GJB2 gene p.V37I point mutation animal model construction method and application

Abstract

The invention relates to the field of animal disease models, and discloses a construction method and application of a GJB2 gene p.V37I point mutation animal model, the GJB2 gene of a normal animal is changed, and the 37th site codon GTG of the GJB2 gene is mutated into ATA, so that the animal model with the same mutation as the human GJB2 gene p.V37I point mutation is obtained. According to the invention, a CRISPR / Cas9 editing technology is utilized, double-base modification is accurately carried out, and a knock-in mouse model of which a specific gene is mutated into p.V37I is constructed instead of pure c.109Ggt; a base mutation is knocked in. Further research finds that the animal model has delayed progressive hearing loss, and the animal model can be used as a novel animal model for exploring the occurrence and development etiology and mechanism of the hearing loss related to p.V37I point mutation of the GJB2 gene; the mutation animal model is closer to actual mutation of human amino acid, the accuracy and correlation of research are improved, and the potential of basic research and clinical application is further improved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a method for constructing a GJB2 gene p.V37I point mutation animal model and its application. Background Art

[0002] Genetic factors are the leading cause of congenital deafness, among which the GJB2 gene is the leading deafness gene. The c.109G>A (p.V37I) mutation of the GJB2 gene has a high carrier rate in East Asian populations and is closely related to hereditary progressive hearing loss. In-depth research on its pathogenic mechanism has important clinical significance.

[0003] Given the unavailability of clinical specimens of the human cochlea, the development of an appropriate animal model is crucial to exploring the potential mechanisms of this mutation. Since GJB2 gene knockout can cause embryonic lethality in pregnant mice, most GJB2 gene studies use conditional knockout (CKO). However, in actual clinical practice, the GJB2 gene has hundreds of mutation types, and different mutation types do not lead to complete inactivation of the GJB2 gene. Using only GJB2 gene knockout mice cannot truly simulate the pathogenesis of hearing loss caused by different types of GJB2 gene point mutations. In addition, due to the different codon compilation of the human and mouse genomes, the single base substitution mutation of c.109G>A at the same base position causes the p.V37I mutation in the human genome, while in mice it causes the amino acid mutation of p.V37M ( Figure 2 ). At present, studies have reported a knock-in mouse model similar to the GJB2 gene c.109G>A site, which leads to the amino acid mutation p.V37M, but does not completely mimic the p.V37I mutation at the amino acid level. Summary of the invention

[0004] In order to obtain a p.V37I mutation that is closer to the same as that of clinical human patients, the present invention provides a method for constructing a double-base knock-in mouse model of the p.V37I mutation of the GJB2 gene and its use, by simultaneously replacing two bases in the corresponding codon region, thereby mutating the codon GTG at the 37th position to ATA, achieving the knock-in of the p.V37I mutation, and obtaining a double-base knock-in mouse model that is the same as the clinical human GJB2 gene c.109G>A (p.V37I) mutation. This solves the problem of low accuracy of subsequent research in the existing animal model construction due to the same base mutation but different amino acid mutation from humans.

[0005] In a first aspect, the present invention provides a method for constructing an animal model of a GJB2 gene p.V37I point mutation.

[0006] a) injecting a CRISPR / Cas9 system and a homologous recombination vector into an animal fertilized egg for homologous recombination, wherein the CRISPR / Cas9 system includes Cas9 mRNA and sgRNA, wherein the sgRNA specifically targets the GJB2 gene; the homologous recombination vector is designed based on a target point based on the sgRNA; the homologous recombination vector includes a donor DNA, wherein the codon GTG at position 37 of the GJB2 gene is mutated to ATA on the donor DNA;

[0007] b) transplanting the surviving fertilized eggs after recombinant into female mice, waiting for pregnancy and giving birth to obtain F0 generation mice, and the F0 generation mice naturally reproduce to obtain F1 generation mice;

[0008] c) The mice carrying the p.V37I homozygous point mutation were screened out from the F1 generation of mice to obtain the GJB2 gene homozygous point mutation mouse model.

[0009] In an optional embodiment, the method for constructing the GJB2 gene p.V37I point mutation animal model comprises: changing the GJB2 gene of a normal animal so that the codon GTG at the 37th position of the GJB2 gene mutates to ATA to obtain a hearing loss animal model.

