Method for constructing autism spectrum disorder mouse model by BAF155 conditional knockout mode

Abstract

The invention belongs to the field of animal models, and particularly discloses a method for constructing an autism spectrum disorder mouse model through a BAF155 conditional knockout mode. According to the application disclosed by the invention, the influence of BAF155 knockout on OL development and myelin sheath formation is observed by constructing a specific BAF155 knockout mouse. A behavioral experiment further proves that deletion of BAF155 in oligodendrocyte precursor cells can cause a mouse to have a core and characteristic ASD-like behavior phenotype taking social preference disorder and repeated engraving behaviors as main behaviors. Therefore, a new gene molecule defect ASD animal model is successfully constructed, and the method has the advantages that the target is clear, ASD can be researched by inducing single gene molecule defects, an appropriate and matched animal model is provided for ASD caused by clinical BAF155 mutation, and accurate diagnosis and formulation of subsequent intervention strategies are facilitated.

Description

Technical Field

[0001] This invention belongs to the field of animal model technology. More specifically, it relates to a method for constructing a mouse model of autism spectrum disorder using a BAF155 conditional knockout pattern. Background Technology

[0002] Autism Spectrum Disorder (ASD) symptoms and causes are highly heterogeneous, with over 1000 ASD-related genes identified to date. Animal models are crucial for ASD research, and gene-molecular defect ASD animal models offer the advantage of a clear target, allowing for the study of ASD by inducing defects in a single gene. Animal models can be used to summarize the associations between ASD genotypes and different phenotypes, exploring the optimal intervention efficacy for different genotypes. However, while existing research has identified many ASD-related risk genes, in most cases these genes are difficult to induce typical ASD-like behavioral phenotypes, or can only explain the influence of a single symptom triggering complex ASD manifestations, failing to effectively explain the complex overall symptoms of ASD and its underlying etiological mechanisms.

[0003] Therefore, researching and developing animal models of ASD that can replicate a series of core symptoms and accompanying symptoms is of great significance for the study of ASD, the development of therapeutic drugs, and the exploration of treatment options. Summary of the Invention

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

[0005] The first objective of this invention is to provide the application of the BAF155 gene in constructing a mouse model of autism spectrum disorder.

[0006] A second objective of this invention is to provide a method for constructing a mouse model of autism spectrum disorder.

[0007] The third objective of this invention is to provide a method for constructing a BAF155 conditional gene knockout mouse model.

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

[0009] The above-mentioned objective of this invention is achieved through the following technical solution:

[0010] By constructing a novel specific knockout transgenic mouse, we found that knocking out BAF155 in oligodendrocyte precursor cells (OPC) leads to impaired myelination development in multiple ASD-related brain regions. Behavioral experiments also confirmed that the mice exhibited ASD-like behavioral phenotypes, primarily characterized by social preference disorder and repetitive stereotyped behaviors.

[0011] Therefore, the present invention claims protection for the following solutions:

[0012] The application of the BAF155 gene in constructing a mouse model of autism spectrum disorder, wherein the NCBIGene ID of the BAF155 gene is 20588.

[0013] This invention provides a method for constructing a mouse model of autism spectrum disorder by knocking out the BAF155 gene in mice or reducing the expression of the BAF155 gene in mice, wherein the NCBI Gene ID of the BAF155 gene is 20588.

[0014] As an alternative implementation, the knockout mouse BAF155 gene is a sequence that knocks out exon 4.

[0015] This invention provides a method for constructing a BAF155 conditional gene knockout mouse model, wherein the exon 4 of the mouse BAF155 gene is modified with flux to obtain flux-modified mice; the flux-modified mice are bred with Cre tool mice, and the offspring are bred with each other to obtain a BAF155 gene conditional knockout mouse model.

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

[0017] As an alternative approach, flox modification of exon 4 of the mouse BAF155 gene is performed 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.

[0018] As an optional approach, 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.35.

[0019] As an optional approach, 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.

[0020] As an alternative approach, primer pairs for identifying flox-modified mouse genotypes 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;

[0021] 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.

