dosage selection, and animal model validation of subsequent anti-anxiety drugs, and have important scientific and applied value. Attached Figure Description

[0021] Figure 1 In Example 1 Exosc9 A schematic diagram illustrating the construction method of a gene knockout animal model and the knockout validation results. In the figure, A represents... exosc9 Methods for constructing gene knockout zebrafish models; B is... exosc9 Gene sequencing results of homozygous knockout zebrafish; C represents Exosc9 Methods for constructing gene knockout mouse models; DE is a Western blot detection method. Exosc9 EXOSC9 protein expression in gene knockout mice was measured, with Tubulin as an internal control. Figure D shows representative bands, and Figure E shows the results of grayscale quantitative analysis. Experimental data are expressed as mean ± standard error (Mean ± SEM). Results were analyzed using one-way ANOVA. p<0.01, p<0.001, p<0.0001, ns p>0.05 indicates a significant difference; Figure 2 In Embodiment 2 of the present invention, exosc9 The morphological changes of mutant zebrafish and the movement speed of juveniles under different conditions were shown in the figures. Figure A shows the body length of juveniles (10 days post-fertilization); B shows the head width of juveniles; C shows the top-down measurement of juvenile zebrafish under a microscope after imaging; D shows the body length of adult zebrafish (90 days post-fertilization); E shows the brain size of adult zebrafish (90 days post-fertilization); F shows a schematic diagram of adult body length measurement; G shows a schematic diagram of adult brain size measurement; H shows the average speed of juvenile zebrafish collected under light conditions for 10 minutes; I shows the average speed of juvenile zebrafish per unit time under light conditions; J shows the average speed of juvenile zebrafish collected under light conditions for 10 minutes; K shows the average speed of juvenile zebrafish per unit time under light conditions; L shows the speed change of zebrafish per unit time with light stimulation; M shows the average speed of zebrafish under each light stimulation. Experimental data are expressed as mean ± standard error (Mean ± SEM), and the results were analyzed using one-way ANOVA. p<0.05, p<0.01, p<0.001, p<0.0001, ns p>0.05 indicates a significant difference; Figure 3 In embodiment 3 of the present invention, exosc9 The movement time and frequency of mutant 7dpf zebrafish juveniles under different experimental conditions are shown in the figure. A represents the distribution of zebrafish juveniles in the open field experimental area; B represents the exploration time of zebrafish in the central area; C represents the exploration time of zebrafish in the peripheral area; D represents the activity level of zebrafish; E represents the distribution of zebrafish juveniles in the light and dark chamber experimental area; F represents the exploration time of zebrafish in the illuminated area; G represents the exploration time of zebrafish in the dark area; and H represents the number of times zebrafish traverse between the light and dark areas. Experimental data are expressed as mean ± standard error (Mean ± SEM). The results were analyzed using one-way ANOVA. p<0.01, p<0.001, p < 0.0001 indicates a significant difference; Figure 4 In embodiment 4 of the present invention, exosc9The movement time and frequency results of adult zebrafish with mutant 3mpf under different experimental conditions are shown in the figure. A is the distribution map of the open field experimental area of ​​adult zebrafish; B is the statistical chart of the exploration time of zebrafish in the central area; C is the statistical chart of the exploration time of zebrafish in the peripheral area; D is the distribution map of the diving experimental area of ​​adult zebrafish in the new aquarium; E is the statistical chart of the exploration time of zebrafish in the top, middle and bottom areas; F is the statistical chart of the latency period of zebrafish entering the top and middle areas for the first time; G is the distribution map of the light and dark box experimental area of ​​adult zebrafish. H represents the exploration time of zebrafish in the illuminated area; I represents the number of times zebrafish traversed between the light and dark areas; J represents the distribution of adult zebrafish in the mirror attack experiment area; K represents the dwell time of zebrafish in the attack area; L represents the number of times zebrafish traversed the attack area; M represents the distribution of adult zebrafish in the three-box social experiment area; N represents the dwell time of zebrafish in the social area; O represents the number of times zebrafish traversed the social area. Experimental data are expressed as mean ± standard error (Mean ± SEM). Results were analyzed using one-way ANOVA. p<0.05, p<0.01, p<0.0001, ns p>0.05 indicates a significant difference; Figure 5 In embodiment 5 of the present invention, Exosc9 The results of the movement time and number of entries of mutant mice under different experimental conditions are shown in the figure. A is the distribution map of the open field experimental area for mice; B is the statistical graph of the total distance traveled by mice in the total open field area; C is the statistical graph of the number of times mice entered the central area; D is the statistical graph of the exploration time of mice in the central area; E is the distribution map of the light-dark box experimental area for mice; F is the statistical graph of the exploration time in the illuminated area; G is the distribution map of the elevated cruciform maze experimental area for mice; H is the statistical graph of the number of times mice entered the open arm; I is the statistical graph of the exploration time of mice in the open arm; J is a schematic diagram of the tail suspension experiment for mice; K is a statistical graph of the time mice remained still in the tail suspension state; L is a schematic diagram of the forced swimming experiment for mice; M is a statistical graph of the time mice remained still in the swimming state. Experimental data are expressed as mean ± standard error (Mean ± SEM). The results were analyzed by one-way ANOVA. p<0.05, p<0.01, ns p>0.05 indicates the significance of the difference. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of the present invention clearer, embodiments of the present invention are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0023] definition In this invention, the term "anxiety disorder" encompasses the following clinical diagnostic entities and their corresponding preclinical phenotypes: (a) Generalized anxiety disorder It refers to a disorder that meets the DSM-5-TR diagnostic criteria 300.02 (F41.1), whose core feature is excessive anxiety and worry about a variety of events or activities (such as work or academic performance), which the patient has difficulty controlling, and is accompanied by at least three of the following symptoms: restlessness or tension, depression, difficulty concentrating, irritability, muscle tension, and sleep disturbance.