[0010] In an optional embodiment, the amino acid sequence of p.V37I containing the mutation site after the GJB2 gene point mutation is SEQ ID NO.1:

[0011] MDWGTLQSILGGVNKHSTSIGKIWLTVLFIFRIMILIVAAKEVWGDEQADFVCNTLQPGCKNVCYDHHFPISHIRLWALQLIMVSTPALLVAMHVAYRRHEKKRKFMKGEIKN EFKDIEEIKTQKVRIEGSLWWTYTTSIFFRVIFEAVFMYVFYIMYNGFFMQRLVKCNAWPCPNTVDCFISRPTEKTVFTVFMISVSGICILLNITELCYLFVRYCSGKSKRPV;

[0012] The amino acid sequence corresponding to the original wild type is SEQ ID NO.2:

[0013] MDWGTLQSILGGVNKHSTSIGKIWLTVLFIFRIMILVVAAKEVWGDEQADFVCNTLQPGCKNVCYDHHFPISHIRLWALQLIMVSTPALLVAMHVAYRRHEKKRKFMKGEIKN EFKDIEEIKTQKVRIEGSLWWTYTTSIFFRVIFEAVFMYVFYIMYNGFFMQRLVKCNAWPCPNTVDCFISRPTEKTVFTVFMISVSGICILLNITELCYLFVRYCSGKSKRPV;

[0014] In an optional embodiment, the GJB2 gene point mutation gRNA sequence is Gjb2-B-L1 (SEQ ID NO. 3): GCCACACACTTAACCCTATGG;

[0015] Or, Gjb2-BR (SEQ ID NO. 4): GCCACCATTTGAAGTCCCTGG.

[0016] In an optional embodiment, in step c), the screening process is performed based on sequencing primers designed based on the p.V37I point mutation sequence.

[0017] In an optional embodiment, the nucleotide sequence of the p.V37I point mutation sequencing primer is SEQ ID NO.5 (F: TTCTCCAGTGCCAACCATCCA), SEQ ID NO.6 (R: AGGTGGCAGTTGTCGGCATATC).

[0018] The present invention provides a method for constructing a mouse model of a GJB2 gene p.V37I mutation point. The mouse model is achieved by double base knock-in of the point mutation of the GJB2 gene, rather than knocking out the GJB2 gene in the entire sequence of the GJB2 gene. The two base knock-ins are performed simultaneously, avoiding the c.109G>A mutation that causes p.V37M in the mouse model, thereby achieving a hearing loss model that is closer to the GJB2 gene p.V37I point mutation carried by patients in the clinic. The model can more accurately simulate the hearing phenotype, inner ear pathological changes and pathogenesis of congenital deafness caused by the homozygous point mutation of the GJB2 gene p.V37I, thereby providing a more suitable animal model for the future development of inner ear targeted drugs and the treatment of deafness caused by GJB2 gene mutations, and the hearing of the model is close to that of patients carrying the same type of mutations in the clinic.

[0019] In a second aspect, the present invention also provides an application of the construction method in constructing an animal model.

[0020] Optionally, the animal model is an animal model of hereditary hearing loss;

[0021] The animal model is an animal model caused by the p.V37I point mutation of the GJB2 gene.

[0022] In a third aspect, the present invention also provides an animal model with homozygous GJB2 gene p.V37I double base knock-in obtained by the construction method.

[0023] In a fourth aspect, the present invention provides a use of the animal model in any of the following aspects:

[0024] (1) To explore the causes and mechanisms of hearing loss associated with the GJB2 gene p.V37I point mutation;

[0025] (2) Use in screening diagnostic targets for hearing loss associated with the GJB2 gene p.V37I point mutation;

[0026] (3) Use in screening therapeutic targets for hearing loss associated with the GJB2 gene p.V37I point mutation;

[0027] (4) Use in screening or preparing drugs for diagnosis or treatment of hearing loss associated with the GJB2 gene p.V37I point mutation.

[0028] In a fourth aspect, the present invention provides a pharmaceutical composition for preventing or treating hearing loss, the pharmaceutical composition comprising an agent for mutating the codon ATA at position 37 of the GJB2 gene to GTG.

