A system and device for constructing an attention deficit animal model

Abstract

The present invention discloses a system and apparatus for constructing an attention deficit disorder animal model. The present invention discovers for the first time that the Calhm2V136G point mutation is a key molecule affecting attention, and that a mouse model carrying the Calhm2V136G point mutation exhibits attention deficit disorder. Based on this, the present invention creatively develops a system and apparatus for constructing an attention deficit disorder animal model carrying the Calhm2V136G point mutation. The present invention provides an effective animal model for studying the mechanism of action of attention deficit disorder or related diseases and for drug screening, and has broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biology, and in particular, relates to an animal model, and more particularly, to a system and device for constructing an attention deficit animal model. Background Art

[0002] Attention deficit disorder, also known as Attention Deficit Disorder (ADD), is a common neurodevelopmental disorder characterized by inattention, impulsivity, and hyperactivity. This condition usually appears in childhood and may persist into adulthood in a few cases. The causes of attention deficit disorder are diverse and may be related to biological, social, and psychological factors. Attention deficit disorder has a significant impact on the patient's life and learning. Patients often experience problems such as distraction, learning difficulties, and difficulty completing tasks in their studies and work. In severe cases, it can also affect their social and family life.

[0003] Attention is the cornerstone that enables people to function effectively in a complex and information-rich world. A deeper understanding of the mechanisms underlying attention deficit is crucial. Animal models play an important role in summarizing and studying the mechanisms underlying different diseases or conditions and are an important transitional stage for human clinical research. However, the complex mechanisms underlying attention deficit make the development of preclinical animal models of attention deficit challenging. Therefore, there is currently a lack of animal models for studying the mechanisms of action and drug screening for attention deficit or related diseases. Summary of the Invention

[0004] The present invention aims to address the technical problem of the lack of an attention deficit animal model in the prior art for studying the mechanism of action of attention deficit or related diseases and for drug screening. To this end, the present invention provides a system and apparatus for constructing an attention deficit animal model.

[0005] Calcium homeostasis modulator (Calhm) is a functional protein primarily involved in the regulation of calcium ions and adenosine 5'-triphosphate (ATP). Calhm2, a member of the Calhm family, is highly expressed in the central nervous system and functions as an ATP release channel. Knocking out Calhm2 in Alzheimer's disease (AD) mice significantly improves spatial learning and memory.

[0006] Currently, there are no studies or reports linking the presence of the Calhm2 V136G point mutation to attention deficit. The present invention discovers for the first time that the Calhm2 V136G point mutation is a key molecule affecting attention, and that a mouse model carrying the Calhm2 V136G point mutation exhibits attention deficit. Based on this, the present invention provides a system and device for constructing an animal model of attention deficit in the field.

[0007] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0008] A first aspect of the present invention provides a system for constructing an attention deficit animal model.

[0009] Furthermore, the system includes the following units:

[0010] Design sequence units and design sgRNA sequences that recognize the Calhm2 genome;

[0011] In the first processing unit, the sgRNA, Cas9 mRNA, and mutant oligonucleotide are microinjected into mouse fertilized eggs for homologous recombination, and then transplanted into surrogate mother mice to obtain F0 generation mice;

[0012] The second processing unit performs PCR genotyping and sequence analysis on the F0 generation mice to obtain positive F0 generation mice carrying the Calhm2V136G point mutation;

[0013] The third processing unit is to breed the positive F0 generation mice carrying the Calhm2 V136G point mutation with wild-type mice for one generation to obtain F1 generation mice with stable inheritance;

[0014] a fourth processing unit, performing PCR genotyping and sequence analysis on the F1 generation mice to obtain positive F1 generation mice carrying the Calhm2V136G point mutation;

[0015] The fifth processing unit is to hybridize the positive F1 generation mice carrying the Calhm2 V136G point mutation, and screen homozygous mutant mice carrying the Calhm2 V136G point mutation using PCR genotyping and sequence analysis technology, which are attention deficit disorder animal models.

[0016] Furthermore, the target site of the sgRNA is exon 2 of the Calhm2 gene;

[0017] The mutant oligonucleotide comprises a V136G (GTG to GGT) mutation site.

[0018] Furthermore, the system also includes a sixth processing unit for verifying the attention phenotype of the attention deficit animal model.

[0019] Furthermore, the verification includes testing the attention of the attention deficit animal model using a five-hole attention test system.

[0020] Furthermore, if the verification results show that the attention level is significantly reduced, it indicates that the attention deficit animal model is successfully constructed.

[0021] A second aspect of the present invention provides a device for constructing an attention deficit animal model.