[0024] (b) Social anxiety disorder It refers to a disorder that meets the DSM-5-TR diagnostic criteria 300.23 (F40.10), characterized by a significant and persistent fear of one or more social situations in which the patient fears that their words or actions or the anxiety symptoms they exhibit will lead to negative evaluation.

[0025] (c) Panic disorder It refers to a disorder that meets the DSM-5-TR diagnostic criteria 300.01 (F41.0), characterized by recurrent, unpredictable panic attacks, accompanied by persistent worry about recurrence or behavioral changes.

[0026] (d) Acute stress disorder (ASD) It refers to a disorder that meets the DSM-5-TR diagnostic criteria 308.3 (F43.0), belonging to the category of "trauma and stress-related disorders", characterized by intrusive memories, negative mood, dissociative symptoms, avoidance symptoms and arousal symptoms within 3 days to 1 month after exposure to a traumatic event.

[0027] (e) Stress-related pathological avoidance behaviors This refers to stressor-induced inhibitory phenotypes of exploratory behavior observed in animal models, including but not limited to: In the elevated cross maze experiment, the number of times the arms were opened and / or the time spent in the arms was shortened. The number of times the central region is entered in the open field experiment is reduced and / or the time spent in the central region is shortened. In the novel environment feeding inhibition experiment, the feeding latency was prolonged.

[0028] This phenotype maps to situational avoidance symptoms in human anxiety disorders, but does not constitute a clinical diagnostic entity in itself.

[0029] This invention provides a method for utilizing Exosc9 Methods and applications of behavioral research on anxiety disorders in non-human mammals with gene mutations. Exosc9 Gene mutations in non-human mammals are known model organisms, and their forms of mutation include knockout, knock-in, or point mutations, commonly used in genetic and neurobehavioral research. This invention, through systematic experiments, is the first to discover... Exosc9 The genetically mutant animals exhibited behavioral phenotypes associated with anxiety disorders in multiple behavioral experiments, providing a reliable experimental basis for anxiety disorder research.

[0030] In some implementations, such as Exosc9 In the zebrafish model of gene mutation, the juvenile fish showed reduced basic kinetic ability and reduced adaptability to light stimuli under both light and dark conditions, accompanied by anxiety-like behavior; the adult fish also exhibited anxiety-like behavior. Exosc9 The mouse model with the gene mutation exhibits anxiety-like behavior. These characteristics make zebrafish and mice with this gene mutation ideal for constructing animal models of anxiety-related mental disorders, providing an experimental vehicle that closely resembles the physiological state for in vivo research on the pathogenesis of anxiety disorders.

[0031] In terms of extending diagnostic and treatment applications, based on Exosc9 The behavioral abnormalities exhibited by gene knockout animal models can be used to screen drugs for the treatment of anxiety disorders. These models can be used to evaluate the restorative effects of candidate drugs on motor function and the improvement effects on anxiety-like behaviors, thus providing an efficient screening tool for the development of clinical therapeutic drugs.