[0029] The technical solution of the present invention has the following advantages:

[0030] 1. A method for constructing a double-base knock-in mouse model of a GJB2 gene p.V37I mutation provided by the present invention comprises: simultaneously changing two bases of the GJB2 gene of a normal animal, so that the codon GTG at the 37th position of the GJB2 gene is mutated to ATA, so as to obtain a corresponding p.V37I mutation animal model. The present invention has found that the animal model obtained by mutating the codon GTG at the 37th position of the GJB2 gene to ATA can be used as a new animal model for exploring the etiology and mechanism of the occurrence and development of hearing loss caused by the GJB2 gene p.V37I mutation, namely, the GJB2 gene p.V37I model; compared with the previously reported p.V37M model that only changes one base at the mouse c.109G>A site, the present p.V37I mutation model is closer to the actual mutation of human amino acids, which can improve the accuracy and relevance of the research, and further improve its potential in basic research and clinical application.

[0031] 2. The present invention provides a CRISPR / Cas9 system for GJB2 gene editing, comprising Cas9 mRNA, the homologous recombination vector, and the sgRNA sequence. Using CRISPR / Cas9 editing technology, double base modification is performed accurately to construct a knock-in mouse model of a specific gene mutation, and the site knock-in is accurate. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the specific implementation methods of the present application or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0033] Figure 1 A flowchart of the invention for introducing a donor sequence into a mutant mouse genome;

[0034] Figure 2 Schematic diagram of wild-type, p.V37I and p.V37M mutant bases and amino acids of GJB2 gene;

[0035] Figure 3 The audiometric results of the F1 generation mice with the GJB2 gene p.V37I mutation of the present invention; 8K is the hearing condition at a frequency of 8000HZ; 16K is the hearing condition at a frequency of 16000HZ; 24K is the hearing condition at a frequency of 24000HZ; *p<0.05;

[0036] Figure 4 The sequencing results of homozygous GJB2 gene p.V37I mutant mice obtained by the present invention; DETAILED DESCRIPTION

[0037] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0038] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0039] Example 1

[0040] like Figure 1As shown, this embodiment provides a method for constructing a GJB2 gene p.V37I point mutation animal model, comprising the following steps:

[0041] Step 1: According to the p.V37I mutation of the GJB2 gene, sgRNA is designed, constructed, and in vitro transcribed, and a homologous recombination vector is designed according to the target sequence of the sgRNA, wherein the homologous recombination vector comprises a donor sequence, wherein the donor sequence comprises a mutation of the codon GTG at the 37th position of the GJB2 gene to ATA (GATCCTCGTG->GATCCTCATA); the codon GTG at the 37th position of the GJB2 gene is mutated to ATA;

[0042] Step 2: Collect fertilized eggs and prepare pseudo-pregnant female mice for transplantation of fertilized eggs;

[0043] Step 3: Inject cas9, sgRNA and homologous recombination vector into mouse fertilized eggs by microinjection for homologous recombination;

[0044] Step 4: Transplant the fertilized eggs that survive the injection into pseudo-pregnant female mice, and wait for them to become pregnant and give birth to obtain F0 generation mice;

[0045] Step 5: Determine the genotype through PCR sequencing and screen out mice carrying the GJB2 gene p.V37I mutation;

[0046] Step 6: The F0 generation GJB2 gene p.V37I mutant mice were naturally bred to obtain the F1 generation, and the genotypes were identified to screen out homozygous GJB2 gene p.V37I mutant mice.

[0047] The above construction method is described in detail below in conjunction with specific embodiments.

[0048] 1. Design sgRNA and homologous recombination vector

[0049] Design of sgRNA and construction of homologous recombination vector. In this experiment, the wild-type nucleotide sequence of GJB2 gene is SEQ ID NO.7: ( is the codon at the mutation site):

[0050]

[0051]

[0052] The wild-type and p.V37M, P.V37I local mutation base sequences and local amino acid sequences of GJB2 gene are shown in Figure 2 .