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

[0023] As an alternative, the flux-modified mice are bred with Cre tool mice, and the resulting offspring are then crossbred. The specific method for crossbreeding these offspring is as follows:

[0024] (1) F2 generation mice were obtained by crossing the F1 generation mice with the Cre tool mice;

[0025] (2) Cross-breed the Cre-positive flox heterozygous mice in the F2 generation to obtain the F3 generation mice. Select the Cre-positive flox homozygous mice in the F3 generation mice to obtain the BAF155 gene knockout mouse model.

[0026] The present invention also provides a method for using the above-mentioned mouse model in the preparation of drugs for treating diseases related to BAF155 gene defects.

[0027] Specifically, the diseases associated with BAF155 gene deficiency include autism spectrum disorder.

[0028] The present invention has the following beneficial effects:

[0029] This invention constructs a BAF155 conditional knockout mouse model of autism spectrum disorder (ASD). When bred with mice carrying the flux sequence, this model produces Tamoxifen-induced Cre-mediated targeted deletion tool mice. Furthermore, it demonstrates that the absence of BAF155 in oligodendrocyte precursor cells leads to core and characteristic ASD-like behavioral phenotypes in mice, primarily characterized by social preference impairment and repetitive, stereotyped behaviors. Therefore, this invention successfully constructs a novel animal model of ASD with a specific genetic defect. Its advantage lies in its clear target; ASD can be studied by inducing a single gene defect. This provides a suitable and matched animal model for clinically induced ASD caused by BAF155 mutations, contributing to accurate diagnosis and the development of subsequent intervention strategies.

[0030] Furthermore, current ASD interventions are mostly based on behavioral improvement, while future treatment directions tend to focus on molecular therapies targeting different genotypes to achieve optimal intervention efficacy. This model lays the foundation for more effective development and selection of treatment options for ASD patients with BAF155 mutations in clinical settings, further enriches animal models of autism spectrum disorders with genetic and molecular defects, and provides experience for research and treatment of other ASD genotypes. Attached Figure Description

[0031] Figure 1 The principles for constructing a target carrier.

[0032] Figure 2 This is a schematic diagram of the Southern blot screening strategy for targeted mice.

[0033] Figure 3 A map showing the construction of the Cas9 / sgRNA vector for this invention.

[0034] Figure 4 This is the result of sgRNA activity assay.

[0035] Figure 5 The results of electrophoresis analysis of sgRNA transcribed in vitro ("-" indicates an empty vector).

[0036] Figure 6 This is a map of the target carrier constructed according to the present invention.

[0037] Figure 7 The results are for enzyme digestion identification of the targeting vector.

[0038] Figure 8 Principles for primer design for PCR identification of F0 generation mouse genotypes.

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

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

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

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

[0043] Figure 13 Southern blot results for F1 generation positive homologous recombinant mice.

[0044] Figure 14 This diagram illustrates the principle of tissue-specific knockout of a target gene by mating a floxed heterozygous mouse with a tissue-specific Cre mouse.

[0045] Figure 15 This is a schematic diagram of the mating protocol between F1 generation heterozygous mice and tissue-specific Cre mice.

[0046] Figure 16 This is a schematic diagram of the mating scheme for heterozygous mice (fl / +, Cre / +).

[0047] Figure 17 Figure A shows the results of validating the knockout efficiency of the transgenic mouse model. Figure B shows the PDGFRα-CreER and the schematic diagram of the construction of the BAF155flox / flox transgenic mouse model. Figure B shows the brain slices of mice at P14 and the immunofluorescence staining results of OPC purified in vitro by immunoopanning, as well as the identification of the BAF155 knockout efficiency of the transgenic mice. N=3. Statistical analysis was performed using an independent samples t-test. The statistical results are expressed as mean ± standard error (SEM). ns represents no statistically significant difference; * represents p<0.05, indicating a statistically significant difference; ** represents p<0.01, indicating a statistically significant difference; *** represents p<0.001, indicating a highly significant difference.

[0048] Figure 18The results show the effects of OPC stage-specific BAF155 knockout on OPC differentiation and proliferation. Figure A shows the immunofluorescence staining and statistical results of PDGFRα in the mPFC region of mice in the control and BAF155 knockout groups at P14. Figure B shows the immunofluorescence staining and statistical results of Olig2 and Ki67 in the corpus callosum region of mice in the control and BAF155 knockout groups at P14. Figure C shows the immunofluorescence staining and statistical results of CC1 in the mPFC region of mice in the control and BAF155 knockout groups at P14. Figure D shows the results of in situ hybridization of mice in the control and BAF155 knockout groups. MAG-positive cells in the mPFC region of BAF155 knockout mice and their statistical results; independent samples t-test was used for statistical analysis, and the statistical results are expressed as mean ± standard error (SEM). In immunofluorescence staining: NWT=5, NKO=5; in in situ hybridization experiment: NWT=3, NKO=3. ns represents no statistically significant difference; * represents p<0.05, statistically significant difference; ** represents p<0.01, statistically significant difference; *** represents p<0.001, highly significant difference.

