A method for constructing a mouse model of autism spectrum disorder by BAF155 conditional knockout mode

Abstract

The present application belongs to the field of animal models, and more specifically, a method for constructing an autism spectrum disorder mouse model by BAF155 conditional knockout mode is disclosed. By constructing a specific knockout BAF155 mouse, the effect of knockout BAF155 on OL development and myelination is observed. Further proved by the behavior experiment, the deletion of BAF155 in oligodendrocyte precursor cells will lead to the appearance of the core and characteristic ASD-like behavior phenotype of mice mainly with social preference disorder and repetitive stereotyped behavior. Therefore, the present application successfully constructs a new genetic molecular defect ASD animal model, and the advantage lies in that the target is clear, a single genetic molecular defect can be induced to study ASD, and a suitable and matched animal model for ASD caused by mutation of BAF155 is provided, which is helpful for precise diagnosis and subsequent intervention strategy.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of animal models. More particularly, it relates to a method for constructing an autism spectrum disorder mouse model by using a BAF155 conditional knockout mode. BACKGROUND

[0002] Autism spectrum disorder (ASD) has high heterogeneity in symptoms and causes, and more than 1000 ASD-related genes have been found. Animal models are essential for the study of ASD. The advantage of a gene molecular defect ASD animal model is that it has a clear target and can study ASD by inducing a single gene molecular defect. Through animal models, the correlation between ASD genotypes and different phenotypes can be summarized to explore the best intervention effect of different genotypes. However, although many ASD-related risk genes have been found in existing research, it is difficult to induce typical ASD-like behavior phenotypes in most cases, or only a single symptom that triggers complex ASD performance can be explained, and the complex overall symptom performance of ASD and its underlying etiological mechanisms have not been effectively explained.

[0003] Therefore, it is of great significance to study and prepare an ASD animal model that can replicate a series of ASD-related core symptoms and associated symptoms for the exploration of ASD-related research, treatment drugs, and treatment programs. SUMMARY

[0004] The present application aims to provide a new preparation method of a genotype autism spectrum disorder mouse model that can replicate a series of ASD-related core symptoms and associated symptoms.

[0005] The first object of the present application is to provide the application of BAF155 gene in constructing an autism spectrum disorder mouse model.

[0006] The second object of the present application is to provide a method for constructing an autism spectrum disorder mouse model.

[0007] The third object of the present application is to provide a method for constructing a BAF155 conditional gene knockout mouse model.

[0008] The fourth object of the present application is to provide a mouse model prepared by the above method and its application.

[0009] The above objects of the present application are achieved by the following technical solutions:

[0010] We found that knockout of BAF155 in oligodendrocyte precursor cells (OPC) leads to myelination defects in multiple brain regions related to ASD, and the behavioral experiments also confirmed that the mice appeared ASD-like behavior phenotype mainly with social preference disorder and repetitive stereotyped behavior.

[0011] Therefore, the present application claims the following scheme:

[0012] The application provides a method for constructing an autism spectrum disorder mouse model, and the BAF155 gene of the mouse is knocked out or the expression of the BAF155 gene of the mouse is reduced, wherein the NCBI Gene ID of the BAF155 gene is 20588.

[0013] The application provides a method for constructing an autism spectrum disorder mouse model, and the BAF155 gene of the mouse is knocked out or the expression of the BAF155 gene of the mouse is reduced, wherein the NCBI Gene ID of the BAF155 gene is 20588.

[0014] As an alternative embodiment, the knockout mouse BAF155 gene is a sequence knocked out on the 4th exon.

[0015] The application provides a method for constructing a BAF155 conditional gene knockout mouse model, wherein the 4th exon of the mouse BAF155 gene is modified by flox to obtain a flox modified mouse; the flox modified mouse is mated with a Cre tool mouse, and the offspring obtained are mated with each other to obtain a BAF155 gene conditional knockout mouse model.

[0016] Specifically, the Cre enzyme of the Cre tool mouse is specifically expressed in oligodendrocytes.

