Construction system and device of attention deficit animal model

Abstract

The invention discloses a system and a device for constructing an attention deficit animal model, and finds that Calhm < 2 > V136G point mutation is a key molecule influencing attention for the first time, and a mouse model carrying the Calhm < 2 > V136G point mutation shows attention deficit, and on the basis of the Calhm < 2 > V136G point mutation, the attention deficit animal model has the attention deficit function. The invention creatively develops the system and the device for constructing the attention deficit animal model carrying Calhm < 2 > V136G point mutation, provides an effective animal model for action mechanism research and drug screening of attention deficit or related diseases thereof, and has a wide application prospect.

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 construction system and a device for an attention deficit animal model. Background Art

[0002] Attention deficit disorder, also known as attention deficit (ADD), is a common neurodevelopmental disorder, mainly characterized by inattention, impulsivity and hyperactivity. This condition usually occurs in childhood and in a few cases continues into adulthood. The causes of attention deficit are diverse and may be related to biological, social and psychological factors. Attention deficit has a significant impact on the patient's life and study. Patients often show 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 ensures people function effectively in a complex and information-rich world. A deep understanding of the mechanisms behind attention deficit is crucial. Animal models play an important role in summarizing and studying the mechanisms related to different diseases or conditions, and are also an important transitional stage in human clinical application research. However, the complex mechanisms of attention deficit also make the development of preclinical attention deficit animal models challenging. Therefore, there is currently a lack of an attention deficit animal model in the field for studying the mechanism of action and drug screening of attention deficit or its related diseases. Summary of the invention

[0004] The present invention aims to solve the technical problem that there is still a lack of an attention deficit animal model in the prior art for studying the mechanism of action of attention deficit or its related diseases and screening drugs. To this end, the present invention provides a system and device for constructing an attention deficit animal model.

[0005] Calcium Homeostasis Modulator (Calhm) is a functional protein mainly associated with the regulation of calcium ions and adenosine 5'-triphosphate (ATP). Calhm2, as a member of the Calhm family, is highly expressed in the central nervous system and is an ATP release channel. Knocking out Calhm2 in Alzheimer's disease (AD) model mice can significantly improve the spatial learning and memory function of AD mice.

[0006] At present, there is no research or report on the relationship between the existence of Calhm2 V136G point mutation and attention deficit. The present invention discovers for the first time that Calhm2 V136G point mutation is a key molecule affecting attention, and the mouse model carrying Calhm2 V136G point mutation exhibits attention deficit. Based on this, the present invention provides a system and device for constructing an attention deficit animal model in the art.

[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 comprises the following units:

[0010] Design sequence unit, design sgRNA sequence that recognizes Calhm2 genome;

[0011] The first processing unit is to microinject the sgRNA, Cas9 mRNA, and mutant oligonucleotide into mouse fertilized eggs for homologous recombination, and then transplant them 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 to obtain F1 generation mice with stable inheritance;

[0014] The fourth processing unit is to perform 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 animal models.

[0016] Further, 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 using a five-hole attention test system to detect the attention of the attention deficit animal model.

[0020] Furthermore, the verification results showed that the attention level was significantly reduced, indicating that the attention deficit animal model was successfully constructed.

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

[0022] Further, the device comprises:

[0023] A single or multiple processors, and a memory, the memory being used to store a single or multiple computer programs, which when executed by the single or multiple processors implement:

[0024] Operation 1, designing sgRNA sequence that recognizes 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 mice carrying the Calhm2 V136G point mutation with wild-type mice to obtain F1 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, hybridizing the positive F1 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 animal models.

[0030] Further, 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 also includes operation 7, verifying the attention phenotype of the attention deficit animal model.

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

[0034] The verification results 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 discovers for the first time that the Calhm2 V136G point mutation is a key molecule that affects attention. The mouse model carrying the Calhm2 V136G point mutation exhibits attention deficit. Based on this, the present invention creatively develops a system and a device for constructing an attention deficit animal model carrying the Calhm2 V136G point mutation. The present invention provides an effective animal model for the study of 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 drawings required for use in the description of the embodiments will be briefly introduced below. 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 is the result graph 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 social ability of mice;

[0045] Figure 6 This is the result graph corresponding to the fact that there is no difference in learning ability between the attention-deficit mice carrying the Calhm2 V136G point mutation and the wild-type mice;

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

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

[0048] Fig. 9 The results of the graphs are the corresponding ATP release in the PFC and hippocampus of the attention-deficient mice carrying the Calhm2 V136G point mutation and the wild-type mice;

[0049] Fig.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] Fig.11 The graph shows the results of ATP release in the anterior cingulate region 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 above-mentioned drawings, multiple operations appearing in a specific order are included, but it should be clearly understood that these operations may not be executed in the order in which they appear in this document or may be executed in parallel. The serial numbers of the operations, such as 101, 102, 103, etc., are only used to distinguish different operations, and the serial numbers themselves do not represent any execution order. In addition, these processes may include more or fewer operations, and these operations may be executed in sequence or in parallel.