[0022] Furthermore, the device comprises:

[0023] A single or multiple processors, and a memory for storing a single or multiple computer programs, which, when executed by the single or multiple processors, implement:

[0024] Operation 1, designing sgRNA sequences that recognize the Calhm2 genome;

[0025] Operation 2: microinjecting the sgRNA, Cas9 mRNA, and mutant oligonucleotide into mouse fertilized eggs for homologous recombination, and transplanting them into surrogate mother mice to obtain F0 generation mice;

[0026] Operation 3, performing PCR genotyping and sequence analysis on the F0 generation mice to obtain positive F0 generation mice carrying the Calhm2V136G point mutation;

[0027] Operation 4, breeding the positive F0 generation mice carrying the Calhm2 V136G point mutation with wild-type mice to obtain F1 generation mice with stable inheritance;

[0028] Operation 5, performing PCR genotyping and sequence analysis on the F1 generation mice to obtain positive F1 generation mice carrying the Calhm2V136G point mutation;

[0029] Operation 6: hybridize the positive F1 generation mice carrying the Calhm2 V136G point mutation, and screen homozygous mutant mice carrying the Calhm2 V136G point mutation using PCR genotyping and sequence analysis technology, which are animal models of attention deficit disorder.

[0030] Furthermore, the target site of the sgRNA is exon 2 of the Calhm2 gene;

[0031] The mutant oligonucleotide comprises a V136G (GTG to GGT) mutation site.

[0032] Furthermore, the apparatus further includes operation 7 of verifying the attention phenotype of the attention deficit animal model.

[0033] Furthermore, the verification includes testing the attention of the attention deficit animal model using a five-hole attention test system;

[0034] The results of the verification show that the attention level is significantly reduced, indicating that the attention deficit animal model is successfully constructed.

[0035] The third aspect of the present invention provides the use of the mutant gene Calhm2 V136G in constructing an attention deficit animal model;

[0036] Optionally, the attention deficit animal model is an attention deficit mouse model.

[0037] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0038] The present invention discovered for the first time that the Calhm2 V136G point mutation is a key molecule affecting attention. The mouse model carrying the Calhm2 V136G point mutation exhibits attention deficit. Based on this, the present invention creatively developed a system and device for constructing an attention deficit animal model carrying the Calhm2 V136G point mutation. The present invention provides an effective animal model for studying the mechanism of action and drug screening of attention deficit or related diseases, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0040] Figure 1 A schematic diagram of a system for constructing an attention deficit animal model provided by an embodiment of the present invention;

[0041] Figure 2 A schematic diagram of a device for constructing an attention deficit animal model provided by an embodiment of the present invention;

[0042] Figure 3 This figure shows the results corresponding to the increase in the number of buried beads in attention-deficit mice carrying the Calhm2 V136G point mutation;

[0043] Figure 4 This is the result graph corresponding to the fact that the Calhm2 V136G point mutation has no effect on the motor ability of mice;

[0044] Figure 5 This is the result graph corresponding to the fact that the Calhm2 V136G point mutation has no effect on the sociability of mice;

[0045] Figure 6 This figure shows the results that there is no difference in learning ability between attention-deficit mice carrying the Calhm2 V136G point mutation and wild-type mice;

[0046] Figure 7 The graphs show the results of attention tests on two groups of mice: attention-deficient mice carrying the Calhm2 V136G point mutation and wild-type mice;

[0047] Figure 8 The graph shows the corresponding results of Calhm2 protein expression in the PFC and hippocampus of two groups of mice with attention deficit disorder and wild-type mice carrying the Calhm2 V136G point mutation;

[0048] Figure 9 The graph shows the corresponding results of ATP release in the PFC and hippocampus of two groups of mice with attention deficit disorder and wild-type mice carrying the Calhm2 V136G point mutation;

[0049] Figure 10 This is the result diagram corresponding to the activity levels of neurons at different Bregma points in the anterior cingulate gyrus after the attention test;

[0050] Figure 11 The graph shows the corresponding results of ATP release in the anterior cingulate gyrus of two groups of mice: attention-deficit mice carrying the Calhm2 V136G point mutation and wild-type mice. DETAILED DESCRIPTION

[0051] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention.

[0052] In some of the processes described in the specification and claims of the present invention and the accompanying drawings, multiple operations are included in a specific order. However, it should be understood that these operations may not be performed in the order in which they are presented herein or may be performed in parallel. Operation numbers such as 101, 102, 103, etc. are merely used to distinguish between different operations and do not represent any order of execution. In addition, these processes may include more or fewer operations, and these operations may be performed sequentially or in parallel.

[0053] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making any creative efforts shall fall within the scope of protection of the present invention.