[0053] The amino acid sequence of p.V37I containing the mutation site after the GJB2 gene point mutation is SEQ ID NO.1:

[0054] MDWGTLQSILGGVNKHSTSIGKIWLTVLFIFRIMIL I VAAKEVWGDE

[0055] QADFVCNTLQPGCKNVCYDHHFPISHIRLWALQLIMVSTPALLVAMHVA

[0056] YRRHEKKRKFMKGEIKNEFKDIEEIKTQKVRIEGSLWWTYTTSIFFRVIFE

[0057] AVFMYVFYIMYNGFFMQRLVKCNAWPCPNTVDCFISRPTEKTVFTVFMISVSGICILLNITELCYLFVRYCSGKSKRPV;

[0058] The amino acid sequence corresponding to the original wild type is SEQ ID NO.2:

[0059] MDWGTLQSILGGVNKHSTSIGKIWLTVLFIFRIMIL V VAAKEVWGDE

[0060] QADFVCNTLQPGCKNVCYDHHFPISHIRLWALQLIMVSTPALLVAMHVA

[0061] YRRHEKKRKFMKGEIKNEFKDIEEIKTQKVRIEGSLWWTYTTSIFFRVIFE

[0062] AVFMYVFYIMYNGFFMQRLVKCNAWPCPNTVDCFISRPTEKTVFTVFMISVSGICILLNITELCYLFVRYCSGKSKRPV;

[0063] The gRNA sequences designed according to the GJB2 gene point mutation were Gjb2-BL (SEQ ID NO.3): GCCACACACTTAACCCTATGG; or Gjb2-BR (SEQ ID NO.4): GCCACCATTTGAAGTCCCTGG;

[0064] The nucleotide sequences of the primers for sequencing the p.V37I point mutation in the knock-in mouse model were SEQ ID NO.5 (F: TTCTCAGTGCCAACCATCCA) and SEQ ID NO.6 (R: AGGTGGCAGTTGTCGGCATATC).

[0065] 2. Fertilized egg collection

[0066] Select 8-week-old female mice with C57BL / 6J background and use hormones to induce ovulation. Immediately place the hormone-induced ovulation female mice into the cages of 12-16-week-old healthy male mice in a 1:1 ratio for mating. On the second day after mating, a white vaginal plug was observed at the vaginal opening of the female mouse, and the female mouse was killed by cervical dislocation.

[0067] 3. Fertilized egg microinjection and gene editing

[0068] Cas9 mRNA and gRNA (Gjb2-BL: GCCACACACTTAACCCTATGG) were obtained by in vitro transcription; a homologous recombination vector (donor vector) was constructed by conventional methods in the art, such as In-Fusion cloning.

[0069] The homologous recombination vector (Donor DNA plasmid) contains a DNA fragment, and the DNA fragment contains a 5' homology arm and a 3' homology arm from upstream to downstream. The sequence of the DNA fragment is specifically SEQ ID NO: 8:

[0070] CTCTTCATCTTCCGCATCATGATCCTCGTGGTGGCTGCAAAGGAG GTGTGGGGAGA

[0071] The DNA fragment shown in SEQ ID NO:8 was inserted into the plasmid to synthesize the homologous recombination vector. Under the observation of a stereo inverted phase contrast microscope, cas9 mRNA, sgRNA and homologous recombination vector were mixed in an equimolar ratio and injected into the fertilized eggs of C57BL / 6J background mice, with an injection volume of 20pL. The injected cas9 mRNA, sgRNA and homologous recombination vector will edit the wild-type GJB2 gene of the fertilized egg, resulting in the fertilized egg carrying a point mutation in the GJB2 gene.

[0072] 4. Fertilized egg transplantation after injection

[0073] Use vasectomized C57BL / 6J male mice and female mice in a 1:1 ratio to make the female mice pseudo-pregnant. Check the vaginal plug the next morning, and the pseudo-pregnant female mice with vaginal plugs can be used for fertilized egg transplantation. Transplant the fertilized eggs into the uterus of the pseudo-pregnant female mice and wait for the birth of F0 mice.

[0074] 5. Genotyping of offspring mice

[0075] The F0 generation mice born by the recipient female mice were naturally bred with the F0 generation GJB2 gene p.V37I mutant mice to obtain the F1 generation. The toe and tail tip tissues were cut 5 days after birth, and the genomic DNA was extracted for sequencing and identification to confirm the genotype. The sequencing results are shown in Figure 4 Then, a stable genetic GJB2 gene point mutation mouse strain was established. When the GJB2 point mutation-positive mice were 6-8 weeks old, they were mated with wild-type mice to screen out homozygous GJB2 gene p.V37I mutation mice, thus obtaining stable genetic F1 generation mice.