[0017] As an alternative, the 4th exon of the mouse BAF155 gene is modified by flox by using CRISPR / Cas9 technology in combination with Cre / LoxP technology, wherein the sgRNA used in the CRISPR / Cas9 technology comprises sgRNA5 and sgRNA8; the sequence of the sgRNA5 is shown in SEQ ID NO. 1, and the sequence of the sgRNA8 is shown in SEQ ID NO. 2.

[0018] As an alternative, the targeting vector used in the CRISPR / Cas9 technology comprises a 5' homologous arm fragment, a BAF155 fragment and a 3' homologous arm fragment, and the sequences are shown in SEQ ID NO. 3-5, respectively.

[0019] As an optional solution, a specific method for flox modification of the 4th exon of the mouse BAF155 gene is as follows: sgRNA, Cas9 mRNA, and a targeting vector are injected into a mouse zygote, and then a surrogate mother is transplanted to obtain F0 generation mice, i.e., the flox modified mice.

[0020] As an optional solution, a primer pair for identifying the genotype of the flox modified mice includes an EGE-ZLF-022-L-GT-F / CKO-3'-D0-R primer pair and a CKO-5'-DO-F / EGE-ZLF-022-R-GT-R primer pair.

[0021] The sequence of the EGE-ZLF-022-L-GT-F is shown in SEQ ID NO. 6, the sequence of the CKO-3'-D0-R is shown in SEQ ID NO. 7, the sequence of the CKO-5'-DO-F is shown in SEQ ID NO. 8, and the sequence of the EGE-ZLF-022-R-GT-R is shown in SEQ ID NO. 9.

[0022] Specifically, after obtaining the F0 generation mice, gene identification is performed, and the positive mice containing the flox sequence in the F0 generation mice are crossed with wild type mice to obtain F1 generation mice.

[0023] As an optional solution, the flox modified mice are crossed with Cre tool mice, and the specific method for crossbreeding the offspring obtained from each other is as follows:

[0024] (1) The flox heterozygous mice in the F1 generation mice are crossed with the Cre tool mice to obtain F2 generation mice.

[0025] (2) The Cre positive flox heterozygous mice in the F2 generation mice are crossbred with each other to obtain F3 generation mice, and the Cre positive flox homozygous mice in the F3 generation mice are screened, i.e., the BAF155 gene knockout mouse model is obtained.

[0026] The application further provides an application scheme of the above mouse model in the preparation of a medicine for treating a BAF155 gene defect related disease.

[0027] Specifically, the BAF155 gene defect related disease includes autism spectrum disorder.

[0028] The application has the following beneficial effects:

[0029] The present application constructs an autism spectrum disorder (ASD) mouse model of BAF155 conditional knockout mode, and when the mouse carrying the flox sequence is mated, a Cre-mediated targeted deletion tool mouse induced by Tamoxifen is generated, and it is proved that the deletion of BAF155 in oligodendrocyte precursor cells can cause the mouse to have core and characteristic ASD-like behavior phenotypes mainly in social preference disorder and repetitive stereotyped behavior. Therefore, the present application successfully constructs a new genetic molecular defect ASD animal model, which has the advantages of clear target and can study ASD by inducing single genetic molecular defect, and provides a suitable and matched animal model for ASD caused by mutation of BAF155, which is helpful for precise diagnosis and subsequent intervention strategy.

[0030] In addition, the current intervention therapy for ASD is mainly based on improving behavior, and the future treatment direction tends to carry out molecular therapy by targeting different genotypes to achieve the best intervention effect. The model lays a foundation for more effectively developing and selecting treatment schemes for ASD patients with partial BAF155 mutation in clinic, further enriches the animal model of genetic molecular defect autism spectrum disorder, and provides experience for the research and treatment of other genotypes of ASD. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 Principle for constructing the targeting vector.

[0032] Figure 2 Schematic diagram of the Southern blot screening strategy for the targeting mouse.

[0033] Figure 3 Diagram of the Cas9 / sgRNA vector constructed in the present application.

[0034] Figure 4 Activity detection results of sgRNA.

[0035] Figure 5 In vitro transcription sgRNA electrophoresis detection results (“-” mark is empty vector)

[0036] Figure 6 Diagram of the targeting vector constructed in the present application.

[0037] Figure 7 Enzyme digestion identification results of the targeting vector.

