Construction method and application of DFNB1 type deafness mouse model with GJB2 gene frameshift mutation

Abstract

The invention discloses a construction method and application of a DFNB1 type deafness mouse model with GJB2 gene frameshift mutation. The construction method comprises the following steps: (1) designing two sgRNA plasmids on a No.2 exon of a mouse Gjb2 gene; (2) screening out mouse embryonic stem cells of which the Gjb2 gene carries a c.299300delAT mutation site; (3) forming a recombinant embryo, transplanting the recombinant embryo into the uterus of the pregnant female mouse, and waiting for pregnancy and farrowing to obtain an F0-generation mouse; and (4) screening the F0 generation mice in the step (3) to obtain the mouse model carrying the c.299300delAT homozygous point mutation of the GJB2 gene. The method has the advantages that the problem of mouse embryo metaphase energy supply disorder lethal caused by Gjb2 homozygous mutation is solved, the embryo survival rate is increased, and the bottleneck of embryo lethal in model construction of a traditional gene editing technology is broken through.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a method for constructing a DFNB1 type deaf mouse model of GJB2 gene frameshift mutation and application thereof. BACKGROUND

[0002] Genetic deafness is a high-incidence sensory system disease worldwide, and the GJB2 gene c.299_300delAT homozygous frameshift mutation is the main pathogenic type of non-syndromic hearing loss in Asian populations. This mutation causes a premature termination codon (PTC) by disrupting the gene reading frame, triggering the "nonsense-mediated mRNA decay (NMD)" mechanism, resulting in complete loss of connexin 26 (Cx26) in cochlear tissue, failure to form gap junction channels, and interruption of intercellular communication, ultimately leading to congenital severe to profound hearing impairment. Constructing a mouse model that closely matches this clinical mutation is a key prerequisite for analyzing the pathogenic mechanism of the GJB2 gene and developing targeted treatment methods. However, there are still key gaps in the existing technical system.

[0003] Currently, there are five publicly available Gjb2 mouse models (such as the Gjb2 2tm1Alb model constructed by Y. Hui Jiang in 1998, the Gjb2 tm1Yelg model by S. Silva-santos in 2015, the Gjb2 tm1.1Bdph model by M. C. C. C. Judson in 2016, the Gjb2 tm2Yelg model by T. Wang in 2017, and the Gjb2 em1Yelg model by R. Avagliano Trezza in 2019). None of these models can meet the needs of clinical research. Most of them are heterozygous mutations or conditional knockouts, the gene mutation sites do not match c.299_300delAT, and they all involve large fragment modifications such as exon deletion. Due to the bottleneck of precise repair of large fragment deletion in existing technology, these models cannot simulate the phenotype improvement after gene editing repair, making it difficult to support the evaluation of gene therapy effects.

[0004] The prior art CN118308425A discloses a method for constructing a GJB2 gene homozygous point mutation mouse model, which introduces p.F142L (TTC→CTG at the 142th site) or p.D50N (GAT→AAC at the 50th site) homozygous point mutations by injecting a CRISPR / Cas9 system and a homologous recombination vector into mouse zygotes through homologous recombination. Although the patent realizes the survival of GJB2 homozygous mutation mice, the core limitation is significant: both p.F142L and p.D50N are missense mutations, only changing the type of amino acid while retaining part of the Cx26 protein function. The purpose is to avoid embryonic lethality, which is significantly different from the pathological mechanism and phenotype of c.299_300delAT (frameshift mutation, complete loss of Cx26 function), and cannot simulate the characteristics of clinically severe hearing loss. Moreover, it still relies on the traditional path of zygote injection + homologous recombination, with a homologous recombination efficiency of less than 1%, and does not solve the fundamental problem of Gjb2 homozygous mutation embryonic lethality. It only avoids technical difficulties by using "compromise mutation types".

[0005] Neither the above-mentioned five models nor the patent technology disclosed in CN118308425A can construct a c.299_300delAT homozygous mutation mouse. The core is restricted by the dual constraints of embryonic lethality and editing efficiency: on the one hand, there are species differences in glucose metabolism between mice and human embryos during the embryonic period. Human embryos rely on multiple energy supplies to compensate for transport disorders, while mouse embryos have almost no energy reserves and completely rely on the specific glucose transport pathway between the placenta and the embryo. The placenta has no compensatory ability, and Gjb2 homozygous mutation will directly block this pathway, leading to the death of the embryo due to energy deficiency during the middle of pregnancy (E10.5-E12.5), and it cannot survive to be screened after birth. CN118308425A passively avoids this problem by selecting point mutations that retain function; on the other hand, traditional CRISPR / Cas9 mainly relies on non-homologous end joining (NHEJ) repair, which is prone to random insertion / deletion (indel) and difficult to accurately reproduce the 2-base deletion of c.299_300delAT. Even if homologous recombination (such as CN118308425A) is used, the efficiency is extremely low (<1%), and it cannot regulate the functional compensation mechanism of GJB2 during embryonic development, and cannot avoid the systemic functional defects caused by homozygous mutation.

