Construction method and application of a Foxg1 p.Tyr392X point mutation mouse model

Abstract

The present invention provides a method for constructing a Foxg1p.Tyr392X point mutation mouse model and its application, belonging to the field of animal models. The method comprises the following steps: constructing a targeting vector; designing the sgRNA sequence and preparing it into microinjection RNA; injecting Cas9, microinjection RNA, and the targeting vector into the fertilized eggs of mice, and developing them into F0 generation mice; mating the F0 generation mice with wild-type mice to obtain F1 generation mice and performing genotype identification and Southern blot identification on them, etc., to confirm the successful construction of the Foxg1 gene point mutation mouse model. The Foxg1 point mutation mouse model constructed based on the mutation position and type of clinical patients can more appropriately reproduce the occurrence and development process of real diseases, and is of great significance for personalized exploration of the pathogenic mechanism, treatment methods, drug screening, etc. of FOXG1 syndrome.

Description

Technical Field

[0001] The present invention relates to a method for constructing a Foxg1 p.Tyr392X point mutation mouse model and its application, belonging to the field of animal models. Background Art

[0002] Heterozygous mutations in the forkhead box transcription factor FOXG1 cause FOXG1 syndrome, and patients show core symptoms of autism such as developmental delay, cognitive deficit, social disorder, and motor disorder. Such neurodevelopmental diseases seriously affect the physical and mental health of patients, bringing heavy mental and economic burdens to patients' families and society.

[0003] The human FOXG1 gene is located on chromosome 14q12. Currently, multiple FOXG1 mutations have been identified. Existing data show that different FOXG1 mutation sites result in different clinical symptoms in patients. Specifically, the full-length human FOXG1 protein contains 489 amino acids, including an N-terminal forkhead domain (FBD, amino acids 181 - 275), a Groucho-binding domain (GBD, amino acids 307 - 317), a JARID1B-binding domain (JBD, amino acids 383 - 406), and a C-terminal domain. Among them, the JBD domain can recruit other transcriptional repressors (such as JARID1B, a demethylase) to jointly regulate downstream target genes with FOXG1. Clinically, mutations from the JBD to the C-terminal domain are mainly nonsense mutations (with a proportion of about 3 / 4).

[0004] Patients with the FOXG1 p.Tyr400X nonsense mutation show typical autistic-like behaviors, such as social disorder, language disorder, motor disorder, eye contact disorder, and repetitive stereotyped hand behaviors. Due to species differences, the corresponding mutation position in mice is Foxg1 p.Tyr392X. There have been few previous research reports on mutations at this site, and currently there is only one case and it is an experiment conducted in cell lines. However, there has never been a research report on the function of Foxg1 p.Tyr392X in vivo, and currently, neither gene overexpression nor knockout mice can accurately simulate the symptoms of clinical patients and cannot reflect the biological functions caused by the nonsense mutation of the FOXG1 protein at tyrosine 392. Therefore, it is very necessary to construct a Foxg1 p.Tyr392X nonsense mutation mouse model. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art, in order to more accurately simulate the phenotypes of FOXG1 syndrome patients, the purpose of the present invention is to provide a method for constructing a Foxg1 p.Tyr392X point mutation mouse model and its application. Specifically, the purpose of the present invention can be achieved by the following technical solutions:

[0006] Design the sgRNA sequence according to the gene sequence of mouse Foxg1 before mutation;

[0007] Synthesize oligos according to the designed sgRNA sequence, and ligate the oligos into the pCS-4G vector containing Cas9 by Gibson Assembly method;

[0008] Design primers to construct the targeting vector;

[0009] Microinject Cas9, sgRNA and the targeting vector into mouse fertilized eggs to obtain F0 generation mice, and design primers to sequence the F0 generation mice;

[0010] Mate the F0 generation mice with wild-type mice to obtain F1 generation mice, and design primers to sequence the F1 generation mice.

[0011] Furthermore, the gene sequence of mouse Foxg1 before mutation includes the sequence shown in SEQ ID NO.25.

[0012] Furthermore, the sgRNA includes the sequences shown in SEQ ID NO.1-8 at the 5' target site and the sequences shown in SEQ ID NO.9-16 at the 3' target site.

[0013] Preferably, the sgRNA at the 5' target site includes the sequence shown in SEQ ID NO.7, and the sgRNA sequence at the 3' target site includes the sequence shown in SEQ ID NO.16.

[0014] Furthermore, the pCS-4G sequence includes the sequences shown in SEQ ID NO.27 and SEQ ID NO.28.

[0015] Furthermore, the gene sequence of the targeting vector includes the sequence shown in SEQ ID NO.26.

[0016] Furthermore, the primer sequences for identifying and sequencing the genotype of F0 generation mice include the sequences shown in SEQ ID NO.17-20.