[0038] Figure 8 Principle for designing the genotype PCR identification primer of F0 generation mouse.

[0039] Figure 9PCR genotyping results of F0 mice (primers used: EGE-ZLF-022-L-GT-F / cKO-3'-DO-R).

[0040] Figure 10 PCR genotyping results of F0 mice (primers used: cKO-5'-DO-F / EGE-ZLF-022-R-GT-R).

[0041] Figure 11 PCR genotyping results of F1 mice (primers used: EGE-ZLF-022-L-GT-F / cKO-3'-DO-R).

[0042] Figure 12 PCR genotyping results of F1 mice (primers used: cKO-5'-DO-F / EGE-ZLF-022-R-GT-R).

[0043] Figure 13 Southern blot results of F1 mice with positive homologous recombination.

[0044] Figure 14 Principle diagram of floxed heterozygous mice mating with tissue-specific Cre mice to achieve tissue-specific knockout of target genes.

[0045] Figure 15 Schematic diagram of mating scheme of F1 heterozygous mice with tissue-specific Cre mice.

[0046] Figure 16 Schematic diagram of mating scheme of heterozygous mice (fl / +, Cre / +).

[0047] Figure 17 Knockout efficiency verification results of transgenic mice; Part A is PDGFRα-CreER; BAF155 flox / flox transgenic mouse model construction diagram; Part B is the brain slice of P14 mice and the in vitro purification of OPC by immunopanning method, immunofluorescence staining results, and identification of the knockout efficiency of BAF155 transgenic mice; N=3, statistical analysis was performed by independent sample t test, the statistical results were expressed in the form of mean ± standard error (SEM), n.s. represents no significant difference in statistical results; * represents p<0.05, the statistical results have statistical difference; ** represents p<0.01, the statistical results have significant difference; *** represents p<0.001, the statistical results have extremely significant difference.

[0048] Figure 18Figure 11 shows the results of the effect of BAF155 knockout on OPC differentiation and proliferation capacity. Part A shows the results of immunofluorescence staining and statistics of PDGFRa in mPFC of control and BAF155 knockout mice at P14. Part B shows the results of immunofluorescence staining and statistics of Olig2 and Ki67 in corpus callosum of control and BAF155 knockout mice at P14. Part C shows the results of immunofluorescence staining and statistics of CC1 in mPFC of control and BAF155 knockout mice at P14. Part D shows the results of in situ hybridization and statistics of MAG positive cells in mPFC of control and BAF155 knockout mice. Statistical analysis was performed using independent sample t test, and the results were expressed as mean ± SEM. In immunofluorescence staining, NWT=5, NKO=5; in in situ hybridization, NWT=3, NKO=3. n.s. represents no significant difference; * represents p<0.05, statistically significant difference; ** represents p<0.01, significant difference; *** represents p<0.001, extremely significant difference.

[0049] Figure 19 Figure 12 shows the results of the effect of BAF155 knockout on myelin development in ASD related brain regions. Part A shows the results of immunofluorescence staining of MBP in mPFC, striatum, corpus callosum, hippocampus and cerebellum of control and BAF155 knockout mice at P14. Part B shows the results of statistics of the difference fold of MBP area. Statistical analysis was performed using independent sample t test, and the results were expressed as mean ± SEM. NWT=5, NKO=5. n.s. represents no significant difference; * represents p<0.05, statistically significant difference; ** represents p<0.01, significant difference; *** represents p<0.001, extremely significant difference.

[0050] Figure 20Figure 6. The behavioral test of the core symptoms of ASD in BAF155 knockout mice at the OPC stage. Part A is a diagram of the three-chamber social interaction test mode. Part B is the proportion of the sniffing time of the control group and the BAF155 knockout group mice to the total sniffing time (the total sniffing time of the left and right side of the tethered cage) in the first stage of the three-chamber social interaction test (social preference test), NWT=12, NKO=13. Part C is the proportion of the sniffing time of the control group and the BAF155 knockout group mice to the total sniffing time (the total sniffing time of the left and right side of the tethered cage) in the second stage of the three-chamber social interaction test (social novelty preference test), NWT=12, NKO=12. Part D is the self-grooming experiment, the cumulative time of the grooming behavior of the control group and the BAF155 knockout group mice within ten minutes of recording time, NWT=11, NKO=10. Statistical analysis was performed by independent sample t test, and the statistical results were expressed in the form of mean ± standard error (SEM), n.s. represents no significant difference in statistical results; * represents p<0.05, there is a statistically significant difference in the statistical results; ** represents p<0.01, there is a significant difference in the statistical results; *** represents p<0.001, there is a very significant difference in the statistical results.