[0006] The above technical difficulties severely limit the in-depth study of the pathogenesis of GJB2-related deafness and the verification of gene therapy strategies. Therefore, combining CRISPR / Cas editing technology with tetraploid compensation technology is the only feasible path to construct a Gjb2 homozygous frameshift mutation mouse model that accurately simulates clinical mutations, which has irreplaceable significance for elucidating the mechanism of GJB2-related diseases. SUMMARY

[0007] Therefore, the application aims to provide a GJB2 gene frameshift mutation DFNB1 type deafness mouse model construction method and application, solve the problem of energy supply disorder in mouse embryonic middle stage caused by Gjb2 homozygous mutation, improve the embryonic survival rate, and break through the "embryonic lethal" bottleneck in the construction of the model by traditional gene editing technology. The precise deletion of 2 bases of c.299_300delAT is realized, random insertion / deletion (indel) caused by non-homologous end joining (NHEJ) is avoided, and the gene editing precision is high.

[0008] The technical scheme of the application discloses a GJB2 gene frameshift mutation DFNB1 type deafness mouse model construction method, which comprises the following steps: (1) two sgRNA plasmids are designed at the 2nd exon of the mouse Gjb2 gene, and are marked as sgRNA Gjb2 A1, sgRNA Gjb2 A2, sgRNA Gjb2 A1 can recognize the PAM site AGG on the mouse Gjb2 gene, and sgRNA Gjb2 A2 can recognize the PAM site GGG on the mouse Gjb2 gene; (2) the two sgRNA plasmids in step (1), a Cas9 expression vector and a donor vector containing a c.299_300delAT mutation site are co-transfected into mouse embryonic stem cells (ESCs), and after 24-48 h of transfection, the cells are cultured in a double-antibiotic screening medium, and after 3-4 days of culture, the surviving mouse embryonic stem cells are screened by a PCR genotyping method, and mouse embryonic stem cells carrying a c.299_300delAT mutation site of the Gjb2 gene are screened out; (3) the mouse embryonic stem cells screened out in step (2) are injected into the inner cell mass region of a tetraploid blastocyst to form a recombinant embryo, the recombinant embryo is transplanted into the uterus of a surrogate mother mouse, and after pregnancy and childbirth, F0 generation mice are obtained; (4) the F0 generation mice in step (3) are screened, and mice carrying a c.299_300delAT point mutation homozygously are screened out, that is, a mouse model carrying a GJB2 gene c.299_300delAT homozygous point mutation is obtained.

[0009] Further, the donor vector in step (2) comprises Donor-Puro and Donor-BSD; the Donor-Puro comprises 5' arm and 3' arm homologous to sgRNA plasmid of the mouse Gjb2 gene, CDS sequence carrying c.299_300delAT mutation and a screening marker cassette of a puromycin resistance gene flanked by a recognition site of Flpo recombinase; The Donor-BSD comprises 5' arm and 3' arm homologous to sgRNA plasmid of the mouse Gjb2 gene, CDS sequence carrying c.299_300delAT mutation and a screening marker cassette of a blasticidin resistance gene flanked by a recognition site of Flp recombinase.

[0010] Further, the transfection in step (2) is electroporation.

[0011] Further, the final concentration of puromycin in the double antibiotic screening medium in step (2) is 1 μg / mL, and the final concentration of blasticidin is 10 μg / mL; the screening culture conditions comprise a temperature of 37℃ and a carbon dioxide mass concentration of 5%.

[0012] Further, the operation process of genotype identification in step (2) is as follows: the surviving mouse embryonic stem cells are lysed by using a monoclonal identification kit to obtain crude DNA, the crude DNA is subjected to PCR amplification according to designed primers, and then the amplification product is sequenced by using sequencing primers.

[0013] Further, the tetraploid blastocyst in step (3) is obtained by an electric fusion method.

[0014] Further, the screening process in step (4) is detected and screened by using sequencing primers designed based on the c.299_300delAT point mutation sequence.

[0015] The technical scheme of the present application discloses a GJB2 gene frameshift mutation DFNB1 type deaf mouse model.