[0017] Furthermore, the primer sequences for identifying and sequencing the genotype of F1 generation mice include the sequences shown in SEQ ID NO.21-24.

[0018] Advantages of the present invention:

[0019] The present invention provides a Foxg1 p.Tyr392X mouse that can simulate the nonsense mutation and its phenotype of the Foxg1 p.Tyr400X patient in FOXG1 syndrome, provides a suitable tool for the study of the pathogenesis of neurodevelopmental disorders, and provides new ideas for the clinical development and intervention of drugs for FOXG1 syndrome. It is of great significance for the personalized exploration of the pathogenic mechanism, treatment methods, drug screening, etc. of FOXG1 syndrome. In addition, the Foxg1 p.Tyr392X point mutation mouse model is conducive to a more in-depth exploration of the functions of the 392nd amino acid site of Foxg1, the JBD (JARID1B binding domain of Foxg1), and the C-terminal domain, filling the gap in this research direction. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is a schematic diagram of the design scheme of the Foxg1 p.Tyr392X point mutation mouse model of the present invention.

[0021] Figure 2 It is a schematic diagram of the Southern blot screening strategy adopted by the present invention.

[0022] Figure 3 It is the map of the pCS-4G vector into which the sequence shown in SEQ ID NO.7 has been inserted in the present invention.

[0023] Figure 4 It is the map of the pCS-4G vector into which the sequence shown in SEQ ID NO.16 has been inserted in the present invention.

[0024] Figure 5 It is the detection result of the activity of the sgRNA constructed by the present invention.

[0025] Figure 6 It is the map of the targeting vector constructed by the present invention.

[0026] Figure 7 It is the PCR identification diagram of the F0 generation mice of the present invention.

[0027] Figure 8 It is the PCR identification diagram of the F1 generation mice of the present invention.

[0028] Figure 9 It is the Southern blot detection result diagram of the F1 generation PCR positive mice of the present invention.

[0029] Figure 10 It is the gene sequencing diagram (c.1176C>A, p.Tyr392*) of the F1 generation mice of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0030] Next, in combination with the accompanying drawings in the embodiments of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0031] The explanations of the professional terms involved in the present invention are shown in Table 1 below:

[0032] Table 1

[0033]

[0034] Example 1:

[0035] This example discloses a method for constructing a Foxg1 p.Tyr392X point mutation mouse model and its application, including the following steps:

[0036] (1) Construction of the targeting vector:

[0037] Using the CRISPR / Cas9 technology, construct the sgRNA targeting the target gene, transcribe it in vitro into mRNA, and guide the Cas9 protein to cleave the DNA double strand at a specific site. First, analyze the case mutation sites and clinical symptoms of FOXG1 syndrome. After the triple codon corresponding to the 400th amino acid of FOXG1 changes from TAC to TAA (the 1176th base C in the FOXG1 coding sequence mutates to base A), the triple codon encoding tyrosine becomes a stop codon, and its symptoms include intellectual disability, movement disorder, eye contact disorder, stereotyped hand movements, scoliosis, muscle hypotonia, etc. Next, according to the species and gene name, know the coding region of the gene, analyze the corresponding genomic structure of humans, and determine to introduce a stop codon at the 392nd amino acid of the mouse Foxg1 gene. The sgRNA is designed in the non-conserved sequence downstream of IntronⅠ and 3’UTR. The homologous arms at the 5’ end and 3’ end are 1.3 kb and 1.3 kb respectively, and the design is as Figure 1 shown. In order to screen the gene targeting mice with correct recombination, use the PCR and Southern blot methods for verification, and at the same time use the 3’Probe (gene probe at the 3’ end) and LR Probe (gene probe at the 5’ end) to verify the F1 generation positive mice, and the specific design is as Figure 2 shown.

[0038] (2) Design and construction of Cas9 / sgRNA

[0039] Based on the design principle of sgRNA, sgRNA gene sequences shown in SEQ ID NO.1 - 8 and SEQ ID NO.9 - 16 were designed at the 5'-end and 3'-end target regions respectively. Oligos were synthesized according to the designed sgRNA sequences and ligated into the pCS-4G vector by the Gibson method. The vector map is as shown in Figure 3 After the ligation product was correctly sequenced, a universal amplification primer was used to amplify the T7 promoter and the sgRNA nucleotide sequence. Finally, in vitro transcription was carried out using this PCR product as a template to obtain the microinjection RNA of sgRNA7 and sgRNA16; and the reaction condition during its preparation was to react at 65°C for 5 minutes.

[0040] After that, the UCA TM method was used to detect the activity of sgRNA. The activity detection results are as shown in Figure 4 Finally, two sgRNAs, SEQ ID NO.7 designed at the 5'-end target and SEQ ID NO.16 designed at the 3'-end target, were selected for the next experiment. The targeting plasmid is as shown in Figure 5 shown.

