Construction method of Elmod3 gene mutation mouse model

Abstract

The invention relates to the technical field of biology, in particular to a construction method of an Elmod3 gene mutation mouse model. Based on clinical data of hereditary deafness family patients, ELMOD3c.512Agt is found; g abnormal mutation can cause delayed progressive sensorineural deafness of a patient. According to the invention, a molecular marker with Elmod3c.512Agt is constructed; the G point mutation mouse model is consistent with the mutation site of a patient, and after the mouse is born, the disease progresses under the normal feeding environment and the natural growth rule, so that the experiment conditions of the disease research of the model mouse are closer to the real disease development of family patients, and a researcher can deeply research how the ELMOD3 gene mutation causes deafness. And a foundation is laid for functional research and subsequent gene therapy research of the ELMOD3 in an inner ear auditory system.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for constructing an Elmod3 gene mutation mouse model. Background Art

[0002] Deafness is a common sensory impairment. According to the 2023 WHO World Hearing Report, 1.5 billion people currently suffer from varying degrees of hearing loss, making it the third leading cause of disability worldwide. Approximately 60% of cases of deafness are genetic. Hereditary deafness refers to abnormalities in genetic material inherited from parents that can be passed down to offspring, leading to abnormal ear development, metabolic disorders, and abnormalities in cell structure or function, resulting in poor hearing function. Hereditary deafness is primarily non-syndromic, without abnormalities in external ear tissues or systems. Non-syndromic deafness can be further categorized as autosomal dominant, autosomal recessive, mitochondrial, and sex-linked.

[0003] In order to gain a more complete and detailed understanding of the genes associated with hereditary deafness and provide new diagnostic and treatment ideas for hereditary deafness, we still need to further explore and functionally investigate the relevant pathogenic genes and their possible pathogenic sites. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a method for constructing an Elmod3 gene mutation mouse model. The present invention provides a method for constructing a gene knockout mouse model using CRISPR-Cas9 gene editing technology, and further research on hereditary deafness caused by Elmod3 mutation based on the mouse model will better reflect the disease development of family patients, lay the foundation for studying the pathogenic mechanism of hereditary deafness, and provide scientific and effective prevention or treatment methods.

[0005] The present invention provides a nucleic acid molecule targeting the Elmod3 gene, which includes a gRNA and / or a donor oligonucleotide donor DNA targeting the Elmod3 gene, wherein:

[0006] The gRNA has a nucleotide sequence as shown in SEQ ID NO: 1;

[0007] The donor oligonucleotide donor DNA has the nucleotide sequence shown in SEQ ID NO: 2.

[0008] The present invention provides a reagent combination for editing the Elmod3 gene, comprising Cas9 mRNA and the nucleic acid molecule.

[0009] The present invention provides the use of the nucleic acid molecule and / or the reagent combination in constructing an Elmod3 gene mutation mouse model.

[0010] The present invention provides a method for constructing an Elmod3 gene mutation mouse model, comprising the following steps:

[0011] Step 1: injecting the reagent combination into mouse fertilized eggs, and then transplanting the fertilized eggs into pseudo-pregnant female mice to produce offspring, thereby obtaining F0 generation mice;

[0012] Step 2: hybridize the F0 generation mice with wild-type mice, and screen the resulting offspring to obtain positive heterozygous F1 generation mice as heterozygous Elmod3 gene mutation mouse models.

[0013] In some embodiments, in step 1, the injected reagents include gRNA, donor oligonucleotide, donor DNA and Cas9 mRNA in a mass ratio of (0.5-2): (1-4): (1-4).

[0014] In some specific embodiments, the injected reagents include gRNA, donor oligonucleotide, donor DNA and Cas9 mRNA in a mass ratio of 1:2:2.

[0015] In some embodiments, step 1 further includes screening the offspring, and selecting mice with positive mutation sites as F0 generation mice.

[0016] In some embodiments, the method further comprises self-pollinating the positive heterozygous F1 generation mice, and screening the resulting offspring to obtain homozygous mutant F2 generation mice, which are used as homozygous Elmod3 gene mutation mouse models.

[0017] In some embodiments, the screening includes using specific primers to identify the genotype of the mutation site in the mouse, and the specific primers include a forward primer of the nucleotide sequence shown in SEQ ID NO: 3 and a reverse primer of the nucleotide sequence shown in SEQ ID NO: 3.