[0076] 6. ABR test of hearing in F1 homozygous point mutation mice

[0077] The hearing of mice was basically fully developed after the 20th day after birth, and ABR testing (brainstem auditory evoked localization) was performed. The mice were divided into groups. The wild-type group was wild-type C57BL / 6J mice (20 days old) (6 mice), and the mutant (HOMO) group was GJB2 gene p.V37I mutant C57BL / 6J mice (20 days old) (6 mice) obtained in step 5. Pentobarbital solution was injected into the peritoneum of the mice. After the mice were anesthetized and the deep reflex disappeared, they were placed in an acoustic and electrical shielding box. An electric blanket was used to maintain the body temperature of the anesthetized mice. During the hearing test, pay attention to the state of the mice. The mice were stimulated with short sounds (Clcik) and short pure sounds (Toneburst) (8K, 16K, 24K), respectively, and the mouse ABR signals were amplified using an amplifier. The hearing threshold of the mice was detected using the TDT RZ6 auditory physiology workstation.

[0078] Data statistical method: Fisher's exact test was used to compare the differences in ABR thresholds between knock-in mice and wild-type mice.

[0079] The hearing test results of GJB2 gene p.V37I mutant mice are as follows Figure 3 As shown, 8K is the hearing condition at a frequency of 8000HZ; 16K is the hearing condition at a frequency of 16000HZ; 24K is the hearing condition at a frequency of 24000HZ; the hearing test results of this point mutation mouse showed delayed-onset progressive hearing loss at high frequencies, which is similar to the symptoms of clinical patients carrying the same type of mutation.

[0080] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A method for constructing an animal model of GJB2 gene p.V37I point mutation, characterized in that: The following steps are involved: a) injecting a CRISPR / Cas9 system and a homologous recombination vector into an animal fertilized egg for homologous recombination; the CRISPR / Cas9 system includes Cas9 mRNA and sgRNA, and the sgRNA specifically targets the GJB2 gene; the homologous recombination vector is designed based on the target site of the sgRNA, and the homologous recombination vector includes a donor DNA, and the donor DNA includes a mutation of the GTG codon at the 37th position of the GJB2 gene to ATA; b) transplanting the surviving fertilized eggs after recombination into pseudo-pregnant female mice, waiting for pregnancy and giving birth to obtain F0 generation mice, and then naturally breeding the F0 generation mice to obtain F1 generation mice; c) Mice carrying the p.V37I homozygous point mutation were screened out from the F1 generation of mice, thus obtaining homozygous GJB2 gene p.V37I double base knock-in mice.

2. The construction method according to claim 1, characterized in that: The amino acid sequence of p.V37I after the point mutation of the GJB2 gene is SEQ ID NO.1, and the original wild-type amino acid sequence is SEQ ID NO.

2.

3. The construction method according to claim 2, characterized in that The p.V37I gene point mutation sgRNA sequence is: Gjb2-B-L1, as shown in SEQ ID NO.3; Or, Gjb2-B-R2, as shown in SEQ ID NO.

4.

4. The construction method according to claim 1, characterized in that: In step c), the screening process is performed based on the sequencing primers designed based on the p.V37I point mutation sequence.

5. The construction method according to claim 4, characterized in that: The nucleotide sequences of the p.V37I point mutation sequencing primers are SEQ ID NO.5 and SEQ ID NO.

6.

6. Use of the construction method according to any one of claims 1 to 5 in constructing an animal model.

7. An animal model with double base knock-in of the GJB2 gene p.V37I mutation obtained by the construction method according to any one of claims 1 to 5.

8. The animal model according to claim 6, characterized in that: The animal model is a hereditary hearing loss animal model.

9. Use of the animal model according to claim 7 or 8 in any of the following non-diagnostic and non-therapeutic fields: (1) To explore the causes and mechanisms of hearing loss associated with the GJB2 gene p.V37I point mutation; (2) Use in screening diagnostic targets for hearing loss associated with the GJB2 gene p.V37I point mutation; (3) Use in screening therapeutic targets for hearing loss associated with the GJB2 gene p.V37I point mutation; (4) Use in screening or preparing drugs for diagnosis or treatment of hearing loss associated with the GJB2 gene p.V37I point mutation.

10. A pharmaceutical composition for preventing or treating hearing loss, characterized in that: The pharmaceutical composition comprises a reagent for mutating the 37th codon ATA of the GJB2 gene to GTG.

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