[0032] In practice, rodent experimental animals (such as mice or rats) were selected as test subjects. These animals exhibited typical anxiety-like behavioral characteristics in a specific behavioral paradigm, specifically: in the open field test, the frequency or duration of activity in the central area was significantly reduced; in the elevated cross maze test, the number of times or the percentage of time spent entering the open arms was reduced; and overall voluntary behavior was inhibited.

[0033] To improve the sensitivity of phenotypic detection, this invention preferably applies standardized pre-treatment interventions to experimental animals before behavioral testing. These interventions include, but are not limited to, mild social isolation, photoperiod dysregulation, or controlled noise stimulation. These treatments do not impair the animals' basic health but can make the anxiety disorder behavioral phenotype more pronounced, facilitating observation and quantification. Through this approach, this invention provides a systematic framework for behavioral research, supporting the scientific analysis and fundamental mechanism research of anxiety disorder behaviors. It also provides a solid foundation for subsequent drug research, dosage design, and validation of experimental animal models.

[0034] The present invention will now be described in conjunction with specific embodiments. Exosc9 The application of gene mutations in non-human mammals in constructing anxiety disorder models or screening anti-anxiety drugs is explained in detail.

[0035] the term" Exosc9 "equivalent to" exosc9 "Anxiety Disorder Model" is equivalent to "Anxiety Model".

[0036] The term "adult zebrafish" refers to juvenile zebrafish that have been raised for at least 3 months.

[0037] The animal strains used in this invention include: WT wild-type zebrafish, exosc9 + / - mutant zebrafish exosc9 - / - mutant zebrafish, WT wild-type mice, Exosc9 + / - Mutant mice. Among them, WT wild-type zebrafish and... exosc9 - / - The mutant zebrafish was purchased by the Institute of Hydrobiology, Chinese Academy of Sciences; exosc9 + / - Mutant zebrafish are adults exosc9 - / - The mice were obtained by crossing zebrafish with wild-type WT zebrafish; the wild-type WT mice were purchased from Shulaibao. Exosc9 + / - The mutant mice were purchased from Cyagen Biosciences. The mutant animal model exhibits a range of characteristics that closely match the core phenotypes of anxiety disorder-related mental disorders.

[0038] Example 1 Build Exosc9 Gene knockout zebrafish and mice.

[0039] zebrafish exosc9 The exon1 region of the gene is a knockout region; knocking out 11 bases results in normal... exosc9 The gene expression lacks the 362nd amino acid, resulting in the loss of normal expression. exosc9 Gene function (e.g.) Figure 1 (As shown in A). Using zebrafish... exosc9 The exon 4 gene was the knockout region. The specificity of the gRNA target site was determined using the Basic Local Alignment Search (BLAST) tool to align with the zebrafish whole genome sequence. For microinjection, the gRNA and Cas9 protein were mixed and co-injected into single-cell stage zebrafish zygotes. The CRISPR / Cas9 system was used to target the target site. exosc9 Genes are knocked out. exosc9At 12 weeks of age (adult zebrafish) following microinjection, the tail fins of zebrafish were cut off, and DNA was extracted for sequencing and identification, with a WT control used. exosc9 - / - Zebrafish sequencing results as follows Figure 1 As shown in B, the F0 zebrafish gene knockout was confirmed to be successful.

[0040] exosc9 + / - Mutant zebrafish: Selected healthy, sexually mature adults over 12 weeks old, whose genotypes have been confirmed. exosc9 - / - Mutant zebrafish and wild-type WT zebrafish were placed in a sterilized hybridization tank with spawning substrate the evening before hybridization, at a 1:1 ratio. The following morning, natural spawning and fertilization were induced by light, thus obtaining... exosc9 + / - Mutant zebrafish.

[0041] Fertilized oocytes of C57BL / 6 background mice Exosc9 The genes were modified using CRISPR / Cas9 technology, and the fertilized eggs were introduced into surrogate mouse mothers. Exosc9 - / - Mutant embryos were lethal, and the resulting embryos were bred through mating to create a mutant embryo. Exosc9 + / - Mutant mouse models. Among them, [selection / selection / selection] Exosc9 exon3 of gene transcripts 5 represents the knockout region, which is 1813bp in size. Knockout can result in the loss of normal function. Exosc9 The purpose of gene function (e.g.) Figure 1 (As shown in C). Build Exosc9 + / - After the mutant mice were successfully bred, the WT control group was used. Exosc9 + / - The level of EXOSC9 protein in mice was significantly decreased (e.g. Figure 1 (As shown in D and E).