[0051] Figure 21 Figure 7. The detection of anxiety-like behavior in BAF155 knockout mice at the OPC stage. Part A is the track diagram of the control group and the BAF155 knockout group mice in the open field test. Part B is the total activity distance of the control group and the BAF155 knockout group mice in the open field test. Part C is the proportion of the activity distance of the control group and the BAF155 knockout group mice in the central area of the open field to the total distance, NWT=11, NKO=11. Part D is the track diagram of the control group and the BAF155 knockout group mice in the elevated plus maze test. Part E is the proportion of the activity distance of the control group and the BAF155 knockout group mice in the open arm area to the total distance, NWT=12, NKO=11. Statistical analysis was performed by independent sample t test, and the statistical results were expressed in the form of mean ± standard error (SEM), n.s. represents no significant difference in statistical results; * represents p<0.05, there is a statistically significant difference in the statistical results; ** represents p<0.01, there is a significant difference in the statistical results; *** represents p<0.001, there is a very significant difference in the statistical results. DETAILED DESCRIPTION

[0052] The present application will be further described in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present application in any form. Unless otherwise specified, the reagents, methods and equipment used in the present application are conventional reagents, methods and equipment in the technical field.

[0053] Unless otherwise indicated, the reagents and materials used in the following examples are commercially available.

[0054] CRISPR / Cas9 activity detection was performed by Bionasy.

[0055] EGE-ZLF-022-Targeting vector was provided by Bionasy.

[0056] Example 1 Design of BAF155 conditional knockout model mice

[0057] 1. BAF155 gene

[0058] The BAF155 (Smarcc1) gene is located on the positive strand of chromosome 9, with a full length of 108.2 kb. The gene ID of BAF155 on the NCBI website is: 20588. The gene contains 9 transcripts, and the design refers to transcript-001.

[0059] 2. Targeting strategy

[0060] The structure of the BAF155 gene was analyzed, and Exon4 was selected for conditional knockout. The principle of targeting vector construction is shown in Figure 1 The sgRNAs were designed in the non-conserved regions of Intron3-4 and Intron4-5, respectively. The homologous arms at the 5' end and the 3' end were 1.8 kb and 1.2 kb, respectively. The EGE system based on CRISPR / Cas9 developed by Bionasy was used to prepare the model mice.

[0061] 3. Southern blot screening strategy

[0062] In order to screen the gene targeting mice with correct recombination, PCR and Southern blot methods were used for verification. The schematic diagram of Southern blot screening strategy of targeting mice is shown in Figure 2 3' Probe-B and LR Probe were used to verify F1 generation positive mice. The specific design is shown in Table 1. AseI and NcoI were used as Southern blot enzyme digestion sites. 3' Probe-B was used to detect whether correct recombination occurred. If correct recombination occurred, wild type and mutant bands would appear; if correct recombination did not occur, only wild type band would appear. LR Probe was used to detect whether random insertion occurred. If correct recombination occurred, wild type and mutant bands would appear; if random insertion existed, multiple bands would appear.

[0063] Table 1 Southern blot strategy

[0064]

[0065] Example 2 Preparation of BAF155 conditional knockout model mice

[0066] I. Sequencing confirmation of target sequence

[0067] To ensure the efficiency of the designed CRISPR / sgRNA, the C57BL / 6 mouse tail target site sequence was first amplified by PCR and sequenced to ensure that the sgRNA recognition sequence is completely consistent with the DNA sequence of the constructed strain mice. The primer sequences used in PCR are shown in Table 2. The product length of EGE-ZLF-022-5'MSD-F / R primer is 696 bp, and the PCR product length of EGE-ZLF-022-3'MSD-F / R primer is 1508 bp. The C57BL / 6 mouse tail DNA was amplified by PCR and sequenced, and the results showed that the C57BL / 6 mouse tail target sequence was consistent with the sequence given by Genebank and Ensembl.