[0054] Figure 1 1 is a schematic diagram of a system for constructing an attention deficit animal model provided by an embodiment of the present invention. Specifically, the system includes the following units:

[0055] 101: Design sequence unit, design sgRNA sequence to recognize Calhm2 genome;

[0056] 102: First processing unit, microinjecting the sgRNA, Cas9 mRNA, and mutant oligonucleotide into mouse fertilized eggs for homologous recombination, and transplanting them into surrogate mother mice to obtain F0 generation mice;

[0057] 103: a second processing unit, performing PCR genotyping and sequence analysis on the F0 generation mice to obtain positive F0 generation mice carrying the Calhm2 V136G point mutation;

[0058] 104: a third processing unit, breeding the positive F0 generation mice carrying the Calhm2 V136G point mutation with wild-type mice to obtain F1 generation mice with stable inheritance;

[0059] 105: a fourth processing unit, performing PCR genotyping and sequence analysis on the F1 generation mice to obtain positive F1 generation mice carrying the Calhm2 V136G point mutation;

[0060] 106: The fifth processing unit is to hybridize the positive F1 generation mice carrying the Calhm2 V136G point mutation, and screen homozygous mutant mice carrying the Calhm2 V136G point mutation using PCR genotyping and sequence analysis technology, which are attention deficit disorder animal models.

[0061] In a specific embodiment, the present invention uses the following construction method to construct an attention deficit mouse model carrying the Calhm2 V136G point mutation: a C57BL / 6 mouse model with a mouse Calhm2 locus point mutation (V136 G) is established by CRISPR / Cas-mediated genome engineering. The mouse Calhm2 gene (GenBank accession number: NM_133746.5, Ensembl: ENSMUSG00000033033) is located on mouse chromosome 19. Three exons were identified, the ATG start codon is located in exon 2, and the TAA stop codon is located in exon 3. V136 is located in exon 2, so exon 2 was selected as the target site. The V136G (GTG to GGT) mutation site in the donor oligomer was introduced into exon 2 by homology-directed repair. In addition, a silent mutation (TCC to TCG) was introduced to prevent gRNA binding and re-cutting the sequence after homology-directed repair. Cas9 mRNA, sgRNA, and donor oligonucleotides were co-injected into fertilized eggs for the production of KI mice (knock-in mice). The pups were genotyped by PCR, followed by sequence analysis and BstEII restriction analysis. The F0 generation of mice was bred to the next generation (F1), which was subsequently genotyped by PCR and DNA sequencing analysis.

[0062] In one embodiment, the target site of the sgRNA is exon 2 of the Calhm2 gene;

[0063] The mutant oligonucleotide comprises a V136G (GTG to GGT) mutation site.

[0064] In one embodiment, the sgRNA can be designed and synthesized using conventional methods in the art, and the Cas9 mRNA can be purchased from conventional sources. For example, the sequence of the target gene (Calhm2) is obtained from an online database (such as NCBI). The online sgRNA design platform (https: / / design.synthego.com / # / ) can be used to analyze and obtain information on the target sequence (20 bp) of the target gene, from which targets with high editing efficiency scores and low off-target scores are selected. The full-length sgRNA sequence is obtained by chemical synthesis.

[0065] In one embodiment, the mutant oligonucleotide can be designed and synthesized using conventional methods in the art. For example, the mutant oligonucleotide can be prepared using a commercial kit (e.g., Guide-it Long ssDNA Strandase Kit, Takara, Clontech).

[0066] In one embodiment, a 606 bp long product was generated from PCR genotyping using the primers and conditions listed below. The amplicon was then purified and sent for DNA sequencing analysis.

[0067] Mouse Calhm2(V136G)-F:5'-TTCTTCAAGAGCAAGGATGTGATGATTT-3'; Mouse Calhm2(V136G)-R:5'-GGTGAGTCTGTGCTTCCTCGGAC-3';

[0068] Annealing temperature: 60℃.

[0069] In one embodiment, the mRNA sequence transcribed from a target allele having a point mutation is as follows: MAALIAENFRFLSLFFKSKDVMIFNGLVALGTVGSQELFSVVAFHCPCSPARNYLYGLTAIGVPALALFLIGVILNNHTWNLVAECQYRRAKNCSAAPNFLLLSSILGRAAVAPVTWSVISLLRGEAYVCALSEFGDPSSLTAGDKGFPPAHATEVLARFPCGEGPANLSSFREEVSRRLKYESQLFGWLLIGVVAILVFLTKCLKHYCSPLSYRQEAYWAQYRTNEDQLFQRTAEVHSRVLAANNVRRFFGFVALNKDDEELVAKFPVEGTQPRPQWNAITGVYLYRENQGLPLYSRLHKWAQGLTGNGTAPDNVEMALLTA.

[0070] In one embodiment, the system further comprises a sixth processing unit for verifying the attention phenotype of the attention deficit animal model.

[0071] In one embodiment, the validation comprises testing the attention of the attention deficit animal model using a five-hole attention test system.

[0072] In one embodiment, the verification result shows that the attention level is significantly reduced, indicating that the attention deficit animal model is successfully established.