[0049] Figure 19 The results show the effect of BAF155 deletion in OPC on myelin development in brain regions related to ASD. Figure A shows the immunofluorescence staining results of MBP in the medial prefrontal cortex (mPFC), striatum, corpus callosum (CC), hippocampus, and cerebellum at P14 in control and BAF155 knockout mice. Figure B shows the fold change in MBP area. Statistical analysis was performed using independent samples t-test, and the results are expressed as mean ± standard error (SEM). NWT = 5, NKO = 5, and ns represent no statistically significant difference. * represents p < 0.05, indicating a statistically significant difference; ** represents p < 0.01, indicating a statistically significant difference; *** represents p < 0.001, indicating a highly significant difference.

[0050] Figure 20Behavioral tests for ASD core symptoms in BAF155 mice specifically knocked out during the OPC phase; Figure A shows the three-box social experiment model; Figure B shows the proportion of sniffing time for the cage containing the mouse to the total sniffing time (total sniffing time for both left and right cages) in the first phase (social preference test) of the three-box social experiment for both control and BAF155 knockout mice, NWT=12, NKO=13; Figure C shows the proportion of sniffing time for the cage containing the new, unfamiliar mouse to the total sniffing time (total sniffing time for both left and right cages) in the second phase (social novelty preference test) of the three-box social experiment for both control and BAF155 knockout mice. The proportion of total sniffing time in the restraint cage (NWT=12, NKO=12); Figure D shows the self-grooming experiment, the cumulative time of grooming behavior of mice in the control group and BAF155 knockout group within a ten-minute recording period (NWT=11, NKO=10); Statistical analysis was performed using an independent samples t-test, and the statistical results are expressed as mean ± standard error (SEM). ns represents no statistically significant difference; * represents p<0.05, indicating a statistically significant difference; ** represents p<0.01, indicating a statistically significant difference; *** represents p<0.001, indicating a highly significant difference.

[0051] Figure 21 To detect anxiety-like behavior in mice by OPC-specific BAF155 knockout; Figure A shows the trajectories of mice in the control and BAF155 knockout groups in the open field experiment; Figure B shows the total distance traveled by mice in the control and BAF155 knockout groups in the open field experiment; Figure C shows the proportion of the distance traveled by mice in the central region of the open field to the total distance traveled by mice in the control and BAF155 knockout groups, NWT=11, NKO=11; Figure D shows the trajectories of mice in the control and BAF155 knockout groups in the elevated cross maze experiment; Figure E shows the proportion of the distance traveled by mice in the open arm region to the total distance traveled by mice in the control and BAF155 knockout groups, NWT=12, NKO=11. Statistical analysis was performed using an independent samples t-test. Results are expressed as mean ± standard error (SEM). ns indicates no statistically significant difference; * indicates p<0.05, indicating a statistically significant difference; ** indicates p<0.01, indicating a statistically significant difference; *** indicates p<0.001, indicating a highly significant difference. Detailed Implementation

[0052] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

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

[0054] CRISPR / Cas9 activity assays were performed by Biocytogen.

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

[0056] Example 1: Design of BAF155 Conditional Knockout 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. This gene contains 9 transcripts, and this design references transcript-001.

[0059] 2. Target practice plan

[0060] The structure of the BAF155 gene was analyzed, and Exon4 was selected for conditional knockout. The principles for constructing the targeting vector are as follows: Figure 1 As shown, sgRNAs were designed into the non-conserved regions of Intron 3-4 and Intron 4-5, respectively. The 5' and 3' homologous arms are 1.8 kb and 1.2 kb, respectively. Model mice were prepared using the CRISPR / Cas9-based EGE system developed by Biocytogen.