[0018] The present invention provides the use of the nucleic acid molecule, the reagent combination or the construction method to construct an Elmod3 gene mutant mouse model in developing drugs for treating diseases related to Elmod3 gene defects.

[0019] In some embodiments, the disease associated with Elmod3 gene defect includes deafness.

[0020] The beneficial effects of the present invention include: Based on clinical data from patients in families with hereditary deafness, the abnormal ELMOD3c.512A>G mutation was found to cause late-onset progressive sensorineural hearing loss in patients. The present invention constructed a mouse model with the Elmod3c.512A>G point mutation, which is consistent with the mutation site in patients. After birth, the disease progresses in mice under normal breeding conditions and natural growth patterns, making the experimental conditions for disease research in model mice more similar to the actual disease development in family patients. This allows researchers to further study how ELMOD3 gene mutations lead to deafness, laying the foundation for functional research on ELMOD3 in the inner ear auditory system and subsequent gene therapy research. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the Founder mouse gene sequencing peak diagram of the Elmod3 gene c.512A>G point mutation;

[0022] Figure 2 This is the gene sequencing peak diagram of the offspring heterozygous mice with the Elmod3 gene 512 (A>G) point mutation;

[0023] Figure 3 This is the gene sequencing peak diagram of the offspring homozygous mice with the Elmod3 gene 512 (A>G) point mutation;

[0024] Figure 4 The graph shows the auditory brainstem evoked potential test results of wild-type (WT), heterozygous (HET) and homozygous (HOMO) mice with the Elmod3 gene 512 (A>G) point mutation. DETAILED DESCRIPTION

[0025] The present invention provides a method for constructing an Elmod3 gene mutation mouse model. Those skilled in the art can learn from the contents of this article and appropriately improve the process parameters to achieve the desired results. It should be noted that all similar replacements and modifications are apparent to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described by preferred embodiments, and relevant personnel can obviously modify or appropriately alter and combine the methods and applications herein without departing from the content, spirit, and scope of the present invention to achieve and apply the technology of the present invention.

[0026] A missense mutation in ELMOD3 (c.512A>G; p.His171Arg) was first discovered in a deaf family and is associated with autosomal dominant deafness. The ELMOD3 gene is located on the short arm of human chromosome 2, p11.2. ELMOD3 is a phagocytic and cell motility protein that activates the GTPase ARL2, playing a crucial role in primary cilium formation and cargo transport from the Golgi apparatus to the primary cilium. Linkage analysis, Sanger sequencing validation, and studies of knockout mouse models have confirmed that the ELMOD3 (c.512A>G) point mutation is the causative locus for hereditary deafness. However, the molecular mechanism underlying hereditary deafness caused by ELMOD3 gene mutations remains unclear. Therefore, developing a mouse model with the ELMOD3 (c.512A>G) point mutation is crucial for studying the pathogenic mechanisms of hereditary deafness and exploring treatments.

[0027] The test materials used in the present invention are all common commercial products and can be purchased in the market. The present invention will be further described below with reference to the examples.

[0028] Example 1 Construction of a transgenic mouse model with Elmod3512 (A>G) gene point mutation based on CRISPR / Cas9 technology

[0029] Regarding the Elmod3 gene, previous studies have shown that although the gene knockout mice constructed using CRISPR-Cas9 gene editing technology have a hearing impairment phenotype, they show full-frequency hearing loss at 2 months of age. This is not completely consistent with the manifestations of family patients who mainly have high-frequency hearing loss at the onset and gradually progress to full-frequency hearing loss, and cannot fully correspond to the clinical phenotypes manifested by gene mutations in family patients.

[0030] The present invention utilizes CRISPR / Cas9 gene targeting technology to construct gRNA targeting the Elmod3 gene, which is transcribed into mRNA in vitro to guide the Cas9 protein to cleave the DNA double strand at a specific site.

[0031] (1) Target design based on gene information and mutation sites

[0032] Gene name: Elmod3, ENSMUSG00000056698

[0033] Mutation target site: c.512A>G (mutates the 171st amino acid of the protein encoded by the Elmod3 gene from histidine to arginine)

[0034] Design of guide RNA and donor oligo sequences

[0035] According to the object to be gene-edited, the coding region of the mouse Elmod3 gene is searched. This gene has 6 transcripts. This patent targets the transcript Elmod3-202 (ENSMUST00000114069.8), which is 2164bp long, has 13 exons, and encodes an ELMOD3 protein with a length of 381 amino acids.