[0068] Table 2 PCR primer sequences

[0069]

[0070] II. CRISPR / sgRNA design and construction

[0071] 1. Design of sgRNA

[0072] Based on the design principle of sgRNA, 7 sgRNAs were designed in the 5' target site and 3' target site region, respectively. The sgRNA sequences in the 5' target site are sgRNA1-7, and the sgRNA sequences in the 3' target site are sgRNA8-14, as shown in Table 3.

[0073] Table 3 sgRNA sequences

[0074]

[0075] 2. Construction of Cas9 / sgRNA vector.

[0076] According to the designed sgRNA sequence, the corresponding primer was synthesized, and the sgRNA1-14 was connected into the pCS vector (the map is shown in Figure 3 ) by Gibson Assembly, and 14 Cas9 / sgRNA vectors with different sgRNAs were constructed. The ligation product was transformed and sent for sequencing verification after transformation.

[0077] 3. Activity detection of sgRNA

[0078] The sgRNA activity was detected by using CRISPR / Cas9 activity detection method (UCATM method), and the detection results are shown in Figure 4 The sgRNA5 and sgRNA8 were comprehensively selected for the next experiment.

[0079] 4. RNA preparation of sgRNA

[0080] The Cas9 / sgRNA vector connected with sgRNA5 and sgRNA8 sequences was subjected to in vitro transcription to obtain sgRNA for microinjection, and the electrophoretic detection results of the in vitro transcribed sgRNA are shown in Figure 5

[0081] 5. Construction of targeting vector

[0082] According to the targeting scheme of Example 1, the primers were designed to construct the targeting vector. The schematic diagram of the construction of the targeting vector is shown in Figure 6 The LR, A and RR fragments of the mouse BAF155 gene were cloned from the mouse genomic DNA as the template, and the LR, A and RR fragments were sequentially connected to the EGE-ZLF-022-Targeting vector to obtain the specific targeting vector.

[0083] The sequence of the LR fragment is shown in SEQ ID NO. 3:

[0084] ​

[0085] The sequence of the A fragment is shown as SEQ ID NO. 4:

[0086] tgaaggacagtaagaattcatatcactgctacattgttttgactggggtatgtctttacagaaaatttagccttttttttttttaatgtgtgtgtgtctgtgtctgtgtatctgtctgtgtgtaggtcagaggacaatttgggagtctgttatcttcttccactgtggattctagggatgactcatctttggggttatctttctaagccttttaaattcatttaagttaagttccacaatgaaatttccacttgtataaggctgaggataaacttaataaagcttttttttctgttaggcggagatttgatcttcagaacccatcccgaatggatcgtaacgttgaaatgttcatgaacattgagaaaacattggtacaggtaagttggagacttagaccttgttgtctctgtcccactcgctcctttgcctcatctttcctctttcccatctgtcatgttgtagatagggcttctttgatccaggtcagcctaaaatttctgcctcaggagtccctattcctaaatctagatgcataagtcactacctcaacttaaaagtttattttaccctaatatacttctaattctttattagattgcaaaagataaagcgaggaactttactgaaatacgcaattttactgtttttatgtgtattgggattagagctgtaattg.

[0087] The sequence of the RR fragment is shown as SEQ ID NO. 5:

[0088]

[0089] The constructed targeting vector is identified by enzyme digestion and verified by sequencing, and the enzyme digestion identification result is shown in Figure 7 .

[0090] 6. Microinjection of zygotes

[0091] Cas9 RNA, sgRNA, and targeting vector are microinjected into mouse zygotes, and the birth of F0 generation mice after injection is shown in Table 4.

[0092] Table 4. Birth of F0 generation mice

[0093]

[0094] 7. Genotype identification of F0 generation mice

[0095] The present application uses the method of injecting zygotes with Cas9 / sgRNA to prepare conditional knockout mice. Since the F0 generation mice obtained by zygote injection may be chimeras / heterozygotes / homozygotes, the genotype of the F0 generation mice obtained by genotype identification of the mouse tail of the F0 generation mice is only for reference, and does not represent that the gene mutation type has a germ line inheritance. The heritable genotype needs to be determined by F1 generation mice.