[0073] In one embodiment, the animals are mice. The mice used in the experiment were Calhm2V136G mutant mice with a C57BL / 6 background and wild-type control mice of the same age, all bred and maintained in the laboratory. The breeding and husbandry of mice strictly adhered to the relevant regulations of the Beijing Municipal Laboratory Animal Management Office. All animal-related experiments were reviewed and approved by the Animal Welfare Committee of the Academy of Military Medical Sciences.

[0074] In one embodiment, this study first conducted a series of autism-like behavior tests on Calhm2 V136G adult mice. The results showed that Calhm2 V136G adult mice did not show typical autism-like behaviors, but only had mild stereotyped behaviors ( Figure 3-5 ).

[0075] In one embodiment, the autism-like behavior detection method in a mouse model mainly includes the following aspects: social interaction and social communication detection, stereotyped movement and narrow interest activity detection.

[0076] In one embodiment, the social interaction and social communication detection is one of the core methods for evaluating the behavior of autistic mouse models. As a highly social animal, mice have rich and varied social behaviors. The autistic mouse model shows symptoms similar to humans in social interaction, such as playful behavior (such as chasing, fighting), social exploration (such as contact, sniffing), sexual activity, and aggressive behavior. Specific detection methods include: young mouse social interaction detection and three-box social experiment.

[0077] Juvenile Mouse Social Interaction Assay: 21-24-day-old mice are tested in standard mouse cages or other locations where detailed observation of mouse interactions is possible. After being isolated and housed for one hour, the mice are paired according to gender and age and placed in an observation area. Video recording is performed for 10 minutes, followed by analysis of behavioral parameters such as nose-to-nose sniffing, directional movement, and following.

[0078] Three-box social experiment: A social preference task test is conducted using a three-box apparatus to record the time the test mice spend with the stranger mouse and the non-social object on the other side. Normal test mice should prefer to spend time with the stranger mouse, while autism model animals spend less time near the stranger mouse.

[0079] In one embodiment, the autistic-like behavior mice also exhibit stereotyped movements and narrow interest activities. Specific methods for detecting the stereotyped movements and narrow interest activities include: stereotyped grooming behavior and fixed pattern (learning reversal) test.

[0080] Stereotyped grooming behavior: The test can be conducted in a standard mouse cage or open field. After the mice become familiar with the cage environment for 10 minutes, the number of grooming behaviors or the duration of grooming behaviors are recorded by video for 10-15 minutes (or continuously for 30 minutes or 2 hours) to evaluate stereotyped repetitive movements in mice with autistic-like behavior.

[0081] Stereotype (learning reversal) testing: This test assesses the mice's stereotypic thinking by observing their fixed patterns in daily behavior and their performance in specific tasks. For example, a T-maze or water maze is used to test the mice's flexibility in switching from established habits to new ones.

[0082] In a specific implementation scheme, the open field experiment includes the following steps: (1) before starting the experiment, the mouse is placed in the behavior room to acclimate for more than 1 hour; (2) a 50 cm × 50 cm × 20 cm open field box is used, and the mouse is gently placed in the middle of the box bottom at the beginning. The behavioral software is started to record the spontaneous activity of the mouse within 5 minutes. After each test, the box is wiped clean with alcohol before the next mouse is tested; (3) the total movement distance of the mouse and the activity time in the center area are exported by the software and the data are analyzed.

[0083] In a specific implementation scheme, the novel object recognition experiment includes the following steps: (1) an open field is used as an adaptation prerequisite for novel object recognition, at least once, and the experiment is conducted in an open field box of 50 cm × 50 cm × 20 cm; (2) before the start of the experiment, the mice are transferred to the experimental room in advance to adapt to the environment for more than 1 hour; (3) the first stage: two old objects are fixed in appropriate positions in each box, and the mice are placed in the middle of the end far from the objects. The mice are recorded exploring the two old objects for 5-10 minutes, the mice are removed, and the box and objects are wiped with alcohol to remove odor; (4) the second stage: 1-2 hours later, before the start, one of the old objects is replaced with another new object with different color and shape, and the mice are recorded exploring the new and old objects for 5-10 minutes; (5) cognitive index = new object exploration time / (new object exploration time + old object exploration time) × 100%.

[0084] In a specific embodiment, the sugar water preference experiment includes the following steps: (1) before the start of the experiment, the mice are housed in a single cage and adapted for 1-2 days. The experiment lasts for one week, during which time they are given sufficient water and food; (2) on the first day, two bottles of 1% sugar water are provided to the mice for one day of adaptation, and the water weight is not weighed; (3) on the second day, one of the bottles of sugar water is replaced with pure water, and the water weight is weighed before being placed; (4) the mice are raised in the above manner for 3 days, the positions of the two bottles of water are changed every day, and the water weight is weighed every 24 hours. The content of each bottle of sugar water and pure water is 200-250 mL; (5) the sugar water preference percentage (sugar water consumption / total liquid consumption × 100%) is continuously measured for 7 days, and the average value is taken as the sugar water preference value of the mice.