[0042] Example 2 exosc9 The absence of these features leads to abnormal development in zebrafish, as well as abnormal basic motor behavior and adaptability in juvenile fish.

[0043] First, the study exosc9 The impact of deficiency on zebrafish growth and development, as well as the basic kinetic behavior and adaptability of zebrafish juveniles.

[0044] 1) Measurement of growth and development The body length of 10 dpf juveniles and 3 mpf adult fish were measured, and the brain of the adult fish was measured microscopically. The results showed ( Figure 2 ), exosc9- / - The body length and head width of juvenile fish were significantly smaller than those in the WT group (p<0.05); the brain volume of adult fish was significantly smaller than that in the WT group (p<0.05). These results indicate that... exosc9 These are key genes essential for the normal growth and brain development of zebrafish.

[0045] 2) Measurement of basic motor behavior Specifically, in order to evaluate exosc9 The effect of absence on zebrafish behavior, compared to wild-type zebrafish (wt). exosc9 + / - mutants and exosc9 - / - After the mutant embryos were cultured to 7 days post-flop (dpf), they were placed in 24-well plates, with one zebrafish in each well, and 500 μL of E3 culture medium was added to each well. The 24-well plates were placed under the camera of the DanioVision behavioral tracking system, and the aperture and camera focus were adjusted to ensure appropriate light intake and accurate focus. The experimental environment temperature was set to 28℃ using a temperature control system. In the light-conditioning exercise experiment, the lighting was turned on and the brightness was set to 100%, allowing the zebrafish larvae to adapt to the light environment for 90 minutes. Then, the tracking instrument was used to record the movement trajectory of the zebrafish larvae over 10 minutes to analyze their movement speed under light conditions. In the dark-conditioning exercise experiment, the lighting was turned off and the brightness was set to 0%, allowing the zebrafish larvae to adapt to the dark environment for 90 minutes. Then, the tracking instrument was used to record the movement trajectory of the zebrafish larvae over 10 minutes to analyze their movement speed under dark conditions. The results are shown in the figure.

[0046] from Figure 2 As can be seen from the data, under light conditions, compared with the control group WT, exosc9 - / - The mutation group showed a significantly reduced movement rate (p < 0.0001) (e.g. Figure 2 (as shown by H and I in the figure), while under dark conditions compared to the control group WT, exosc9 - / - The mutant group had a slightly lower motility rate (p=0.1069) (e.g. Figure 2 (As shown in J and K).

[0047] 3) Measurement of adaptability The zebrafish flashing stimulation experiment was used to assess the adaptability of zebrafish. This experiment also used 7 dpf zebrafish. The experimental environment temperature was set to 28℃ using a temperature control system. The program was set to a dark, unstimulated environment, allowing the juvenile zebrafish to acclimatize for 90 minutes. After the acclimatization period, white light flashing was introduced. The white light intensity was set to 100%, and flashes lasting 5 minutes were performed every 5 minutes, for a total of 5 flashes. The changes in the zebrafish's movement speed during this period were recorded, and the corresponding velocity curves were plotted.

[0048] from Figure 2 As can be seen, when exposed to light stimuli, compared with the control group WT, exosc9 - / - Except for the first stimulus, the mutant juveniles showed a significant increase in speed during the subsequent four stimuli (p < 0.001). Figure 2 (As shown by L and M in the diagram).

[0049] The above results indicate that exosc9 Zebrafish with homozygous deletion of genes exhibit slow growth and development, reduced basic motor skills in juveniles, and weakened adaptability to light stimulation.

[0050] Example 3 exosc9 The absence of these features leads to anxiety-like behaviors in juvenile zebrafish.

[0051] Secondly, the study exosc9 Does the absence of this feature lead to anxiety-like behavior in juvenile zebrafish?

[0052] 1) Open field experiment for juvenile fish The zebrafish juvenile open field test was used to assess anxiety behavior in zebrafish. Seven-day-field (dpf) zebrafish were placed in six-well plates filled with E3 medium, with one juvenile zebrafish in each well. The ambient temperature was set to 28°C using a temperature control system, allowing the juvenile zebrafish to acclimatize for 5 minutes. After acclimatization, the swimming trajectory and speed of the zebrafish were recorded for a total of 10 minutes. Each well was divided into an inner and outer observation area, and the time the zebrafish spent in each area was recorded. The results are shown below. Figure 3 As shown.