[0061] 3. Southern blot screening strategy

[0062] To screen for gene-targeted mice that have undergone correct recombination, we used PCR and Southern blot methods for verification. A schematic diagram of the Southern blot screening strategy for targeted mice is shown below. Figure 2 As shown in Table 1, 3'Probe-B and LR Probe were used to verify F1 generation positive mice. The specific design was as shown in Table 1. AseI and NcoI were used as Southern blot restriction sites. 3'Probe-B was used to detect whether correct recombination occurred. If correct recombination occurred, two bands (wild-type and mutant) would appear; if incorrect recombination did not occur, only a wild-type band would appear. LR Probe was used to detect whether random insertions were present. If correct recombination occurred, two bands (wild-type and mutant) would appear; if random insertions were present, multiple bands would appear.

[0063] Table 1. Southemn blot strategy

[0064]

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

[0066] I. Sequencing confirmation of the target sequence

[0067] To ensure the efficiency of the designed CRISPR / sgRNA, the target site sequence of the C57BL / 6 mouse tail first needs to be amplified by PCR and sequenced for verification, ensuring that the sgRNA recognition sequence is completely consistent with the DNA sequence of the constructed mouse strain. The primer sequences used for PCR are shown in Table 2. The PCR product length of the EGE-ZLF-022-5'MSD-F / R primer is 696 bp, and the PCR product length of the EGE-ZLF-022-3'MSD-F / R primer is 1508 bp. PCR amplification and sequencing of the C57BL / 6 mouse tail DNA showed that the C57BL / 6 mouse tail target sequence is consistent with the sequences provided 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 principles of sgRNA, seven sgRNAs were designed in the 5' and 3' target sites, respectively. The sgRNA sequences at the 5' target site are sgRNA1-7, and the sgRNA sequences at the 3' target site are sgRNA8-14, as shown in Table 3.

[0073] Table 3 sgRNA Sequences

[0074]

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

[0076] Primers were synthesized according to the designed sgRNA sequence, and sgRNAs 1-14 were ligated into the pCS vector using seamless cloning (Gibson Assembly) (see diagram). Figure 3 As shown in the figure, 14 Cas9 / sgRNA vectors with different sgRNAs were constructed and obtained. The ligation products were transformed and sequenced to verify that they were correct.

[0077] 3. sgRNA activity detection

[0078] The activity of sgRNA was detected using the CRISPR / Cas9 activity assay (UCATM method), and the results are as follows: Figure 4As shown, sgRNA5 and gRNA8 were selected for the next experimental step.

[0079] 4. RNA preparation of sgRNA

[0080] The Cas9 / sgRNA vector containing sgRNA5 and sgRNA8 sequences was transcribed in vitro to obtain sgRNA for microinjection. The electrophoretic detection results of the transcribed sgRNA are shown below. Figure 5 As shown.

[0081] 5. Construction of the target carrier

[0082] Based on the targeting scheme in Example 1, primers were designed to construct a targeting vector. A schematic diagram of the targeting vector construction is shown below. Figure 6 As shown, using mouse genomic DNA as a template, the LR, A, and RR fragments of the mouse BAF155 gene were cloned, and the LR, A, and RR fragments were sequentially ligated into the EGE-ZLF-022-Targeting vector to obtain a specific targeting vector.

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

[0084]

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

[0086] tgaaggacagtaagaattcatatcactgctacattgttttgactggggtatgtctttacagaaaatttagccttttttttttttaatgtgtgtgtgtctgtgtctgtgtatctgtctgtgtgtaggtcagaggacaatttgggagtctgttatcttcttccactgtggattctagggatgactcatctttggggttatctttctaagccttttaaattcatttaagttaagttccacaatgaaatttccacttgtataaggctgaggataaacttaataaagcttttttttctgttaggcggagatttgatcttcagaacccatcccgaatggatcgtaacgttgaaatgttcatgaacattgagaaaacattggtacaggtaagttggagacttagaccttgttgtctctgtcccactcgctcctttgcctcatctttcctctttcccatctgtcatgttgtagatagggcttctttgatccaggtcagcctaaaatttctgcctcaggagtccctattcctaaatctagatgcataagtcactacctcaacttaaaagtttattttaccctaatatacttctaattctttattagattgcaaaagataaagcgaggaactttactgaaatacgcaattttactgtttttatgtgtattgggattagagctgtaattg.