[0016] Further, the hearing of the model is close to the hearing of a patient carrying the same type of mutation in clinic.

[0017] The technical scheme of the present application discloses a GJB2 gene frameshift mutation DFNB1 type deaf mouse model in the research of hearing phenotype, inner ear pathology or pathogenesis of congenital deafness caused by GJB2 gene c.299_300delAT site mutation.

[0018] Advantages of the present application: 1. The application discloses a kind of GJB2 gene frame shift mutation DFNB1 type deaf mouse model construction method, CRISPR / Cas9 mediated homologous directed repair technology is combined with tetraploid embryo compensation technology, solve the energy supply disorder of Gjb2 homozygous mutation caused in mouse embryo middle stage (E10.5-E12.5) lethality problem, make embryo survival rate improve, break through the "embryo lethal" bottleneck in the model construction of traditional gene editing technology.And by designing for mouse Gjb2 gene 2 exons double sgRNA (sgRNA-Gjb2-A1, sgRNA-Gjb2-A2) and the donor vector (Donor-Puro / Donor-BSD) containing precision mutation, realize the precision deletion of 2 bases of c.299_300delAT, avoid the random insertion / deletion (indel) caused by non-homologous end joining (NHEJ), and the precision of gene editing is high.

[0019] 2. The application discloses a kind of GJB2 gene frame shift mutation DFNB1 type deaf mouse model, model phenotype is highly consistent with clinical, i.e. the mutation site and human autosomal recessive 1 type deafness (DFNB1) core pathogenic mutation c.299_300delAT are completely identical, trigger nonsense-mediated mRNA degradation (NMD) by frame shift mutation, lead to cochlear tissue connexin 26 (Cx26) completely missing, accurately simulate the pathological chain of "Cx26 function loss-gap junction channel interruption-congenital severe deafness" of human patient.Auditory brainstem response (ABR) test shows that the hearing threshold of model mouse high frequency band (16-32kHz) is greater than or equal to 90dB, and is completely matched with the phenotype of severe to extremely severe hearing loss of clinical patients.

[0020] 3. The application discloses a kind of GJB2 gene frame shift mutation DFNB1 type deaf mouse model and its application, fill the blank in the field: current lack of animal model that can accurately simulate human Gjb2 c.299_300delAT mutation, the application provides a unique in-vivo research tool for analyzing the pathogenesis (such as cochlear cell communication disorder, NMD pathway activation) of the mutation.

[0021] Support multidimensional research needs: can be used for drug screening (such as gap junction modulator, gene therapy vector optimization), treatment target verification (such as NMD pathway inhibitor) and hearing function repair mechanism research, lay a foundation for the transformation from "symptomatic intervention" to "gene cure" of genetic deafness. BRIEF DESCRIPTION OF DRAWINGS

[0022] In order to make the technical solutions in the embodiments of the present application or the prior art clearer, the accompanying drawings needed in the embodiments or prior art description will be briefly introduced below. Obviously, the accompanying drawings in the following description only constitute some embodiments of the present application, and other accompanying drawings can be obtained by those skilled in the art without any creative effort on the basis of these accompanying drawings.

[0023] Figure 1 Strategy diagram for constructing Gjb2: c.299_300delAT homozygous frameshift mutation mice; Figure 2 Strategy diagram for constructing donor vectors Donor-Puro and Donor-BSD containing c.299_300delAT mutation, wherein (a) is a strategy diagram for constructing donor vector Donor-Puro, and (b) is a strategy diagram for constructing donor vector Donor-BSD; Figure 3 PCR product and PCR product sequencing map results of Gjb2: c.299_300delAT homozygous frameshift mutation embryonic stem cells, wherein (a) is a PCR product electrophoresis verification diagram, and (b) is a Sanger sequencing verification diagram; Figure 4 Strategy diagram for constructing Gjb2: c.299_300delAT homozygous mice by using tetraploid complementation technology; Figure 5 PCR product and PCR product sequencing map results of F0 generation Gjb2: c.299_300delAT homozygous frameshift mutation mice, wherein (a) is a PCR product electrophoresis verification diagram, and (b) is a Sanger sequencing verification diagram; Figure 6 Western blot detection results of Cx26 protein in cochlea and liver tissues of wild-type mice and Gjb2: c.299_300delAT homozygous frameshift mutation mice, wherein (a) is the Western blot detection results of Cx26 protein in cochlea tissues of mice, and (b) is the Western blot detection results of Cx26 protein in liver tissues of mice; Figure 7 ABR waveform representative diagram of auditory brainstem evoked potential experiment of wild-type and Gjb2: c.299_300delAT homozygous frameshift mutation mice; Figure 8 ABR waveform ANOVA variance test analysis schematic diagram of auditory brainstem evoked potential experiment of wild-type and Gjb2: c.299_300delAT homozygous frameshift mutation mice; Figure 9Figure 6 shows the DPOAE waveform chart of the wild type and Gjb2: c.299_300delAT homozygous frameshift mutation mouse aberrant product. DETAILED DESCRIPTION