[0096] (1) Design of genotype identification primers

[0097] The design principles of F0 generation mouse genotype PCR identification primers are shown in Figure 8 , and the specific identification primer sequences are shown in Table 5, and the reaction conditions of the PCR identification primers are shown in Table 6.

[0098] Table 5. Identification primer sequences

[0099]

[0100] Table 6. Reaction conditions of PCR identification primers

[0101]

[0102] (2) Genotype identification results of F0 generation mice

[0103] The genotype of the F0 generation mice is identified by PCR experiment, and the identification results are shown in Figure 9 and Figure 10 , and the results show that EF22-1, EF22-3, EF22-5, EF22-15, and EF22-20 are F0 generation positive mice.

[0104] 8. Genotype identification of F1 generation homologous recombination mice

[0105] (1) The positive F0 mice EF22-1, EF22-3, EF22-5, EF22-20 were mated with wild type mice to obtain F1 generation, and the mating results are shown in Table 7. The genotype of F1 mice was identified by PCR experiment (primers are shown in Table 5), and the PCR results are shown in Figure 11 and Figure 12 .

[0106] (2) Southern blot detection was performed on F1 generation of positive homologous recombination mice. The tail DNA of the F1 mice identified as positive by PCR was extracted for Southern blot detection, and the detection results are shown in Figure 13 .

[0107] According to the PCR results and Southern blot detection results, 1EF22-7, 1EF22-9, 1EF22-10, 1EF22-11, 1EF22-12, 1EF22-13 and 1EF22-14 are F1 generation positive mice, correct recombination, and no random insertion.

[0108] Table 7 Mating results of positive F0 mice and wild type mice

[0109]

[0110] Example 3 BAF155 conditional knockout model mouse mating scheme

[0111] I. Obtain conditional gene knockout mice

[0112] F1 generation floxed heterozygous mice were mated with tissue-specific Cre mice to achieve tissue-specific knockout of the target gene, and the principle is shown in Figure 14 .

[0113] Step 1: floxed heterozygous mice were mated with tissue-specific Cre mice (Ts-Cre) to obtain fl heterozygous mice, and the mating scheme is shown in Figure 15 . This step can only obtain heterozygous mice (fl / +, Cre / +), and homozygous mice (experimental mice) need to be further mated to obtain. Mice with genotypes (+ / +, Cre / +), (fl / +, + / +), and (+ / +, + / +) can be used as controls.

[0114] Step 2: The obtained heterozygous mice (fl / +, Cre / +) were mated with each other to obtain fl homozygous mice (fl / fl, Cre / +), and the mating scheme is shown in Figure 16 . Mice with genotype (fl / fl, Cre / +) belong to the experimental group, and mice with other genotypes belong to the control group.

[0115] II. Genotype detection

[0116] PCR was used to identify the genotype of mice, and finally the experimental group mice with genotype (fl / fl, Cre / +) were screened. The PCR identification primers are shown in Table 12.

[0117] Table 12 PCR identification primer sequences

[0118]

[0119] Example 4 Verification of transgenic mice

[0120] I. Verification of knockout efficiency of transgenic mice

[0121] The experimental group mice obtained in Example 3 were continuously administered tamoxifen from the 4th to the 8th day after birth and were taken for immunofluorescence staining on the 14th day. In addition, we also purified oligodendrocyte precursor cells (OPC) in vitro culture by immunopanning and performed cell climbing sheet immunofluorescence staining. F1 generation floxed homozygous mice were used as a control group (i.e. BAF155 flox / flox transgenic mice without Cre enzyme).