[0036] The designed guide RNA sequence is:

[0037] 5'-GACAGCCAAAACCCAACGCA-3'(SEQ ID NO.1)

[0038] The designed donor oligo sequence is:

[0039]

[0040] The single-underlined bases are synonymous mutations made to prevent secondary cutting by gRNA, and the double-underlined bases are the mutated bases.

[0041] (2) Superovulation of embryo donor mice (C57BL / 6J)

[0042] Donor female C57BL / 6J mice were treated with pregnant mare serum gonadotropin (PMSG) and injected with human chorionic gonadotropin (hCG) 48 hours later. They were then mated with male mice in the same cage. The next day, fertilized eggs were collected to obtain pronuclear embryos for subsequent microinjection and embryo transfer.

[0043] (3) Microinjection and embryo transfer

[0044] Cas9 mRNA and guide RNA were obtained by in vitro transcription, and oligodonor DNA was obtained by synthesis. 5 μL of a mixture containing 20 ng / μL Cas9 mRNA, 10 ng / μL gRNA, and 20 ng / μL donor DNA was microinjected into the pronuclear embryos obtained above. The pronuclear embryos were then transplanted into the fertilized eggs of pseudopregnant female mice. Once the mice were born, F0 generation mice were obtained.

[0045] Example 2 Genotype identification of a transgenic mouse model with an Elmod3512 (A>G) gene point mutation constructed based on CRISPR / Cas9 technology

[0046] (1) Founder mouse identification

[0047] After the embryo-transferred mice are born, their toes are clipped to extract DNA, and PCR reaction is performed based on the designed primers to identify the mouse genotype.

[0048] The forward and reverse primer sequences used for genotyping are:

[0049] Forward: 5'-TGCTCAGTGTGAGTGTGAGG-3'(SEQ ID NO.3)

[0050] Reverse: 5'-GTGCTGCTCCTGAGGTCCTA-3'(SEQ ID NO.4)

[0051] PCR reaction system:

[0052] 2X RapidTaqMastermix: 12.5μL

[0053] ddH2O: 8 μL

[0054] Primer forward: 1 μL

[0055] Primer reverse: 1 μL

[0056] DNA: 2.5 μL

[0057] PCR amplification procedure: (1) pre-denaturation at 95°C for 5 min; (2) denaturation at 95°C for 30 sec; annealing at 60°C for 30 sec, extension at 72°C for 30 sec; 35 cycles in total; (3) extension at 72°C for 5 min.

[0058] The PCR amplification products were sequenced by Sanger sequencing to identify the mouse genotype. The sequencing diagram of the Founder mouse mutation site is shown in the figure Figure 1 shown.

[0059] (2) Identification of offspring mice

[0060] After the founder mice reach sexual maturity, they are mated with wild mice of the opposite sex. The toes of the mice are cut to extract DNA. After PCR amplification as above, the mouse genotype is identified by Sanger sequencing. If F1 generation point mutation-positive mice are born, it means that the mutation has been integrated into the germ cells, indicating that the strain has been successfully constructed.

[0061] After the F1 generation of positive mice reached sexual maturity, the male and female mice were mated together. After the offspring were born, DNA was extracted for genotyping. The sequencing diagram of the offspring heterozygous point mutation mice is shown in Figure 2. Figure 2 As shown, the sequencing diagram of the offspring homozygous point mutation mice is as follows Figure 3 shown.

[0062] Example 3 Phenotypic Identification of a Transgenic Mouse Model with Elmod3512 (A>G) Gene Point Mutation Constructed Based on CRISPR / Cas9 Technology

[0063] In this example, the auditory brainstem response detection method was used to perform hearing tests on three genotype mice obtained in the above embodiment: wild type, heterozygous mice with Elmod3 gene 512 (A>G) point mutation, and offspring homozygous mice with Elmod3 gene 512 (A>G) point mutation.

[0064] (1) The specific experimental steps are as follows:

[0065] Before audiometry, mice were anesthetized with 100 mg / kg of sodium pentobarbital injected intraperitoneally. Once fully anesthetized, they were placed in a soundproof box and maintained at body temperature with a warming blanket. ABR recording electrodes were inserted subcutaneously at the midpoint of a line connecting the anterior margins of the auricles on both sides of the mouse. A reference electrode was inserted subcutaneously behind the test ear. A ground electrode was inserted subcutaneously on the back of the mouse. A sound speaker was placed approximately 10 cm from the opening of the external auditory canal of the test ear. ABR stimuli were presented using click and toneburst sounds, with a bandpass filter of 300–3000 Hz, a notch filter of 50 Hz, 512 overlaps, and a sweep time of 10 ms. The sound frequencies were click, 4, 8, 12, 16, 24, and 32 kHz, respectively. The sound intensity of each frequency was gradually decreased from 90dB SPL at intervals of 10dB SPL. When no repeatable ABR waveform was detected, the sound intensity was increased by 5dB SPL on the basis of this sound intensity for further testing. The stimulus sound intensity at which a repeatable ABR waveform was recorded was recorded as the ABR threshold of the mouse at this frequency.