[0041] (3) Superovulation of embryo donor mice (C57BL / 6)

[0042] Donor female mice were treated with PMSG (pregnant mare serum gonadotropin). 48 hours later, hCG (human chorionic gonadotropin) was injected. They were caged with male mice for mating. The fertilized eggs were taken the next day for microinjection.

[0043] (4) Microinjection

[0044] The microinjection RNAs of Cas9 and sgRNA and the targeting vector were microinjected into mouse fertilized eggs to obtain F0 generation mice.

[0045] (5) Genotype identification of F0 generation mice

[0046] Since the cleavage rate of embryos is very fast in the early stage, the obtained F0 generation mice are chimeras. Primers with sequences shown in SEQ ID NO.17 - 20 were designed to perform PCR identification on the tails of F0 generation mice. The genotypes of the obtained F0 generation mice are for reference only. The identification results are as shown in Figure 6 shown. However, the mice obtained this time do not necessarily represent a heritable gene mutation type. The heritable genotype needs to be determined after the genotype identification of F1 generation mice.

[0047] (6) Genotype and Southern blot identification of F1 generation mice

[0048] The above-mentioned F0 generation mice were mated with wild-type mice, and the offspring mice were F1 generation mice. Primers with the sequences shown in SEQ ID NO. 21-24 were designed, and the DNA of the F1 generation mice identified as positive by PCR (mice with gene mutations) was subjected to Southern blot detection and sequencing. The results are shown in Figures 7, 8, and 9, confirming the correct recombination of the mutant allele and no random insertion, indicating the successful construction of the Foxg1 p.Tyr392X point mutant mouse model.

[0049] (7) Performance of Foxg1 p.Tyr392X point mutant mice

[0050] The Foxg1 p.Tyr392X point mutant mice were observed at the adult stage, and it was found that the mice had characteristics such as intellectual disability, movement disorder, repetitive stereotyped movements, social disorder, and insufficient muscle tone.

[0051] The Foxg1 p.Tyr392X point mutant mouse model obtained by the present invention can simulate the pathogenesis of FOXG1 syndrome, providing a good visual animal model for accurately developing therapeutic drugs for patients with FOXG1 p.Tyr400X mutations, deeply exploring the pathogenic mechanism of FOXG1 syndrome, and exploring the methods and feasibility of gene therapy.

[0052] The above embodiments have detailed the technical solutions and beneficial effects of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not used to limit the present invention. Any modifications, supplements, equivalent replacements, etc. made within the scope of the principles of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for constructing a Foxg1 p.Tyr392X point mutation mouse model, characterized in that, Comprising the following steps: Design an sgRNA sequence according to the gene sequence of mouse Foxg1 before mutation; Synthesize oligos according to the designed sgRNA sequence, and ligate the oligos into the pCS-4G vector containing Cas9 by the Gibson Assembly method; Design primers to construct a targeting vector, in which the 1176th base C in the gene coding sequence is mutated to base A; Microinject Cas9, sgRNA and the targeting vector into mouse fertilized eggs to obtain F0 generation mice, and design primers to sequence the F0 generation mice; Mate the F0 generation mice with wild-type mice to obtain F1 generation mice, design primers to sequence the F1 generation mice, confirm the correct recombination of the mutant allele and no random insertion, indicating the successful construction of the Foxg1 p.Tyr392X point mutant mouse model.

2. The construction method according to claim 1, characterized in that The gene sequence of mouse Foxg1 before mutation includes the sequence shown in SEQ ID NO.

25.

3. The construction method according to claim 1, characterized in that, The sgRNA includes sequences designed at the 5' target site and the 3' target site respectively. The sgRNA sequence at the 5' target site includes any one of the sequences shown in SEQ ID NOs. 1-8, and the sgRNA sequence at the 3' target site includes any one of the sequences shown in SEQ ID NOs. 9-16.

4. The construction method according to claim 1, characterized in that The sequence of the pCS-4G vector includes the sequences shown in SEQ ID NO.27 and SEQ ID NO.

28.

5. The construction method according to claim 1, characterized in that The primer sequences for identifying the gene sequence of the F0 generation mice include the sequences shown in SEQ ID NOs. 17-20.

6. The construction method according to claim 1, characterized in that The primer sequences for genotyping and sequencing the F1 generation mice include the sequences shown in SEQ ID NOs. 21-24.

7. The construction method according to claim 1, wherein The sequence of the targeting vector includes the sequence shown in SEQ ID NO.

26.

8. Use of the Foxg1 p.Tyr392X point mutant mouse model according to claim 1 in the field of studying FOXG1 syndrome.

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