[0053] from Figure 3 It can be seen from this that Figure 3 In the diagram, A represents a model of an open-field experiment for zebrafish juveniles, compared to the control group WT. exosc9 - / - The mutant group juveniles spent significantly less time exploring the central region (p<0.001) (e.g. Figure 3 As shown in B); the exploration time in the peripheral region was significantly increased (p<0.01) (as shown in B). Figure 3(as shown in C in the text). Figure 3 In this context, D represents the zebrafish juvenile vitality level, calculated as zebrafish activity time / total test time, compared to the control group WT. exosc9 - / - The vitality level of the mutant group juvenile fish was significantly reduced.

[0054] 2) Light and dark chamber experiment for juvenile fish The zebrafish juvenile light-dark chamber experiment was used to assess anxiety-like behavior and phototaxis / dark-taxis preferences in zebrafish. Seven-day-fed zebrafish were placed in 24-well plates filled with E3 medium, with one juvenile zebrafish in each well. Half of each well (bottom and sidewalls) was pre-painted with matte black paint, creating a semi-black, semi-white independent light-dark zone. The experimental environment was set to 28°C using a temperature control system, with uniform illumination (100% brightness) throughout the experiment, without any light-dark transitions. The zebrafish juveniles were allowed 5 minutes to acclimatize. After acclimatization, their activity trajectories and speeds were recorded over 10 minutes. Each well was designated as a dark zone and a light zone, and the time spent in the dark zone, the number of times they moved between zones, and their movement speed in each zone were recorded.

[0055] from Figure 3 It can be seen from this that Figure 3 E in the diagram represents the light and dark chamber test pattern for zebrafish juveniles, compared to the control group WT. exosc9 - / - The mutant group of juvenile fish spent significantly less time exploring the illuminated area (p<0.01). Figure 3 As shown in F); the exploration time in dark areas increased significantly (p<0.01) (as shown in F in the figure). Figure 3 As shown in G); the shuttle frequency in the bright and dark boxes decreased significantly (p<0.0001) (as shown in G). Figure 3 (as shown by H in the diagram).

[0056] The above results indicate that exosc9 Zebrafish juveniles with homozygous deletion of the gene exhibited significant anxiety-like behavior.

[0057] Example 4 exosc9 The absence of these features leads to anxiety-like behaviors in adult zebrafish.

[0058] At the same time, the study exosc9 Does the absence of this feature lead to anxiety-like behavior in adult zebrafish?

[0059] 1) Open field experiment of adult fish The zebrafish open field test is a method for evaluating the spontaneous exploratory behavior and anxiety levels of laboratory animals in a novel environment. This experiment used 3-month-old adult zebrafish as experimental materials, and a transparent acrylic aquarium measuring 40×40×20 cm as the experimental setup. During the experiment, a zebrafish was placed in the open field test tank and allowed 3 minutes for acclimatization. The zebrafish's trajectory was then tracked and recorded by a camera over 5 minutes. The experiment was conducted daily from 9:00 AM to 3:00 PM. To quantify the zebrafish's behavioral characteristics, the aquarium was divided into a central area and a peripheral area, and the time spent in each area was statistically analyzed.

[0060] from Figure 4 It can be seen from this that Figure 4 In the diagram, A represents a schematic of an open-field experiment with adult zebrafish. Compared to the control group (WT), exosc9 - / - The mutant fish spent significantly less time exploring the central region (p<0.05) (e.g. Figure 4 As shown in B); the exploration time in the peripheral region was significantly increased (p<0.01) (as shown in B). Figure 4 (as shown in C).

[0061] 2) Diving experiment of adult fish in a new aquarium The zebrafish diving experiment in a new aquarium was used to assess anxiety behavior in zebrafish. Adult zebrafish with a 3 mpf size were used as experimental subjects, and a transparent acrylic aquarium measuring 28 × 20 × 5 cm was used as the experimental setup. To ensure consistent experimental conditions, square light panels were installed on the back and bottom of the aquarium to provide uniform illumination and maintain a constant background environment. During the experiment, the zebrafish were placed in the aquarium, and their behavior was recorded immediately using a side-mounted camera. Each individual was recorded for 10 minutes, and the experiment was conducted daily from 9:00 AM to 5:00 PM. The aquarium was vertically divided into three areas: top, middle, and bottom. The dwell time in each area and the latency period for the first entry into the middle and top areas were statistically analyzed.