[0024] The application will be further described in conjunction with the drawings, which can be further understood by referring to the following specific examples, which are presented herein for the purpose of illustration only and are not intended to limit the scope of the application. Various modifications and changes can be made to the application without departing from the spirit and scope thereof, and it is intended to include all such modifications and changes in the scope of the claims.

[0025] Example 1: Construction strategy of Gjb2: c.299_300delAT homozygous frameshift mutation mouse ESC 1. Determine the basic information of knockout gene (1) The name of the target gene (GenBank accession number): Gjb2 (NM_008125.3).

[0026] (2) The number of the target gene in the Ensembl database: ENSMSUG00000046352.

[0027] (3) The scheme corresponds to the transcript (Ensembl number): Gjb2-201 (ENSMUSG00000046352.8). The sequence of Gjb2-201 is shown in SEQ ID NO: 1.

[0028] (4) Mutation site: Deletion AT (299_300) of Mouse Gjb2.

[0029] 2. Construction strategy of Gjb2: c.299_300delAT homozygous frameshift mutation mouse ESC, as shown in Figure 1

[0030] (1) Using CRISPR / Cas9 gene editing system, 2 sgRNAs were designed for the 5' arm and 3' arm regions (close to 3UTR) of Gjb2 gene, wherein sgRNA Gjb2 The 5'-3' sequence of A1 is shown in SEQ ID NO: 2; sgRNA Gjb2 ​The 5'-3' sequence of A2 is shown in the sequence listing SEQ ID NO: 3, which guides the Cas9 nuclease to generate double-stranded DNA breaks (DSBs) at the above two sites, respectively, to form clear gaps on both sides of the target fragment containing the c.299_300 site in the Gjb2 wild-type allele, so as to initiate the subsequent homologous directed repair (HDR) process, and provide precise recombination boundaries for the donor vector carrying the c.299_300delAT mutant sequence to replace the wild-type fragment.

[0031] (2) Two donor vectors are constructed, respectively named Donor-Puro and Donor-BSD, and the structures are shown in Figure 2 (a) and Figure 2 (b), respectively, the sequence of Donor-Puro is shown in the sequence listing SEQ ID No. 6, and the sequence of Donor-BSD is shown in the sequence listing SEQ ID No. 7.

[0032] a) Both donor vectors contain 5arm sequences and 3arm sequences homologous to the Gjb2 gene fracture region, which are used to mediate HDR; b) A preset nucleotide mutation Gjb2:c.299_300delAT is introduced in the coding sequence (CDS) of the Gjb2 gene; c) Different selection marker cassettes are inserted between the CDS and the 3' untranslated region (3UTR): Donor-Puro vector: "Frt-Puro-Frt" cassette flanked by Flp recombinase recognition sites (Frt) and connected with the puromycin resistance gene (Puro) is inserted; Donor-BSD vector: "Frt-BSD-Frt" cassette flanked by Frt sites and connected with the blasticidin resistance gene (BSD) is inserted.

[0033] (3) The above two donor vectors (Donor-Puro, Donor-BSD) are mixed with CRISPR / Cas9 expression plasmid and sgRNA expression plasmid respectively, and are co-delivered into mouse ESC cells by electroporation; 24-48h after electroporation, the sgRNA generated by transcription of the sgRNA expression plasmid guides the Cas9 protein to generate double-stranded breaks (DSBs) at the preset sites of the Gjb2 wild-type (WT) allele, and at the same time, the donor vector undergoes homologous recombination with the fractured WT allele by the HDR mechanism, and finally two kinds of targeted alleles are obtained respectively: a) Recombination with Donor-Puro, to obtain ESC of Targeted allele1: its Gjb2 gene CDS with preset mutation c.299_300delAT, and "Frt-Puro-Frt" cassette inserted between CDS and 3UTR; b) Recombination with Donor-BSD, to obtain ESC of Targeted allele2: its Gjb2 gene CDS with preset mutation c.299_300delAT, and "Frt-BSD-Frt" cassette inserted between CDS and 3UTR.