[0085] In a specific implementation plan, the bead-burying experiment includes the following steps: (1) placing the mouse in the behavioral room for 1-2 hours in advance, laying a 5-cm thick bedding in a rectangular opaque mouse cage, and placing 20 glass beads of the same color and size evenly spaced on each bead after leveling; (2) placing the mouse in the middle of the cage at the beginning, covering it with a lid, and conducting the experiment for 30 minutes; (3) removing the mouse, and following a uniform standard throughout the process, if more than 2 / 3 of the glass beads are buried, it is considered buried, and the number of buried beads is recorded and statistically analyzed.

[0086] In a specific implementation scheme, the three-box social interaction includes the following steps: (1) the mice are placed in a behavioral room to adapt for 1-2 hours. The three-box social interaction box is a rectangle of 60cm×40cm×422cm, divided into three chambers. There is a 4cm×4cm middle channel at the bottom of each partition to connect the three chambers and allow the experimental animals to move freely in the three chambers; (2) in the first stage, the channels on both sides are blocked, the mice are placed in the middle chamber, and nothing is placed in the chambers on both sides. The mice explore for 10 minutes; (3) in the second stage, a small hollow cage that can hold a strange mouse is placed on each side, but no mouse is placed in it, and then the channel is opened to allow the mice to explore for 10 minutes; (4) in the third stage, a strange mouse is placed in the cage on the right, and the cage on the left is still empty. The channel is opened and the mice explore for 10 minutes; (5) in the fourth stage, strange mice are placed in cages on both sides, and the mice explore for 10 minutes; the data are exported and the communication time between the mice and the cages in each stage is statistically analyzed.

[0087] In a specific embodiment, all data were analyzed and analyzed using GraphPad and ImageJ software. Adobe Photoshop was used to adjust the contrast of the images, and the Unpaired Student's two-tailed t test was used to determine statistical significance. In all experiments, p < 0.05 was considered statistically significant. For ns > 0.05, p < 0.05 is indicated as *, p < 0.01 is indicated as **, p < 0.001 is indicated as ***, and p < 0.0001 is indicated as ****.

[0088] In one embodiment, we used the classic five-hole attention behavior paradigm (The 5-Choice Serial Reaction Time Task, 5-CSRTT) to test their learning ability and attention. The results showed that the learning ability of Calhm2 V136G mice was not statistically different from that of the control group mice, but the attention level was significantly reduced ( Figure 6-7 ), this result intuitively proves that the mouse model carrying the Calhm2 V136G point mutation is an attention deficit mouse model.

[0089] In one embodiment, the 5-CSRTT was originally designed to study the deficits shown by children with attention-deficit / hyperactivity disorder (ADHD) at the preclinical level. In the rodent version of the task, five holes in the task apparatus are presented with a brief stimulus light in a pseudo-random manner, and the animal is required to make a nose touch response at the correct spatial location (stimulus light is on) to obtain a reward. In order to complete this task, the animal is required to maintain attention and monitor a row of five holes arranged horizontally, and not to make a nose touch response before the stimulus light is on, but to respond after the stimulus light is on. In general, the accuracy of stimulus discrimination provides an indicator of attention ability, while premature responses generated before the stimulus is presented are considered a form of impulsivity, representing a failure of inhibitory control. Therefore, this task can detect attention and impulsive behavior at the same time. The 5-CSRTT provides the possibility of testing the effects of various neural, pharmacological and behavioral operations on behavioral control measures, including response accuracy, impulsive responses, forced responses and response latency, etc.

[0090] The basic task format of the 5-CSRTT is well suited to assessing deficits and / or improvements in sustained and spatially divided attention in animals induced by pharmacological and neural manipulations. In the 5-CSRTT, sustained active attention can be assessed by varying the inter-trial interval (ITI). In addition, the attentional load of the task can be controlled by reducing or increasing the duration of the visual stimulus (stimulus duration, SD). The 5-CSRTT requires animals to allocate their limited attentional resources across different sensory channels and spatial locations (spatially divided attention) in order to achieve optimal performance levels. In addition, this modified basic task structure can test selective (focused) attention by inserting irrelevant and distracting stimuli (most commonly auditory patterns, inserted self-noise interference) during the ITI when the animal must attend to the target visual stimulus.