[0062] from Figure 4 It can be seen from this that Figure 4 In the diagram, D represents the diving experiment model for zebrafish in a new aquarium. Compared with the control group WT, exosc9 - / - The mutant fish exhibited significantly reduced exploration time in the top region (p<0.0001) and significantly increased exploration time in the bottom region (p<0.0001). Figure 4 (as shown in E in the diagram). exosc9 - / - The latency period for the first entry of the mutant fish into the top region was significantly increased (p<0.05) (e.g. Figure 4 (as shown by F in the diagram).

[0063] 3) Adult fish light and dark chamber experiment The zebrafish light-dark chamber experiment is primarily used to assess anxiety-like behaviors and phototaxis / dark-taxis preferences in adult zebrafish. It is designed based on the innate behavioral characteristic of zebrafish, which tend to avoid light and remain in dark areas when anxious. This experiment used adult zebrafish with a 3mpf size as the experimental material. The experimental setup consisted of a 30×15×15 cm acrylic aquarium, half transparent and half black along its length. An opaque acrylic panel divided the aquarium into equal-sized light and dark zones, with a 3 cm high passageway at the boundary for the zebrafish to move freely. The light zone was uniformly illuminated by a light panel (brightness set to 100%), while the darkness was kept below 5 lux. The light panel provided uniform illumination and maintained a constant background environment. During the experiment, a single adult zebrafish was gently placed in the center of the aquarium and allowed 5 minutes for acclimatization. After acclimatization, its behavior was recorded by a top-mounted camera for 10 minutes per individual. The experiment was conducted daily from 9:00 AM to 3:00 PM. Statistical analysis was conducted on the time zebrafish spent in the light zone and the number of times they moved between the light and dark zones.

[0064] from Figure 4 It can be seen from this that Figure 4 G in the diagram represents the experimental setup of adult zebrafish in a light-dark chamber, compared to the control group WT. exosc9 - / - The mutant fish spent significantly less time exploring the illuminated area (p<0.05) (e.g. Figure 4 (As shown in H); the shuttle frequency between the light and dark areas decreased significantly (p<0.05) (as shown in H). Figure 4 As shown in I).

[0065] 4) Mirror attack experiment on adult fish The zebrafish mirror test is primarily used to detect aggressive behavior in zebrafish and can also reflect the animal's anxiety and cognitive abilities. This experiment used adult zebrafish with a 3 mpf size as the experimental material, and a transparent acrylic aquarium measuring 18×4.5×15cm as the experimental setup. A light panel provided uniform illumination and maintained the background environment. During the experiment, after placing the zebrafish in the aquarium, they were allowed 5 minutes to adapt to the environment. Their behavior was then recorded using a side-mounted camera, with each individual recorded for 10 minutes. The experiment was conducted daily from 9:00 AM to 3:00 PM. The time the zebrafish spent in the aggressive zone and the frequency of transitions between different zones were statistically analyzed.

[0066] from Figure 4 It can be seen from this that Figure 4 J in the diagram represents the experimental model of mirror attack in adult zebrafish, compared to the control group WT. exosc9 - / -The mutant fish spent significantly less time in the attack area (p<0.01) (e.g. Figure 4 (As shown in K).

[0067] 5) Three-box social experiment with adult fish The three-box experiment was primarily used to detect social preferences in zebrafish. Social preference behavior is an innate tendency in animals to observe, imitate, and approach other animals of the same species. This simple and relatively primitive social behavior usually appears early in an individual's life, laying the necessary foundation for the complex social functions that develop later. This experiment used adult zebrafish at 3 mpf as experimental material, employing a transparent acrylic fish tank measuring 37×15×15 cm as the experimental setup. The tank was divided into three sections by a transparent acrylic panel. Opaque, removable inserts and slots were located at the junctions of the middle and the two sides of the tank. The central 20 cm section was used to observe and record the behavioral trajectory of the zebrafish being measured, while the other two sides contained only water on one side and a school of zebrafish on the other, simulating different social environments. A light panel provided uniform illumination and maintained the background environment. During the experiment, an opaque partition was first inserted to allow the zebrafish to adapt to their new environment without knowing what was on either side. After 5 minutes of adaptation, the partition was removed. At this point, the zebrafish were positioned with a school of fish on one side and water on the other. Their behavior and preferences were recorded using a side-mounted camera. Each individual was recorded for 10 minutes, and the experiment was conducted daily from 9:00 AM to 3:00 PM. The duration of the zebrafish's stay in the social zone and the frequency of transitions between different zones were statistically analyzed.