[0034] (4) Homozygous mutation screening of cells: a) Mixed culture of the above-mentioned ESCs recombined with single donor vectors, and replacement with double antibiotic screening medium containing puromycin (final concentration 1 μg / mL) + bleomycin (final concentration 10 μg / mL); the screening culture conditions include: temperature 37°C, carbon dioxide mass concentration 5%.

[0035] b) Single clone picking of ESC clones surviving double antibiotic screening, i.e. using single clone identification kit to lyse the surviving mouse embryonic stem cells, obtaining crude DNA, and performing PCR amplification on the crude DNA according to the designed primers, and then sequencing the amplification product using sequencing primers, wherein the primers used are shown in SEQ ID No. 4 and SEQ ID No. 5; the sequencing primers used are SEQ ID No. 4.

[0036] PCR reaction system:

[0037] PCR reaction conditions:

[0038] By designing specific primers, the Gjb2 gene Exon 2 region of mouse ESC cells was PCR amplified, and the product was added to 1.5% agarose gel, and electrophoresis was performed at 120V for 30min, and the results were observed. The results are shown in Figure 3 (a), the length of the amplified fragment is 503bp, corresponding to the Gjb2 gene Exon 2 and its flanking region. Different lanes in the electrophoresis map represent different genotypes of mouse ESC cells, and the presence or absence and size of the band can preliminarily screen the positive cells of Gjb2 gene Exon 2 homozygous deletion, thereby completing the preliminary positive confirmation of the genotype, and screening the positive reaction confirmed mouse ESC cells Gjb2 gene Exon 2 homozygous deletion AT (299_300). To clarify the sequence accuracy of gene editing, the PCR positive product was subjected to Sanger sequencing and compared with the wild type sequence, and the results are as followsFigure 3 (b), the results show that there is a homozygous deletion mutation of c.299_300delAT in the Exon 2 region of the Gjb2 gene, which directly proves from the sequence level that the Exon 2 of the Gjb2 gene in the mouse ESC cells has achieved homozygous deletion, and finally verifies the effectiveness of gene editing.

[0039] 3. Flpo-mediated screening marker deletion Introducing Flpo recombinase into the homozygous mutant ESCs described above to mediate recombination between the two Frt sites, thereby deleting the screening marker: (1) For Targeted allele1, Flpo recombinase deletes the Puro gene in the “Frt-Puro-Frt” cassette, leaving only a single Frt site; (2) For Targeted allele2, Flpo recombinase deletes the BSD gene in the “Frt-BSD-Frt” cassette, leaving only a single Frt site.

[0040] (3) Finally, homozygous mutant ESCs are obtained, in which both alleles of the Gjb2 gene carry “Gjb2: c.299_300delAT + single Frt site”.

[0041] Example 2: Preparation of Gjb2: c.299_300delAT homozygous mouse based on tetraploid complementation technology Figure 4 As shown in the following steps: 1. Construction of tetraploid blastocysts (1) Select female mice of appropriate age (C57BL / 6N strain) and perform superovulation treatment by intraperitoneal injection of gonadotropin; then mate with male mice, and collect zygotes the next day; (2) Use electrofusion technology to electrically stimulate zygotes to fuse two pronuclei to obtain tetraploid embryos; place the tetraploid embryos in embryo culture medium and incubate at 37°C, 5% CO2 incubator to the blastocyst stage for standby.

[0042] 2. Preparation of embryonic stem cells Recover the Gjb2 gene modified ESC cells carrying Targeted allele1 and Targeted allele2 obtained in the above Example 1, inoculate in culture plates coated with mitomycin C treated feeder cells, use ESC cell complete culture medium for culture, maintain the undifferentiated state of the cells, and when the cells proliferate to an appropriate density, perform subsequent operations.

[0043] The tetraploid blastocyst was removed from the zona pellucida and aggregated with the single-cell digested ESC cells carrying the Gjb2:c.299_300delAT homozygous mutation; the aggregated embryos were placed in embryo culture solution and cultured to the expanded blastocyst stage to ensure good embryo development.

[0044] 3. Formation and development of chimeric embryos A pseudopregnant female mouse (ICR strain) was selected and the aggregated embryos were transplanted into its uterus by surgical operation.

[0045] 4. Generation and confirmation of mouse models After the embryos developed to term in the recipient mouse, the recipient mouse gave birth to F0 generation mice.