[0122] The results of the verification of the knockout efficiency of transgenic mice are shown in Table 3, and the results show that the knockout efficiency of BAF155 in BAF155 knockout mice induced by tamoxifen (i.e. PDGFRα-CreER; BAF155 flox / flox transgenic mice) can reach about 70%, indicating that the transgenic mouse model of specific knockout of BAF155 in OPC stage is successfully constructed. Figure 17 II. Effect of specific knockout of BAF155 in OPC stage on OPC differentiation and proliferation ability

[0123] The experimental group mice were continuously administered tamoxifen from the 4th to the 8th day after birth and were taken for observation on the 14th day. F1 generation floxed homozygous mice were used as a control group (i.e. BAF155 flox / flox transgenic mice without Cre enzyme)

[0124] The results of immunofluorescence staining are shown in Table 4, and the results show that there is no significant difference in the number of OPC specific marker PDGFRα and oligodendrocyte lineage marker Olig2 positive cells between the BAF155 knockout group mice and the control group mice. This indicates that knocking out BAF155 in OPC does not affect the number of cells in the entire oligodendrocyte lineage, nor does it affect the number of OPC.

[0125] Figure 18

[0126] ​​In addition, we further observed the change of the number of cells in the proliferation stage by co-labeling Ki67 and Olig2. The results showed that knocking out BAF155 did not affect the proliferation ability of OPCs. Next, we further observed the expression of mature oligodendrocyte (OL) markers. Immunofluorescence staining and in situ hybridization results showed that, compared with the control group of mice, the number of cells positive for CC1 and MAG, markers of mature OLs, in the BAF155 knockout group of mice was significantly reduced. This shows that knocking out BAF155 leads to differentiation disorders of OPCs to mature OLs.

[0127] The above results fully show that specific knockout of BAF155 in the OPC stage leads to differentiation disorders of OPCs without affecting their proliferation ability.

[0128] Example 5 OPC stage-specific knockout of BAF155 leads to myelination disorders in multiple ASD-related brain regions such as mPFC

[0129] Myelin basic protein (MBP) is one of the most abundant proteins in the CNS and plays an important role in the myelination of nerve cells. We observed the expression of MBP in multiple ASD-related brain regions, including the medial prefrontal cortex (mPFC), striatum, corpus callosum (CC), hippocampus (Hip), and cerebellum. The results, as shown in Figure 19 the results showed that, compared with the control group of mice, the expression of MBP in the BAF155 knockout group of mice in the above multiple brain regions was significantly decreased, indicating that the absence of BAF155 in OPCs leads to widespread myelination disorders.

[0130] Example 6 OPC stage-specific knockout of BAF155 leads to social preference disorders and repetitive stereotyped behavior in mice

[0131] Autism Spectrum Disorder (ASD) has high clinical phenotype heterogeneity, and different patients have very different performances in clinical core symptoms and related concomitant symptoms. The core symptoms usually refer to social interaction disorders and repetitive stereotyped behavior. In addition, some patients also show different degrees of anxiety, depression, cognitive impairment, and other concomitant symptoms.

[0132] To verify whether the absence of BAF155 leads to ASD-like behavioral performance in mice, we induced the mice by giving them tamoxifen continuously from the 4th to the 8th day after birth and tested the behavioral performance of the mice at 6-8 weeks.

[0133] One, three-chamber social interaction experiment

[0134] Three-chamber social test is considered as the most reliable method to detect the social ability of mice. Therefore, we first evaluated the social ability of BAF155 knockout mice by three-chamber social test. The schematic diagram of three-chamber social test is shown in Figure 1A. Figure 20

[0135] After the experimental mice were familiar with the test environment, in the first stage of three-chamber social test, we compared the exploration time of BAF155 knockout mice and control mice to the cage with a stranger mouse and the empty cage. The results showed that the proportion of the exploration time of the cage with a stranger mouse to the total exploration time was significantly lower in the knockout group than in the control group (P < 0.05, Figure 1B). This indicates that the knockout mice have certain social preference disorders. Figure 20

[0136] To further evaluate the social novelty preference ability of mice, in the second stage of three-chamber social test, we compared the exploration time of BAF155 knockout mice and control mice to the cage with a new stranger mouse and the cage with a relatively familiar mouse. The results showed that there was no statistical difference in the proportion of the exploration time of the two cages between the knockout group and the control group (P > 0.05, Figure 1C). The above results show that BAF155 deletion can lead to social preference disorders in mice, but has no significant effect on social novelty preference ability. Figure 20