[0068] from Figure 4 It can be seen from this that Figure 4 M in the diagram represents the three-box social interaction model of adult zebrafish, compared to the control group WT. exosc9 There was no significant difference in the dwell time and switching frequency of the genetically mutated fish in the social zone (p>0.05) (e.g. Figure 4 (As shown in the diagram of N and O).

[0069] The above results indicate that exosc9 Adult zebrafish with homozygous deletion of the gene exhibited significant anxiety-like behavior, but the gene knockout did not significantly affect the social preferences of zebrafish.

[0070] Example 5 Exosc9 The absence of this feature resulted in anxiety-like behavior in the mice.

[0071] Research Exosc9 Does the deletion lead to anxiety-like behavior in mice?

[0072] 1) Open field experiment The open field test is used to assess spontaneous exploratory behavior and anxiety levels in mice in novel, open environments. Six- to eight-week-old mice were used in a transparent acrylic open field box measuring 45×45×45 cm, with the bottom of the box divided into a center, corners, and other areas. The experimental environment was kept quiet and evenly lit. Mice were gently placed in the center of the open field box, and their movement was recorded by a camera over 5 minutes. The total distance traveled, the time spent in the central and corner areas, and the frequency of these activities were statistically analyzed.

[0073] from Figure 5 It can be seen from this that Figure 5 In the diagram, A represents the mouse open field experiment model, compared to the control group WT. Exosc9 Het mutant mice showed no significant change in motor function (p>0.05). Figure 5 (as shown in B in the image). Exosc9 Het mutant mice entered the central region significantly less frequently (p<0.05) (e.g. Figure 5 As shown in C); the exploration time in the central region was significantly reduced (p<0.05) (as shown in C). Figure 5 (as shown in D in the diagram).

[0074] 2) Dark and Light Box Experiment The mouse black-and-white box experiment is used to assess anxiety-like behavior in mice, based on their innate tendency to seek darkness and avoid light. Six- to eight-week-old mice are used in a transparent acrylic box measuring 40×20×25 cm. The box is divided lengthwise into a two-thirds light zone (white inner wall, 300-500 lux illumination) and a one-third dark zone (black inner wall, <5 lux illumination). A passageway is provided at the boundary between the two zones for the mice to move freely. The experimental environment is kept quiet. A single mouse is placed in the center of the light zone, and its behavior is recorded for 6 minutes. The time spent in the light zone is statistically analyzed.

[0075] from Figure 5 It can be seen from this that Figure 5 E in the diagram represents the mouse light-dark chamber experiment schematic, compared to the control group WT. Exosc9 Het mutant mice showed a significantly reduced exploration time in bright areas (p<0.05) (e.g. Figure 5 (as shown by F in the diagram).

[0076] 3) Elevated cross-maze experiment The elevated cross maze test is used to assess anxiety-like behavior in mice, based on their natural avoidance of heights and open areas. Six- to eight-week-old mice were used, employing an elevated cross maze apparatus (arms 30 cm long, 5 cm wide, and 50 cm high), consisting of two open arms and two closed arms (the closed arms had sidewalls 15 cm high). The experimental environment was kept quiet and dimly lit. A single mouse was placed in the center of the maze, with its head facing any open arm, and its behavioral trajectory was recorded over 6 minutes. The time spent in the open and closed arms, and the number of times the mouse entered each arm, were statistically analyzed.

[0077] from Figure 5 It can be seen from this that Figure 5 G in the diagram represents the elevated cruciate maze experiment for mice, compared to the control group WT. Exosc9 Het mutant mice entered the open arm a significantly reduced number of times (p<0.01) (e.g. Figure 5 (as shown by H in the diagram). Exosc9 Het mutant mice showed a significantly reduced exploration time in open arms (p<0.01) (e.g. Figure 5 As shown in I).

[0078] 4) Tail Suspension Test The mouse tail suspension test is used to assess depressive-like behavior in mice. It is designed based on the behavioral characteristics of mice exhibiting desperate struggles followed by immobility when suspended. Six- to eight-week-old mice were used, employing a tail suspension device (30 cm long suspension rope, 50 cm above the ground). The experimental environment was kept quiet with soft lighting. The tip of the mouse's tail was secured to the suspension rope with tape 1 cm above the ground, ensuring the mouse's body was freely suspended without any limb contact with any object. After acclimatization for 1 minute, the mouse's behavior was recorded using a camera over 6 minutes. The duration of immobility and the number of struggles were statistically analyzed.