[0046] Example 3: Genotype identification of mice (1) The primers used are shown in SEQ ID No. 4 and SEQ ID No. 5; (2) The sequencing primer is SEQ ID No. 4; (3) PCR reaction system:

[0047] (4) PCR reaction conditions:

[0048] After the genomic DNA of the target F0 generation mouse to be identified was subjected to PCR amplification of the Gjb2 gene Exon 2 region, the amplification product was added to a 1.5% agarose gel and electrophoresed at 120V for 30 min. The electrophoresis result is shown in Figure 5 (a), which shows the expected positive reaction band, indicating that the mouse may carry a Gjb2 gene Exon 2 related mutation; further combined with the sequencing verification result, as shown in Figure 5 (b), it is determined that the mouse Gjb2 gene Exon 2 region has a deletion of AT base at position c.299_300, and it is finally confirmed that the mouse is a Gjb2:c.299_300delAT homozygous mutation mouse.

[0049] Example 4: Western blotting of Cx26 protein in cochlea and liver of Gjb2:c.299_300delAT mice 1. Tissue samples and protein preparation (1) Experimental animal selection: 8-week-old, 20-25 g Gjb2: c.299_300delAT mice and wild-type mice of the same background were selected, 4 in each group, of which 4 Gjb2: c.299_300delAT mice were M5, M6, M7 and M8, and 4 wild-type mice were WT5, WT6, WT7 and WT8.

[0050] (2) Tissue sampling: After isoflurane anesthesia (induction concentration 5%, maintenance concentration 2%) and decapitation, the mice were immediately placed on ice and operated: a) Cochlea: The skull was cut open, and the bilateral cochlea was separated with microforceps and placed in a pre-cooled 1.5 ml EP tube; b) Liver: Open the abdominal cavity, take about 0.1 g of liver tissue, and also place it in a pre-cooled 1.5 ml EP tube.

[0051] (3) Tissue lysis: Add 200 μl of RIPA lysis buffer containing protease inhibitors (containing 1% Triton X-100, 0.1% SDS) to each tube, cut the tissue into a homogenate state with an ophthalmic scissors, and lyse on ice for 30 minutes.

[0052] (4) Protein extraction and processing a) Centrifuge at 12000 rpm for 15 minutes at 4°C. The upper layer is the fat layer (discarded), and the middle clear liquid is the protein supernatant (carefully aspirate into a new tube to avoid aspirating the lower layer of sediment); b) Use the BC protein quantitative kit to detect the concentration: set a standard gradient of 0-200 μg / ml in a 96-well plate, add 10 μl of sample, add 200 μl of BCA working solution (A liquid B liquid = 50:1), incubate at 37°C for 30 minutes, then measure the absorbance at 562 nm with a microplate reader, and calculate the protein concentration according to the standard curve; c) Mix according to the ratio of "protein supernatant:5x loading buffer=4:1", and boil the sample in a 100°C metal bath for 10 minutes, and cool on ice.

[0053] 2. Electrophoresis and membrane transfer (1) SDS-PAGE electrophoresis a) Gel preparation: 12% separation gel and 5% concentration gel were selected according to the molecular weight of Cx26, and 10% ammonium persulfate and TEMED were added to initiate polymerization during preparation, isopropanol was coated on the surface of the separation gel, and it was left to stand at room temperature for 45 minutes until it was completely solidified; b) Sample loading and electrophoresis: Install the gel into the electrophoresis tank, add 1x Tris-glycine electrophoresis buffer, load 20 μl per well (total protein amount about 20 μg), and add 3 μl of protein Marker to the first well. c) Electrophoresis parameters: 80V constant voltage electrophoresis for 30 minutes, after bromophenol blue enters the separation gel, adjust to 120V constant voltage to continue electrophoresis until bromophenol blue migrates to the bottom of the gel 1cm, turn off the power.

[0054] (2) Transferring membrane a) Transferring membrane preparation: the gel is placed in 1x Tris-glycine transferring buffer for 15 minutes; PVDF membrane is soaked in methanol for 1 minute, and 6 same size filter papers are soaked in transferring buffer; b) Transferring membrane assembly: place in the transferring membrane clamp according to the order of “black sponge pad→3 filter papers→gel→PVDF membrane→3 filter papers→white sponge pad”, and roll each layer with a glass rod to remove air bubbles; c) Transferring membrane operation: place the transferring membrane clamp in the transferring membrane slot, add transferring buffer and put in an ice bag, and transfer at a constant current of 300mA for 1.5 hours.