[0091] In a specific embodiment, the five-hole attention test includes the following steps: (1) Fasting: Take 8-week-old healthy WT and Calhm2 V136G male mice, 14 per group, and house them in single cages. The initial normal weight of the mice is counted; (2) After 1-2 days of adaptation to single cage housing, fast and house them for 1-2 days. After the weight of the mice reaches 90%-80% of their original weight, the weight is controlled within this range by feeding; Handle the mice for 2-4 days; (3) Preparation phase procedure: All five LED screens in the machine light up; after the mouse touches the food trough, the food light goes out, and then the five screens light up at the same time, waiting for the mouse to make a choice. When the mouse touches the lit screen, it is judged to be correct. With a beep, a piece of food falls into the food trough, and the food trough light turns on again. When the food trough light is on, it will not go out unless the mouse touches the food trough, and the trial will not start. When the screen light is on, the screen will not turn off unless the mouse touches the screen, and nothing will happen with it; (4) The parameters and data calculation formulas involved in the training stage of Stage 1-6 are as follows: ①SD: the duration of the LED screen on; LH: the duration of the choice given to the mouse; ITI: the time from the trough light off to the LED screen turning on; TP: the punishment duration; ② the percentage of correct answers in the total number of trials (Correct percent), Correct percent = Correct count / Trial count; ③ Accuracy, Accuracy = Correct count / (Correct count + Error count); ④ Omission rate (Omission), Omission = Omission count / (Correct count + Error count + Omission count); ⑤ Premature, Premature = Premature count / Trial count; (5) Stage 1-6 training stage procedures: ① At the beginning of the exploration experiment, the house light is off, the trough light is on, and the trial begins when the trough is touched; ② After touching the trough, one of the five screens lights up randomly after a specific period of time (ITI).If the mouse pokes the screen before this specific time (ITI), it is judged as impulsive; ③ The mouse needs to make a choice within the time (LH) given to the mouse to make a choice. If it pokes the correct screen, it is judged as correct; if it pokes the wrong screen, it is judged as error; if it does not make a choice after the LH, it is judged as omission; ④ The duration of the LED screen (SD) and the time (LH) given to the mouse to make a choice are two parameters, which means that the screen can be off but the mouse can still make a choice; ⑤ Correct choice: accompanied by a beep, the food trough light lights up and a piece of food falls; ⑥ Wrong choice, omission and impulsive: the house light is on as the punishment time (TP), and then the food light is on again; ⑦ When the food trough light is on, the light will not go out unless the mouse touches the food trough, and the trial will not start; (6) Test phase procedure: The mice that have passed the 6-stage training enter the test phase. The program of this phase is that SD or ITI is randomly displayed with three different values, and other parameters are the same as the basic training phase; (7) The specific values of each parameter in the training and test phases are shown in Table 1 below.

[0092] Table 1 Program parameter settings and passing standards

[0093]

[0094]

[0095] In one embodiment, in order to further study the molecular mechanism by which the Calhm2 V136G point mutation affects attention, we selected the prefrontal cortex involved in attention control for in-depth research. We first used c-Fos, a molecular marker for detecting neuronal activity, to stain the anterior cingulate area (ACA) of Calhm2 V136G mice and control mice after attention testing. The results showed that the amount of c-Fos in the ACA brain region of mice carrying the Calhm2 V136G point mutation was significantly less than that of the control mice ( Figure 10 ).

[0096] In a specific embodiment, the histological and immunostaining methods involved are as follows: After anesthesia with tribromoethanol (200 mg / kg intraperitoneally), mice are perfused transcardially with normal saline (4°C) and then with 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS, 4°C) and post-fixed in 4% PFA for 4 hours. The brain is then removed and incubated first in filtered 20% and then 30% sucrose solution (dissolved in 0.1M PBS) until it sinks, then embedded in an optical tomography and sectioned coronally into 50 μm thick sections at -20°C using a freezer. Sections are collected in flat plates or mounted on gel-coated slides for immunostaining.

[0097] To stain the nuclei, sections were incubated in a 1:5000 DAPI solution (D1306, Invitrogen, USA) for 1 minute or covered with 50% glycerol mounting medium containing 1:2000 DAPI. For immunohistochemical staining, selected sections were collected in a cell culture plate and washed three times with PBS for 15 minutes each. Then, sections were permeabilized three times with PBS containing 0.3% Triton X-100 (PBST) for 10 minutes each. After incubation in blocking solution (3% BSA in PBST) for 90 minutes at room temperature (RT), sections were incubated with antibodies against c-Fos (1:1000, 226 008, SYSY, Germany) and NeuN (1:1000, 93972, Cell Signaling, USA) for 24 hours at 4°C. Then, sections were washed 5-6 times with PBS for 20 minutes each and then incubated with secondary antibodies for 2.5 hours at room temperature. Alexa 488 goat anti-rabbit antibody (1:500, 111-545-003, Jackson ImmunoResearch, USA) was used as the primary antibody for c-Fos, Alexa 647 goat anti-mouse antibody (1:500, ab 150115, AbCam, UK) was used as the primary antibody against NeuN. Finally, Anti-Fade Fluorescence mounting medium without DAPI (ab 104135, AbCam, UK) or ProLong mounting medium with DAPI was used. TM The sections were mounted on slides using Diamond Antifade mounting slides (P36962, Veritas, USA).