[0053] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. 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 creative work are 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: a 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: A fifth processing unit, 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 animal models.

[0061] In a specific embodiment, the present invention adopts 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 is selected as the target site. The V136G (GTG to GGT) mutation site in the donor oligomer is introduced into exon 2 by homology-directed repair. In addition, a silent mutation (TCC to TCG) is introduced to prevent gRNA binding and re-cutting of the sequence after homology-directed repair. Cas9 mRNA, sgRNA and donor oligonucleotide 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 mice were bred to the next generation (F1) and 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 Cas9mRNA can be purchased from conventional purchase channels. For example, the sequence of the target gene (Calhm2) is obtained through an online database (such as NCBI). The online sgRNA design platform (https: / / design.synthego.com / # / ) can be used to analyze the target sequence (20bp) of the target gene, and select targets with high editing efficiency scores and low off-target scores. The full-length sequence of the sgRNA is obtained by chemical synthesis.

[0065] In one embodiment, the mutant oligonucleotide can be designed and synthesized by conventional methods in the art. For example, the mutant oligonucleotide can be prepared by using a commercial kit (eg, 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 the 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 includes 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 constructed.

[0073] In one embodiment, the animal is a mouse. The mice used in the experiment were Calhm2V136G mutant mice with C57BL / 6 background and wild-type control mice of the same age, which were constructed and bred in the laboratory. The breeding and feeding of mice strictly complied with the relevant regulations of the Beijing Laboratory Animal Management Office, and 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-like behavior mouse models. As a highly social animal, mice have rich and varied social behaviors. The autistic-like behavior mouse model shows symptoms similar to humans in social interaction, such as game behavior (such as chasing, fighting), social exploration (such as contact, sniffing), sexual activity and reduction of aggressive behavior. Specific detection methods include: young mouse social interaction detection, three-box social experiment.

[0077] Social interaction test of young mice: The test subjects are 21-24 day old mice. The experimental site can be a standard mouse cage or other place where the interaction of mice can be observed in detail. After the young mice are isolated and raised alone for 1 hour, they are matched in pairs of the same gender and age and placed in the observation area at the same time. After 10 minutes of video recording, parameters such as nose-to-nose sniffing, opposite movement, following and other behaviors are analyzed.

[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. The specific methods for detecting the stereotyped movements and narrow interest activities include: stereotyped grooming behavior, 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 are recorded by video for 10-15 minutes (or continuously for 30 minutes or 2 hours) to evaluate stereotyped repetitive movements of mice with autistic-like behavior.

[0081] Stereotype (learning reversal) test: Assess stereotyped thinking by observing mice’s stereotyped patterns in daily behavior and performance in specific tasks. For example, use a T-maze or water maze to test the flexibility of mice to switch from established habits to new habits.

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

[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 at appropriate positions in each box, and the mice are placed in the middle of the end far from the object, and the mice are recorded to explore the two old objects for 5-10 minutes, and the mice are taken out, and the box and objects are wiped with alcohol to remove the odor; (4) the second stage: after 1-2 hours, one of the old objects is replaced with another new object with different color and shape before the start, and the mice are recorded to explore 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 implementation scheme, the sugar water preference experiment includes the following steps: (1) Before the start of the experiment, the mice are housed in a single cage for 1-2 days to adapt. 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 to adapt for one day without weighing the water; (3) On the second day, one of the bottles of sugar water is replaced with pure water, and the water is weighed before being placed; (4) The mice are housed in the above manner for 3 days, the positions of the two bottles of water are changed every day, and the water 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 tested for 7 days, and the average value is taken as the sugar water preference value of the mice.

[0085] In a specific implementation scheme, the buried bead experiment includes the following steps: (1) placing mice in a behavior room for 1-2 hours in advance, laying bedding about 5 cm thick 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 mice in the middle of the cage at the beginning, closing the lid, and conducting the experiment for 30 minutes; (3) taking out the mice, 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 60 cm × 40 cm × 422 cm, divided into three chambers. Each partition has a 4 cm × 4 cm middle channel at the bottom to connect the three chambers, so that the experimental animals can move freely in the three chambers; (2) in the first stage, the channels on both sides are blocked, and the mice are placed in the middle chamber. Nothing is placed in the chambers on both sides, and 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 mouse 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 mouse explores 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 at each stage is statistically analyzed.

[0087] In a specific embodiment, all data statistics and analysis were performed using GraphPad and ImageJ software. Adobe Photoshop was used to adjust the contrast of the image data, and the Unpaired Student's two-tailed t test was used to determine the significance between the data. In all experiments, p<0.05 was considered to be statistically significant. ns>0.05, p<0.05 is *, P<0.01 is **, p<0.001 is ***, and p<0.0001 is ****.