[0055] 3. Immunoreaction and color development (1) Blocking and primary antibody incubation a) Blocking: after transferring membrane, take out the PVDF membrane and place it in a dish containing 5% skimmed milk powder, and shake slowly on a shaking table at room temperature for 1 hour; b) Primary antibody incubation: wash the membrane with TBST for 3 times, add rabbit anti-mouse Cx26 polyclonal antibody, and incubate on a shaking table at 4°C overnight.

[0056] (2) Secondary antibody incubation a) After primary antibody incubation, wash the membrane with TBST for 3 times; b) Add HRP labeled goat anti-rabbit IgG secondary antibody, and incubate on a shaking table at room temperature for 1 hour; c) After incubation, wash the membrane with TBST for 4 times.

[0057] (3) ECL color development and imaging a) Color development preparation: mix ECL chemiluminescence reagent kit A liquid (containing luminol) and B liquid (containing hydrogen peroxide) at a ratio of 1:1; b) Exposure imaging: place the PVDF membrane protein side up, drop ECL working solution to cover the entire membrane surface, react for 2 minutes, then put it into a chemiluminescence imager, adjust the exposure time according to the brightness of the band, and save the TIFF format image.

[0058] Detection results: Cx26, as a connexin family member, can form a non-covalent homodimer (theoretical molecular weight about 52 kDa, monomer about 26 kDa) through the interaction of the transmembrane domain or the cytoplasmic domain, but this aggregation state has tissue specificity. Cx26 in the cochlea needs to form stable connexin multimers to participate in the rapid transmission of auditory signals. The dimer stability is very strong, and it cannot be dissociated into monomers under conventional extraction conditions. In the liver, Cx26 functions mainly on the basis of intercellular signal transmission, without the need for high polymerization, and can be fully dissociated, retaining only two forms of unmodified monomers (~26 kDa) and phosphorylated monomers (~28-32 kDa). The specific results are as follows Figure 6 As shown in Figs. 6(a) and 6(b), the Cx26 protein dimer band of the cochlea of WT5, WT6, WT7 and WT8 mice is located near 55 kDa, the Cx26 protein of the cochlea of M5, M6, M7 and M8 Gjb2:c.299_300delAT mice is not expressed, and the reference protein (GAPDH) is normally expressed; the two bands of the liver Cx26 protein of WT5, WT6, WT7 and WT8 mice are located between 25-35 kDa, the liver Cx26 protein of M5, M6, M7 and M8 Gjb2:c.299_300delAT mice is not expressed, and the reference protein (GAPDH) is normally expressed. This result shows that the c.299_300delAT defect of the Gjb2 mutant mouse blocks the synthesis of intact protein, resulting in the complete loss of Cx26 protein.

[0059] Example 5: Auditory brainstem evoked potential experiment of Gjb2:c.299_300delAT mice (1) After the mice are adapted to the environment, the control group of wild type mice and the experimental group of Gjb2:c.299_300delAT mice are respectively subjected to auditory brainstem evoked potential (ABR) test.

[0060] (2) The mice are anesthetized by intraperitoneal injection of tri-bromoethanol (400 mg / kg), and the external auditory canal of the mice is cleaned.

[0061] (3) After the mice are deeply anesthetized, the ABR recording electrode is placed in the middle of the mouse's front parietal, penetrating the skin to the bone periosteum.

[0062] (4) The reference electrode and the ground electrode are respectively inserted into the test ear and the contralateral ear subcutaneously, and the sound horn is placed in the external auditory canal of the test ear.

[0063] (5) The ABR stimulating sound is selected as click and short pure tone (4, 8, 16, 32 kHz).

[0064] (6) The stimulation intensity starts from 90 dB SPL and gradually decreases by 10 dB, and when approaching the threshold, it gradually decreases by 5 dB until there is no hearing waveform, and the lowest stimulation intensity that can distinguish the repeatable ABR wave II is used to judge the ABR threshold.

[0065] (7) The ABR threshold is analyzed, that is, the lowest stimulation intensity required to induce a recognizable ABR waveform (such as wave V). The ABR response threshold of normal hearing mice is usually 30 dB, and if the response threshold is elevated, it may indicate hearing loss. The experimental results are as follows Figure 7 : The threshold range of the control group mice at each detection frequency is 10-30 dB, which is within the normal range, and the ABR waveform representative graph threshold is 20 dB; the ABR waveform representative graph threshold of the experimental group mice is 75 dB.

[0066] (8) ANOVA variance analysis shows that compared with the control group, the threshold of the experimental group mice at each detection frequency is significantly elevated, which is statistically significant, as shown in Figure 8 , indicating that the hearing (ABR) threshold of the experimental group mice is significantly elevated at each test frequency, indicating that the Gjb2:c.299_300delAT mice have severe hearing impairment.