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

[0088]

[0089] After the constructed targeting vector was verified by enzyme digestion and sequencing, the construction of the targeting vector was confirmed to be complete. The enzyme digestion results are as follows: Figure 7 As shown.

[0090] 6. Intrauterine Injection of Fertilized Eggs

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

[0092] Table 4. Birth details of F0 generation mice

[0093]

[0094]

[0095] 7. Genotyping of F0 generation mice

[0096] This invention uses Cas9 / sgRNA injection into fertilized eggs to prepare conditional knockout mice. Since F0 generation mice obtained by this method may be chimeric / heterozygous / homozygous, the genotypes of F0 generation mice obtained by genotyping their tails are for reference only and do not represent germline inheritance of their gene mutations. Heritable genotypes need to be determined by testing F1 generation mice.

[0097] (1) Primer design for genotyping identification

[0098] Primer design principles for PCR identification of F0 generation mice, such as... Figure 8 As shown in Table 5, the specific primer sequences for identification are shown in Table 6, and the reaction conditions for PCR identification primers are shown in Table 6.

[0099] Table 5. Primer sequences for identification

[0100]

[0101] Table 6. Reaction conditions for PCR identification primers

[0102]

[0103]

[0104] (2) Genotyping results of F0 generation mice

[0105] Genotyping of F0 generation mice was performed using PCR experiments, and the results are as follows: Figure 9 and Figure 10 As shown, the results indicate that EF22-1, EF22-3, EF22-5, EF22-15, and EF22-20 are F0 generation positive mice.

[0106] 8. Genotyping of F1 generation homologous recombination mice

[0107] (1) F0 mice (EF22-1, EF22-3, EF22-5, EF22-20) were mated with wild-type mice to obtain the F1 generation. The mating results are shown in Table 7. Genotyping of the F1 generation mice was performed by PCR (primers as in Table 5). The PCR results are shown in Table 7. Figure 11 and Figure 12 As shown.

[0108] (2) Southern blot detection was performed on F1 generation positive homologous recombination mice. DNA from the tails of the F1 mice identified as positive by PCR was extracted and subjected to Southern blot detection. The results are as follows: Figure 13 As shown.

[0109] Based on the combined PCR and Southern blot results, 1EF22-7, 1EF22-9, 1EF22-10, 1EF22-11, 1EF22-12, 1EF22-13 and 1EF22-14 were F1 generation positive mice, correctly recombined, and without random insertions.

[0110] Table 7. Results of mating between positive F0 mice and wild-type mice

[0111]

[0112] Example 3: Mating scheme for BAF155 conditional knockout mice

[0113] I. Obtaining Conditional Gene Knockout Mice

[0114] F1 generation fluxed heterozygous mice were mated with tissue-specific Cre mice to achieve tissue-specific knockout of the target gene. The principle is as follows: Figure 14 As shown.

[0115] Step 1: Floxed heterozygous mice are mated with tissue-specific Cre mice (Ts-Cre) to obtain fl heterozygous mice. A schematic diagram of the mating protocol is shown below. Figure 15 As shown, this step only yields heterozygous mice (fl / +, Cre / +). Homozygous mice (experimental mice) require further mating. Mice with genotypes of (+ / +, Cre / +), (fl / +, + / +), and (+ / +, + / +) can all be used as controls.

[0116] Step 2: The obtained heterozygous mice (fl / +, Cre / +) are mated to obtain fl homozygous mice (fl / fl, Cre / +). The mating protocol is as follows: Figure 16As shown in the figure. Mice with the genotype (fl / fl, Cre / +) belonged to the experimental group, while mice with other genotypes served as the control group.

[0117] II. Genotype Testing

[0118] The mouse genotype was identified by PCR using primers, and the experimental group of mice with the genotype (fl / fl, Cre / +) was finally screened out. The PCR primers are shown in Table 12.

[0119] Table 12 Primer sequences for PCR identification

[0120]

[0121] Example 4: Validation of transgenic mice

[0122] I. Verification of the knockout efficiency of transgenic mice

[0123] In Example 3, the experimental group mice were induced with tamoxifen continuously from day 4 to day 8 after birth, and samples were collected on day 14 for immunofluorescence staining. Furthermore, oligodendrocyte precursor cells (OPCs) were purified by immunoopanning and cultured in vitro, followed by cell slide immunofluorescence staining. F1 generation fluxed homozygous mice were used as the control group (i.e., BAF155flox / flox transgenic mice without Cre enzyme).