[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 there was no statistical difference in the learning ability of Calhm2 V136G mice compared with the control group mice in the same litter, 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 displayed 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 on) to obtain a reward. In order to complete this task, the animal is required to maintain attention and monitor a column 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 manipulated by decreasing 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) to achieve optimal performance levels. In addition, this modified basic task structure allows testing of selective (focused) attention by inserting irrelevant and distracting stimuli (most commonly auditory paradigms, insertion of self-noise distractors) during the ITI when the animal must attend to the occurrence of the target visual stimulus.

[0091] In a specific implementation scheme, the five-hole attention test includes the following steps: (1) Fasting: Take healthy and normal WT and Calhm2 V136G male mice aged 8 weeks, and raise them in single cages with 14 mice in each group, and count the initial normal weight of the mice; (2) After 1-2 days of adaptation to single cage raising, fast and raise them for 1-2 days. After the weight of the mice reaches 90%-80% of the original weight, the weight is controlled within this range by feeding; Handle the mice for 2-4 days; (3) Preparation phase procedure: All the LED screens of the five holes 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 choose. When the mouse touches the lit screen, it is judged to be correct, accompanied by a drip, a piece of food falls into the food trough, and the food trough light lights up again. When the food trough light is on, unless the mouse touches the food trough, the light will not go out, 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 mouse's choice; ITI: the time from the trough light off to the LED screen turning on; TP: the punishment duration; ②The percentage of correct answers in the total number of trials (Correct percent), Correct percent = Correct count / Trial count; ③Accuracy, Accuracy = Correct count / (Correct count+Error count); ④Omission rate (Omission), Omission = Omission count / (Correct count+Error count+Omission count); ⑤Premature, Premature = Premature count / Trial count; (5)Stage 1-6 training stage procedure: ①At 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; ②After 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. Poking the correct screen is judged as correct; poking the wrong screen is judged as error; not making a choice after LH 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 impulsiveness: 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, unless the mouse touches the food trough, the light will not go out 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 stage is that SD or ITI appears randomly at three different values, and other parameters are the same as the basic training stage; (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 number 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 ( Fig.10 ).

[0096] In a specific embodiment, the histological and immunostaining methods involved are as follows: After anesthesia with tribromoethanol (200 mg / kg intraperitoneal), mice are perfused transcardially with saline (4°C), then 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. The sections are collected in flat plates or mounted on gel-coated slides for immunostaining.

[0097] To stain the nuclei, sections were incubated in 1:5000 DAPI solution (D1306, Invitrogen, USA) for 1 min or covered with 50% glycerol mounting medium containing 1:2000 DAPI. For immunohistochemical staining, selected sections were collected in cell culture plates and washed three times with PBS for 15 min each. Then, sections were permeabilized three times with PBS containing 0.3% Triton X-100 (PBST) for 10 min each. After incubation in blocking solution (3% BSA in PBST) for 90 min 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 h at 4 °C. Then, sections were washed 5-6 times with PBS for 20 min each and then incubated with secondary antibodies for 2.5 h at RT. 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 for 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 in the whole brain, we selected 84 whole immunostained sections covering each brain sample from 2.53 mm to −5.77 mm of the anterior tectum, and statistical analysis was performed every 100 μm. The c-Fos-positive neurons were counted using an advanced version of the cell counting program from our previous study. The program has the following five steps: Step 1 is image preparation. We used ZEN blue to output the image, and then used Adobe Photoshop to crop and straighten it. Step 2 requires taking the hemisphere images from step 1 and registering them with the Allen reference atlas (https: / / scalablebrainatlas.incf.org / mouse / ABA_v3), and then renaming them according to the numbers in the Allen reference atlas. Then, step 3 was run to match each brain image to the selected Allen reference atlas by performing a reference point-based triangulation segmentation of the slice image followed by a nonlinear affine transformation. In step 4, the 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 calls the results of steps 3 and 4 to complete the cell counting and output the counts and densities 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 indicator designed to assess the monotonic relationship between two variables without assuming linearity or nonlinearity. This coefficient is derived from the ordering of the variables, effectively ignoring 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, in order to further explore how mice carrying the Calhm2 V136G point mutation affect the activity of ACA neurons, we used real-time fluorescence quantitative PCR (Quantitative PCR, QPCR) to detect the Calhm2 protein content, and simultaneously detected the ATP release by in vivo microdialysis and brain tissue ATP determination. The results showed that although the V136G point mutation did not significantly affect the expression of Calhm2 protein in the prefrontal cortex and hippocampus of mice ( Figure 8 ), but it can significantly reduce the amount of ATP released in PFC ( Fig. 9 and Fig.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 the number 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 forward and backward primers with a concentration of 100 μM, and 1 μL of template; (5) Perform the reaction using a PCR instrument, store the reaction product at 4°C, and perform DNA sequencing in a timely manner.