[0079] from Figure 5 It can be seen from this that Figure 5 J in the diagram represents the mouse tail suspension test pattern, compared to the control group WT. Exosc9 There was no significant difference in the time it took for Het mutant mice to remain still (p>0.05) (e.g. Figure 5 (As shown in K).

[0080] 5) Forced swimming test The forced swimming test in mice is used to assess depressive-like behavior in mice. It is based on the despair behavior of mice gradually transitioning from struggling to immobility in an inescapable aquatic environment. Six- to eight-week-old mice were used in a transparent acrylic cylindrical water tank measuring 20×20×30 cm, filled with 25°C clean water (15 cm deep, ensuring the mice could only swim and not stand). The experimental environment was kept quiet with soft lighting. Each mouse was gently placed in the tank, and after acclimatization for 1 minute, its behavior was recorded for 6 minutes using a camera. The immobility time, swimming time, and number of struggles were statistically analyzed.

[0081] from Figure 5 It can be seen from this that Figure 5 The "L" in the diagram represents a schematic of the forced swimming experiment in mice, compared to the control group (WT). Exosc9 Het mutant mice had a significantly reduced resting immobility time (p<0.05) (e.g. Figure 5 (As shown in M ​​in the diagram).

[0082] The above results indicate that Exosc9 Mice with the gene deletion exhibited significant anxiety-like behavior but not depression-like behavior; instead, they showed a tendency toward an antidepressant phenotype.

[0083] In summary, this invention is the first to discover... exosc9 The absence of this feature leads to stunted development in zebrafish, causing basal motor defects, adaptive deficits, and anxiety-like behaviors in juveniles, as well as anxiety-like behaviors in adults. Exosc9 The absence of this feature also induced anxiety-like behavior in mice. This indicates that... EXOSC9 Gene knockout animal models can serve as a new and ideal vehicle for studying animal models of anxiety-related mental disorders.

[0084] For any points not covered above, existing technologies shall apply.

[0085] Although specific embodiments of the present invention have been described in detail by way of examples, those skilled in the art should understand that the above examples are for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications or additions to the described specific embodiments or use similar methods to replace them, without departing from the direction of the invention or exceeding the scope defined by the appended claims. Those skilled in the art should understand that any modifications, equivalent substitutions, improvements, etc., made to the above embodiments based on the technical essence of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for constructing an animal model of anxiety disorder, characterized in that, Will Exosc9 Genetically mutated animals serve as the animal model of the anxiety disorder. Exosc9 The genetically mutated animals are mice or zebrafish.

2. The method for constructing an animal model of anxiety disorder according to claim 1, characterized in that, The Exosc9 Gene mutations include Exosc9 Gene knockout or Exosc9 Point mutations in genes.

3. The method for constructing an animal model of anxiety disorder according to claim 1, characterized in that, The anxiety disorder mentioned includes one or more of the following: generalized anxiety disorder, social anxiety disorder, panic disorder, acute stress disorder, or stress-related pathological avoidance behaviors.

4. A method for screening anti-anxiety drugs, characterized in that, Includes the following steps: Provided as claimed in claim 1 Exosc9 Animals with genetic mutations; To the Exosc9 The candidate drug was administered to genetically mutated animals; The evaluation of the Exosc9 Behavioral and physiological indicators related to anxiety symptoms in genetically modified animals; If the candidate drug can improve the behavioral indicators related to the anxiety symptoms, it is identified as an anti-anxiety candidate drug.

5. The method for screening anti-anxiety drugs according to claim 4, characterized in that, The behavioral indicators include open field experiments, light and dark box experiments, diving experiments, or elevated cross maze experiments, and the physiological indicators include head length or body length measurements.

6. A method for evaluating the efficacy of candidate anti-anxiety drugs, characterized in that, Using the method described in claim 1 Exosc9 Using genetically modified animals as test subjects, the extent to which drugs improve the anxiety-like behavioral phenotypes of the animals was observed.

7. The method for evaluating the efficacy of candidate anti-anxiety drugs according to claim 6, characterized in that, The Exosc9 Gene mutations include Exosc9 Gene knockout or Exosc9 Point mutations in genes.

8. The method for evaluating the efficacy of candidate anti-anxiety drugs according to claim 7, characterized in that, The anxiety disorder mentioned includes one or more of the following: generalized anxiety disorder, social anxiety disorder, panic disorder, acute stress disorder, or stress-related pathological avoidance behaviors.

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