[0066] (2) The experimental results are as follows:

[0067] Figure 4 Results shown are ABR thresholds for wild-type, heterozygous, and homozygous mice harboring the ELMOD3 gene 512 (A>G) point mutation at 3, 5, and 7 months of age. At 3 months of age, there were no significant differences in ABR thresholds across all frequencies among the three genotypes. At 5 months of age, wild-type mice showed an increase in ABR thresholds only at 32 kHz compared to 3 months of age, while heterozygous and homozygous mutant mice showed significant increases at 24 and 32 kHz compared to 3 months of age. At 7 months of age, wild-type mice showed significant increases in ABR thresholds only at click, 8, 12, 24, and 32 kHz compared to 3 months of age. In contrast, heterozygous and homozygous mutant mice showed significant increases in ABR thresholds at all frequencies compared to 3 months of age. Furthermore, compared to 7-month-old wild-type mice, 7-month-old heterozygous mutant mice showed a significant increase in ABR thresholds at 16 kHz, while 7-month-old homozygous mutant mice showed significant increases at all frequencies.

[0068] The construction method or transgenic mice obtained by the construction provided by the present invention are used to explore the deafness-causing mechanism of Elmod3 gene mutation. The results show that both Elmod3 c.512A>G heterozygous and homozygous Elmod3 c.512A>G mutant mice exhibit a hearing phenotype of late-onset progressive sensorineural hearing loss, similar to the phenotype of patients with ELMOD3 gene mutations.

[0069] The above are only preferred embodiments of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A nucleic acid molecule targeting the Elmod3 gene, characterized in that It includes gRNA and / or donor oligonucleotide donor DNA targeting the Elmod3 gene, wherein: The gRNA has a nucleotide sequence as shown in SEQ ID NO: 1; The donor oligonucleotide donor DNA has the nucleotide sequence shown in SEQ ID NO:

2.

2. A reagent combination for editing the Elmod3 gene, characterized in that: Comprising Cas9 mRNA and the nucleic acid molecule of claim 1.

3. Use of the nucleic acid molecule according to claim 1 and / or the reagent combination according to claim 2 in constructing an Elmod3 gene mutation mouse model.

4. A method for constructing an Elmod3 gene mutation mouse model, characterized in that: The steps include: Step 1: injecting the reagent combination described in claim 2 into mouse fertilized eggs, transplanting the fertilized eggs into pseudo-pregnant female mice, producing offspring, and obtaining F0 generation mice; Step 2: hybridize the F0 generation mice with wild-type mice, and screen the resulting offspring to obtain positive heterozygous F1 generation mice as heterozygous Elmod3 gene mutation mouse models.

5. The construction method according to claim 4, characterized in that: In step 1, the injected reagents include gRNA, donor oligonucleotide, donor DNA and Cas9 mRNA in a mass ratio of (0.5-2): (1-4): (1-4).

6. The construction method according to claim 4, characterized in that The step 1 also includes screening the offspring, and selecting mice with positive mutation sites as F0 generation mice.

7. The construction method according to any one of claims 4 to 6, characterized in that: The method also includes self-pollinating the positive heterozygous F1 generation mice, and screening the resulting offspring to obtain homozygous mutation F2 generation mice, which are used as homozygous Elmod3 gene mutation mouse models.

8. The construction method according to any one of claims 4 to 7, characterized in that: The screening includes using specific primers to identify the genotype of the mutation site of the mouse, and the specific primers include a forward primer with a nucleotide sequence as shown in SEQ ID NO: 3 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO:

3.

9. Use of the nucleic acid molecule according to claim 1, the reagent combination according to claim 2, or the Elmod3 gene mutant mouse model constructed by the construction method according to any one of claims 4 to 8 in developing drugs for treating diseases related to Elmod3 gene defects.

10. The use according to claim 9, characterized in that The diseases associated with Elmod3 gene defects include deafness.

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