[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 ( Fig. 9 and Fig.11 ) and a decrease in neuronal activity levels ( Fig.10 ). This study confirmed that Calhm2 V136G is a key molecule that causes attention impairment. It also revealed the potential of ATP in the treatment of attention, and provided 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 comprises:

[0104] A single or multiple processors, and a memory, the memory being used to store a single or multiple computer programs, which when executed by the single or multiple processors implement:

[0105] Operation 1, designing sgRNA sequence that recognizes 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 mice carrying the Calhm2 V136G point mutation with wild-type mice to obtain F1 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, hybridizing the positive F1 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 animal models.

[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 includes testing the attention of the attention deficit animal model using a five-hole attention test system;

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

[0115] In addition, the embodiments of the present invention also provide the use 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] The present invention 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 disease research.

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

[0119] Those skilled in the art can 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 the present 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 only schematic. For example, the division of the units is only a logical function division. There may be other division methods in actual implementation, 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 separated, and the components shown as units may or may not be physical units, that is, they may be located in one place or distributed on multiple network units. Some or all of the units may be selected according to actual needs to achieve the purpose of the solution of this embodiment.

[0122] In addition, each functional unit in each embodiment of the present invention may be integrated into one processing unit, or each unit may exist physically separately, or two or more units may be integrated into one unit. The above-mentioned integrated unit may be implemented in the form of hardware or in the form of software functional units.

[0123] A person of ordinary skill in the art can 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, and the storage medium may include: a read-only memory (ROM), a random access memory (RAM), a disk or an optical disk, etc.

[0124] A person skilled in the art can understand that all or part of the steps in the above-mentioned embodiment method can be completed by instructing related 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 a person skilled in the art, according to the concept of the embodiments of the present invention, there may be changes in the specific implementation method and application scope. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. A system for constructing an attention deficit animal model, characterized in that: The system comprises the following units: Design sequence unit, design sgRNA sequence that recognizes Calhm2 genome; The first processing unit is to microinject the sgRNA, Cas9 mRNA, and mutant oligonucleotide into mouse fertilized eggs for homologous recombination, and then transplant them into surrogate mother mice to obtain F0 generation mice; The second processing unit performs PCR genotyping and sequence analysis on the F0 generation mice to obtain positive F0 generation mice carrying the Calhm2 V136G point mutation; The third processing unit is to breed the positive F0 generation mice carrying the Calhm2 V136G point mutation with wild-type mice to obtain F1 generation mice with stable inheritance; The fourth processing unit is to perform PCR genotyping and sequence analysis on the F1 generation mice to obtain positive F1 generation mice carrying the Calhm2 V136G point mutation; 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 animal models.

2. The system for constructing an attention deficit animal model according to claim 1, characterized in that: The target site of the sgRNA is exon 2 of the Calhm2 gene; The mutant oligonucleotide comprises a V136G (GTG to GGT) mutation site.

3. The system for constructing an attention deficit animal model according to claim 1, characterized in that: The system also includes a sixth processing unit for verifying the attention phenotype of the attention deficit animal model.

4. The system for constructing an attention deficit animal model according to claim 3, characterized in that: The verification includes testing the attention of the attention deficit animal model using a five-hole attention test system.

5. The system for constructing an attention deficit animal model according to claim 4, characterized in that: The verification results show that the attention level is significantly reduced, indicating that the attention deficit animal model is successfully constructed.

6. A device for constructing an attention deficit animal model, characterized in that: The device comprises: A single or multiple processors, and a memory, the memory being used to store a single or multiple computer programs, which when executed by the single or multiple processors implement: Operation 1, designing sgRNA sequence that recognizes 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 Calhm2V136G point mutation; Operation 4, breeding the positive F0 mice carrying the Calhm2 V136G point mutation with wild-type mice to obtain F1 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 Calhm2V136G point mutation; Operation 6, hybridizing the positive F1 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 animal models.

7. The device for constructing an attention deficit animal model according to claim 6, characterized in that: The target site of the sgRNA is exon 2 of the Calhm2 gene; The mutant oligonucleotide comprises a V136G (GTG to GGT) mutation site.

8. The device for constructing an attention deficit animal model according to claim 6, characterized in that: The apparatus further comprises operation 7 of verifying the attention phenotype of the attention deficit animal model.

9. The device for constructing an attention deficit animal model according to claim 8, characterized in that: The verification includes testing the attention of the attention deficit animal model using a five-hole attention test system; The verification results show that the attention level is significantly reduced, indicating that the attention deficit animal model is successfully constructed.

10. Application of the mutant gene Calhm2 V136G in constructing an animal model of attention deficit disorder; Optionally, the attention deficit animal model is an attention deficit mouse model.

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