[0098] To map c-Fos-positive neurons throughout the brain, we selected 84 whole immunostained sections covering the area from 2.53 mm to -5.77 mm for each brain sample, with statistical analysis performed every 100 μm. c-Fos-positive neurons were counted using an advanced version of a cell counting program from our previous study. This procedure has five steps: Step 1 is image preparation. We used ZEN Blue to output images, which were then cropped and straightened using Adobe Photoshop. Step 2 requires taking the hemispheric images from Step 1 and registering them with the Allen Reference Atlas (https: / / scalablebrainatlas.incf.org / mouse / ABA_v3), renaming them according to the numbers in the Allen Reference Atlas. Next, Step 3 was run to match each brain image to the selected Allen Reference Atlas by performing a reference point-based triangulation of the slice image followed by a nonlinear affine transformation. In Step 4, a CNN model was used to detect c-Fos-positive neurons in the output of Step 2. After checking and correcting the results of step 4, step 5 uses the results of steps 3 and 4 to complete the cell count, outputting the count and density of c-Fos-positive neurons in different brain regions. Finally, we extract the data required for hierarchical clustering and perform functional network analysis.

[0099] In a specific embodiment, statistical analysis was performed using LAB and GraphPad Prism version 8 (GraphPad Software, Inc). To compare data between different groups, we used a two-way analysis of variance (VAR) followed by Tukey's multiple comparison test, while for comparisons between two groups, we used an unpaired Student's t test. Data with error bars are presented as mean ± SEM. In the context of assessing correlations, Spearman's rank correlation coefficient (rho) and associated p-values were calculated and reported. The Spearman rank correlation coefficient represents a nonparametric correlation index designed to assess the monotonic relationship between two variables without assuming linearity or nonlinearity. This coefficient is derived from the ranking of the variables and effectively ignores the exact observed values. To assess the correlation between c-Fos expression and mean reaction time, we used the Spearman correlation coefficient of c-Fos density.

[0100] In one embodiment, to further explore how mice carrying the Calhm2 V136G point mutation affect the activity of ACA neurons, we used real-time fluorescence quantitative PCR (QPCR) to detect the level of Calhm2 protein, and simultaneously detected the release of ATP by in vivo microdialysis and brain tissue ATP assay. The results showed that although the V136G point mutation did not significantly affect the expression of Calhm2 protein in the mouse prefrontal cortex and hippocampus ( Figure 8 ), but it can significantly reduce the amount of ATP released in PFC ( Figure 9 and Figure 11 ).

[0101] In a specific embodiment, the mouse genotype identification includes the following method: (1) the toes and tails of mice of appropriate age are removed according to their numbers and placed in EP tubes, and the identification is performed using the Biyuntian Mouse Tail Gene Rapid Identification Kit; (2) a digestion solution consisting of 96 μL DNA Extraction Solution and 4 μL Enzyme Mix is added to each tube, mixed and placed in a 55°C oven overnight; (3) after the overnight mouse tails are placed in a 95°C metal bath for 5 minutes, 100 μL of Stop Solution is added to each sample and mixed; (4) the total reaction system is 20 μL, containing 10 μL of Easy-Load TM PCR Master Mix, 7.4 μL ultrapure water, 0.08 μL of each of the front and back primers with a concentration of 100 μM, and 1 μL of template; (5) PCR instrument was used for the reaction, and the reaction products were stored at 4°C and DNA sequencing was performed in time.

[0102] In summary, we found that the Calhm2 V136G point mutation caused attention deficit in adult mice ( Figure 7 ), accompanied by the release of ATP in the ACA brain region ( Figure 9 and Figure 11 ) and a decrease in neuronal activity levels ( Figure 10 This study confirmed that Calhm2 V136G is a key molecule causing attention impairment. It also revealed the potential of ATP in treating attention, providing a new target for in-depth analysis of the mechanism of attention deficit and its related diseases.

[0103] Figure 2 Schematic diagram of a device for constructing an attention deficit animal model provided by an embodiment of the present invention. Specifically, the device includes:

[0104] A single or multiple processors, and a memory for storing a single or multiple computer programs, which, when executed by the single or multiple processors, implement:

[0105] Operation 1, designing sgRNA sequences that recognize the Calhm2 genome;

[0106] Operation 2: microinjecting the sgRNA, Cas9 mRNA, and mutant oligonucleotide into mouse fertilized eggs for homologous recombination, and transplanting them into surrogate mother mice to obtain F0 generation mice;

[0107] Operation 3, performing PCR genotyping and sequence analysis on the F0 generation mice to obtain positive F0 generation mice carrying the Calhm2V136G point mutation;

[0108] Operation 4, breeding the positive F0 generation mice carrying the Calhm2 V136G point mutation with wild-type mice to obtain F1 generation mice with stable inheritance;

[0109] Operation 5, performing PCR genotyping and sequence analysis on the F1 generation mice to obtain positive F1 generation mice carrying the Calhm2V136G point mutation;

[0110] Operation 6: hybridize the positive F1 generation mice carrying the Calhm2 V136G point mutation, and screen homozygous mutant mice carrying the Calhm2 V136G point mutation using PCR genotyping and sequence analysis technology, which are animal models of attention deficit disorder.