[0124] The results of the knockout efficiency verification of transgenic mice are as follows: Figure 17 As shown, the results indicate that the BAF155 knockout efficiency of tamoxifen-induced BAF155 knockout mice (i.e., PDGFRα-CreER; BAF155 flox / flox transgenic mice) can reach about 70%, indicating that the transgenic mouse model of BAF155 knockout specifically in the OPC stage has been successfully constructed.

[0125] II. Effects of BAF155 knockout specifically at the OPC stage on OPC differentiation and proliferation.

[0126] The experimental group mice were induced with tamoxifen continuously from day 4 to day 8 after birth, and samples were collected for observation on day 14. F1 generation floxed homozygous mice served as the control group (i.e., BAF155 flox / flox transgenic mice without Cre enzyme).

[0127] Immunofluorescence staining results as follows Figure 18 As shown, the results indicated that there was no significant difference in the number of OPC-specific marker PDGFRα and oligodendrocyte lineage marker Olig2-positive cells between BAF155 knockout mice and control mice. Figure 18(See Figures A and B). This indicates that knocking out BAF155 in OPC does not affect the cell count of the entire oligomyolipin lineage, nor does it affect the number of OPC cells.

[0128] Furthermore, we used Ki67 and Olig2 co-labeling to further observe changes in the number of cells in the proliferation phase. The results showed that knocking out BAF155 did not affect the proliferation ability of OPCs. Figure 18 (Figure B). 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 mice, the number of mature OL markers CC1 and MAG-positive cells was significantly reduced in the BAF155 knockout group mice. Figure 18 (See Figures C and D). This demonstrates that knocking out BAF155 leads to impaired differentiation of OPCs into mature OLs.

[0129] These results fully demonstrate that specific knockout of BAF155 at the OPC stage leads to OPC differentiation disorder without affecting its proliferative capacity.

[0130] Example 5: OPC-specific knockout of BAF155 leads to impaired myelination in multiple ASD-related brain regions, including the mPFC.

[0131] Myelin basic protein (MBP) is one of the most enriched proteins in the central nervous system (CNS) and plays an important role in myelination of nerve cells. We observed MBP expression in several brain regions associated with ASD, including the medial prefrontal cortex (mPFC), striatum, corpus callosum (CC), hippocampus, and cerebellum. The results are as follows: Figure 19 As shown, the results indicate that, compared with the control group mice, the expression level of MBP in multiple brain regions of the BAF155 knockout group mice was significantly reduced, indicating that the absence of BAF155 in OPC leads to widespread myelin development disorders.

[0132] Example 6: OPC-specific knockout of BAF155 leads to social preference disorder and repetitive stereotyped behaviors in mice.

[0133] Autism Spectrum Disorder (ASD) exhibits high clinical phenotypic heterogeneity, with significant differences in the presentation of core clinical symptoms and related comorbidities among patients. Core symptoms typically include impairment in social interaction and repetitive, stereotyped behaviors. In addition, some patients may also exhibit varying degrees of comorbid symptoms such as anxiety, depression, and cognitive impairment.

[0134] To verify whether the absence of BAF155 leads to ASD-like behavioral manifestations in mice, we administered tamoxifen to mice from day 4 to day 8 after birth and conducted behavioral tests on the mice at 6-8 weeks to observe their mental and behavioral performance.

[0135] I. Three-Box Social Experiment

[0136] The three-box social test is currently considered the most reliable method for assessing social abilities in mice. Therefore, we first used the three-box social test to evaluate the social abilities of BAF155-deficient mice. A diagram of the three-box social test is shown below. Figure 20 As shown in Figure A.

[0137] After the experimental mice were fully familiar with the test environment, in the first stage of the three-box socialization experiment, we compared the exploration time of BAF155 knockout mice and control mice in cages containing unfamiliar mice and empty cages. The results showed that the knockout group mice spent significantly less time exploring cages containing unfamiliar mice than the control group mice. Figure 20 (See Figure B). This indicates that the knockout mice exhibit some degree of social preference disorder.