[0137] (2) In addition to social disorders, repetitive stereotypic behavior is also one of the main core symptoms of ASD. Therefore, we evaluated whether BAF155 knockout mice have repetitive stereotypic behavior by self-grooming test. The results showed that under the same experimental conditions, the self-grooming time of BAF155 knockout mice was significantly higher than that of control mice. This indicates that BAF155 deletion can lead to a certain degree of repetitive stereotypic behavior in mice. Figure 20

[0138] Example 7 OPC stage-specific knockout of BAF155 leads to anxiety-like ASD common comorbid symptoms in mice

[0139] In addition to social interaction disorders and repetitive stereotypic behavior, ASD patients usually also show some comorbid symptoms, among which the most common comorbid symptoms are anxiety and cognitive impairment.

[0140] Open field test and elevated plus maze test are widely used to detect anxiety-like behavior in mice. Therefore, we used these two experimental methods to detect whether BAF155 knockout mice have anxiety-like behavior.

[0141] ​​​​(1) In the open field test, we found that the BAF155 knockout mice did not show significant difference in the total distance of activity compared with the control mice, which indicated that the BAF155 knockout mice did not have the influence on the movement ability (Fig. 1A). Figure 21 However, we observed that the distance of activity in the central area of the knockout mice was significantly less than that of the control mice. This indicated that the BAF155 knockout mice had more significant anxiety level compared with the control mice.

[0142] (2) In order to further verify this result, we observed the distance of activity in the open arm range of the BAF155 knockout mice in the elevated plus maze test. The results showed that the distance of activity in the open arm range of the knockout mice was significantly less than that of the control mice (Fig. 1B). Figure 21 This result again verified that the BAF155 knockout mice had anxiety-like behavior performance.

[0143] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, and are included in the protection scope of the present application.

Claims

1. A method for constructing a mouse model of autism spectrum disorder, characterized in that, Flux-modified mice were obtained by flux modification of exon 4 of the mouse BAF155 gene. The flux-modified mice were bred with Cre tool mice, and the offspring were bred with each other to obtain BAF155 gene conditional knockout mice. The BAF155 gene conditional knockout mice were induced with tamoxifen continuously from day 4 to day 8 after birth to establish a mouse model of autism spectrum disorder. The Cre enzyme in the Cre tool mice was specifically expressed in oligodendrocytes.

2. The method according to claim 1, characterized in that, The mouse BAF155 gene exon 4 was modified with flox using CRISPR / Cas9 technology combined with Cre / LoxP technology. The sgRNAs used in the CRISPR / Cas9 technology include sgRNA5 and sgRNA8. The sgRNA5 sequence is shown in SEQ ID NO.1 and the sgRNA8 sequence is shown in SEQ ID NO.

2.

3. The method according to claim 2, characterized in that, The targeting vector used in the CRISPR / Cas9 technology includes a 5' homologous arm fragment, a BAF155 fragment, and a 3' homologous arm fragment, the sequences of which are shown in SEQ ID NO.3-5, respectively.

4. The method according to claim 2 or 3, characterized in that, The specific method for flux modification of exon 4 of the mouse BAF155 gene is as follows: sgRNA, Cas9 mRNA, and the targeting vector are injected together into mouse fertilized eggs and then transplanted into surrogate mothers to obtain F0 generation mice, which are the flux-modified mice.

5. The method according to claim 1, characterized in that, Primer pairs for identifying the genotype of flux-modified mice include the EGE-ZLF-022-L-GT-F / CKO-3'-DO-R primer pair and the CKO-5'-DO-F / EGE-ZLF-022-R-GT-R primer pair; The EGE-ZLF-022-L-GT-F sequence is shown in SEQ ID NO.6, the CKO-3'-DO-R sequence is shown in SEQ ID NO.7, the CKO-5'-DO-F sequence is shown in SEQ ID NO.8, and the EGE-ZLF-022-R-GT-R sequence is shown in SEQ ID NO.

9.

6. The use of the mouse model obtained by any one of the methods described in claims 1-5 in the preparation of drugs for treating diseases related to BAF155 gene defects.

7. The application according to claim 6, characterized in that, The diseases associated with BAF155 gene deficiency include autism spectrum disorder.

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