[0111] In one embodiment, the target site of the sgRNA is exon 2 of the Calhm2 gene; and the mutant oligonucleotide comprises a V136G (GTG to GGT) mutation site.

[0112] In one embodiment, the apparatus further comprises operation 7 of verifying the attention phenotype of the attention deficit animal model.

[0113] In one embodiment, the verification comprises testing the attention of the attention deficit animal model using a five-hole attention test system;

[0114] The results of the verification show that the attention level is significantly reduced, indicating that the attention deficit animal model is successfully constructed.

[0115] In addition, the present invention also provides an embodiment of the application of the mutant gene Calhm2 V136G in constructing an attention deficit animal model;

[0116] Optionally, the attention deficit animal model is an attention deficit mouse model.

[0117] This study discovered and verified for the first time that the mutant gene Calhm2 V136G can cause the phenotype of attention deficit in mice, providing a new target for in-depth analysis of the mechanism of attention deficit and its related diseases.

[0118] The validation results of this validation example show that assigning inherent weights to indications can moderately improve the performance of the method relative to the default setting.

[0119] Those skilled in the art will clearly understand that, for the convenience and brevity of description, the specific working processes of the systems, devices and units described above can refer to the corresponding processes in the aforementioned method embodiments and will not be repeated here.

[0120] In the several embodiments provided in this application, it should be understood that the disclosed systems, devices and methods can be implemented in other ways. For example, the device embodiments described above are merely schematic. For example, the division of the units is merely a logical function division. In actual implementation, there may be other division methods, such as multiple units or components can be combined or integrated into another system, or some features can be ignored or not executed. Another point is that the mutual coupling or direct coupling or communication connection shown or discussed can be an indirect coupling or communication connection through some interfaces, devices or units, which can be electrical, mechanical or other forms.

[0121] The units described as separate components may or may not be physically separate, and the components shown as units may or may not be physical units, that is, they may be located in one place or distributed across multiple network units. Some or all of these units may be selected to achieve the purpose of this embodiment according to actual needs.

[0122] In addition, the functional units in the various embodiments of the present invention may be integrated into a single processing unit, each unit may exist physically separately, or two or more units may be integrated into a single unit. The aforementioned integrated units may be implemented in the form of hardware or software functional units.

[0123] Those skilled in the art will understand that all or part of the steps in the various methods of the above embodiments can be completed by instructing related hardware through a program, and the program can be stored in a computer-readable storage medium, which may include: read-only memory (ROM), random access memory (RAM), disk or optical disk, etc.

[0124] Those skilled in the art will understand that all or part of the steps in the above-mentioned embodiment method can be implemented by instructing the relevant hardware through a program, and the program can be stored in a computer-readable storage medium. The above-mentioned storage medium can be a read-only memory, a disk or an optical disk, etc.

[0125] The above is a detailed introduction to a computer device provided by the present invention. For those skilled in the art, according to the concept of the embodiments of the present invention, there may be changes in the specific implementation methods and application scope. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. A device for constructing an attention deficit animal model, characterized in that: The device comprises: A single or multiple processors, and a memory for storing a single or multiple computer programs, which, when executed by the single or multiple processors, implement: Operation 1, designing sgRNA sequences that recognize the Calhm2 genome; Operation 2: microinjecting the sgRNA, Cas9 mRNA, and mutant oligonucleotide into mouse fertilized eggs for homologous recombination, and transplanting them into surrogate mother mice to obtain F0 generation mice; Operation 3, performing PCR genotyping and sequence analysis on the F0 generation mice to obtain positive F0 generation mice carrying the Calhm2 V136G point mutation; Operation 4, breeding the positive F0 generation mice carrying the Calhm2 V136G point mutation with wild-type mice to obtain F1 generation mice with stable inheritance; Operation 5, performing PCR genotyping and sequence analysis on the F1 generation mice to obtain positive F1 generation mice carrying the Calhm2 V136G point mutation; Operation 6, hybridizing the positive F1 generation mice carrying the Calhm2 V136G point mutation, and screening homozygous mutant mice carrying the Calhm2 V136G point mutation using PCR genotyping and sequence analysis technology, which are attention deficit disorder animal models; The target site of the sgRNA is exon 2 of the Calhm2 gene; The mutant oligonucleotide comprises a mutation site that produces a V136G point mutation; The apparatus further comprises an operation 7 of verifying the attention phenotype of the attention deficit animal model; The verification comprises testing the attention of the attention deficit animal model using a five-hole attention test system; The results of the verification show that the attention level is significantly reduced, indicating that the attention deficit animal model is successfully constructed.

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