[0138] To further assess the social novelty preference ability of mice, in the second phase of the three-box social experiment, we compared the exploration time of BAF155 knockout mice and control mice in cages containing new, unfamiliar mice and cages containing relatively familiar mice. The results showed that there was no statistically significant difference in the proportion of exploration time between the knockout group and the control group in the two cages. Figure 20 (Figure C). The above results indicate that BAF155 deficiency leads to social preference disorder in mice, but has no significant effect on social novelty preference.

[0139] (2) Besides social impairment, repetitive and stereotyped behaviors are also one of the main core symptoms of ASD. Therefore, we assessed whether BAF155 knockout mice exhibited repetitive and stereotyped behaviors through a self-grooming experiment. The results showed that, under the same experimental conditions, the repetitive grooming time of the BAF155 knockout group mice was significantly longer than that of the control group mice. This indicates that BAF155 deficiency leads to a certain degree of repetitive and stereotyped behaviors in mice. Figure 20 (D diagram).

[0140] Example 7: OPC-specific knockout of BAF155 leads to the development of anxiety-like symptoms common in ASD in mice.

[0141] In addition to core symptoms such as social interaction impairment and repetitive stereotyped behaviors, patients with ASD often exhibit some accompanying symptoms, the most common of which are anxiety and cognitive impairment.

[0142] Open field test and elevated cross maze test are widely used to detect anxiety-like behaviors in mice. Therefore, we used these two experimental methods to detect whether BAF155-deficient mice exhibit anxiety-like behavioral manifestations.

[0143] (1) In the open field experiment, we found that there was no significant difference in the total distance traveled between the BAF155 knockout group mice and the control group mice, which indicates that the motor ability of BAF155-deficient mice was not affected. Figure 21 (See Figures A and B). However, we observed that the knockout group mice had a significantly shorter activity distance in the central region compared to the control group mice (Figures A and B). Figure 21 (See Figures A and C). This demonstrates that BAF155-deficient mice exhibit significantly higher levels of anxiety compared to the control group.

[0144] (2) To further verify this result, we observed the distance traveled by BAF155 knockout mice within the open arm range in the elevated cross maze experiment. The results showed that the knockout group mice traveled a significantly shorter distance within the open arm range than the control group mice. Figure 21 (Figures D and E). This result further confirms that BAF155 knockout mice exhibit anxiety-like behaviors.

[0145] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. Application of BAF155 gene in constructing an autism spectrum disorder animal model, wherein the NCBI Gene ID of the BAF155 gene is 20588.

2. A method for constructing an autism spectrum disorder mouse model, characterized in that: The BAF155 gene of the mouse is knocked out or the expression of the BAF155 gene of the mouse is reduced, and the NCBI Gene ID of the BAF155 gene is 20588.

3. A method for constructing a BAF155 conditional knockout mouse model, characterized in that: Exon 4 of the mouse BAF155 gene is flox-modified to obtain flox-modified mice; the flox-modified mice are bred with Cre tool mice, and the resulting offspring are bred with each other to obtain a BAF155 gene conditional knockout mouse model.

4. The method according to claim 3, characterized in that: The Cre enzyme of the Cre tool mouse is specifically expressed in oligodendrocytes.

5. The method according to claim 3 or 4, characterized in that: The CRISPR / Cas9 technology is combined with the Cre / LoxP technology to perform flox modification on exon 4 of the mouse BAF155 gene. The sgRNA used in the CRISPR / Cas9 technology includes sgRNA5 and sgRNA8; the sgRNA5 sequence is shown in SEQ ID NO.1, and the sgRNA8 sequence is shown in SEQ ID NO.

2.

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

7. The method according to claim 5 or 6, characterized in that: The specific method for performing flox modification on exon 4 of the mouse BAF155 gene is: injecting sgRNA, Cas9 mRNA, and a targeting vector into a mouse fertilized egg and then transplanting it into a surrogate to obtain F0 generation mice, namely the flox-modified mice.

8. The method according to any one of claims 3 to 7, characterized in that: Primer pairs for identifying flox-modified mouse genotypes included the EGE-ZLF-022-L-GT-F / CKO-3′-D0-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'-D0-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.

9. Use of the mouse model prepared by the method according to any one of claims 2 to 8 in the preparation of drugs for treating diseases related to BAF155 gene defect.

10. The use according to claim 9, characterized in that: The BAF155 gene defect-related diseases include autism spectrum disorders.

Images