[0067] Example 6: Distortion product otoacoustic emissions experiment of Gjb2:c.299_300delAT mice: (1) After the mice adapt to the environment, the control group wild type mice and the experimental group Gjb2:c.299_300delAT mice are tested for distortion product otoacoustic emissions (DPOAE); (2) After the mice are anesthetized, they are placed in a soundproof room and tested with a US TDT RZ6 machine, and a suitable probe is inserted into the mouse's external auditory canal and sealed to ensure that the probe is in the correct position without loosening or displacement; (3) Turn on the test software and set the stimulation signal parameters, including the frequency ratio and intensity of the two initial pure tones f1 and f2, and when the stimulation signal is stable; (4) Start the test, and the software will automatically record and analyze the distortion product otoacoustic emissions signal; (5) Perform DPOAE detection at each frequency from low to high frequency, and observe and record whether DPOAE waveform can be induced at each frequency; (6) By analyzing whether DPOAE waveform can be induced, the hearing level can be evaluated, and the function of outer hair cells and their response to different stimuli can be further understood.

[0068] The experimental results are as follows Figure 9The control group mice can successfully induce DPOAE waveform (marked by red box line), the experimental group mice cannot successfully induce DPOAE waveform, which indicates that the DPOAE of the experimental group mice cannot be induced, and it is shown that the function of the cochlear outer hair cells of the Gjb2:c.299_300delAT mice is significantly damaged.

[0069] The above is the preferred embodiment of the present application, and for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, which should also be considered as the protection scope of the present application.

Claims

1. A method for constructing a DFNB1-type deafness mouse model with a frameshift mutation in the GJB2 gene, characterized in that, It includes the following steps: (1) Two sgRNA plasmids were designed in exon 2 of the mouse Gjb2 gene and labeled as sgRNA. Gjb2 A1, sgRNA Gjb2 A2, sgRNA Gjb2 A1 can recognize the PAM site on the mouse Gjb2 gene as AGG, sgRNA. Gjb2 A2 can recognize the PAM site on the mouse Gjb2 gene as GGG; (2) The two sgRNA plasmids, Cas9 expression vector and donor vector containing c.299_300delAT mutation site described in step (1) were co-transfected into mouse embryonic stem cells. After 24-48 hours of transfection, the cells were screened in a double antibiotic selection medium. After 3-4 days of screening culture, the surviving mouse embryonic stem cells were screened by PCR genotyping to identify mouse embryonic stem cells carrying the c.299_300delAT mutation site in the Gjb2 gene. (3) The mouse embryonic stem cells selected in step (2) are injected into the inner cell mass region of the tetraploid blastocyst to form a recombinant embryo. The recombinant embryo is then transplanted into the uterus of a surrogate mother mouse, and after pregnancy and birth, F0 generation mice are obtained. (4) Screen the F0 generation mice in step (3) and select mice that are homozygous to carry the c.299_300delAT point mutation to obtain the mouse model carrying the c.299_300delAT homozygous point mutation of the GJB2 gene.

2. The method for constructing a DFNB1-type deafness mouse model with a frameshift mutation in the GJB2 gene according to claim 1, characterized in that, The donor vector in step (2) includes Donor-Puro and Donor-BSD; the Donor-Puro contains 5'arm and 3'arm homologous to the sgRNA plasmid of the mouse Gjb2 gene, a CDS sequence carrying the c.299_300delAT mutation, and a selection cassette flanked by the puromycin resistance gene by the Flpo recombinase recognition site. The Donor-BSD contains 5'arm and 3'arm homologous to the sgRNA plasmid of the mouse Gjb2 gene, a CDS sequence carrying the c.299_300delAT mutation, and a selection marker cassette flanked by the Flp recombinase recognition site linking the bleomycin resistance gene.

3. The method for constructing a DFNB1-type deafness mouse model with a frameshift mutation in the GJB2 gene according to claim 1, characterized in that, The transfection in step (2) is electrotransfection.

4. The method for constructing a DFNB1-type deafness mouse model with a frameshift mutation in the GJB2 gene according to claim 1, characterized in that, In step (2), the final concentration of puromycin in the dual antibiotic screening medium is 1 μg / mL, and the final concentration of bleomycin is 10 μg / mL. The screening culture conditions include: temperature of 37℃ and carbon dioxide concentration of 5%.

5. The method for constructing a DFNB1-type deafness mouse model with a frameshift mutation in the GJB2 gene according to claim 1, characterized in that, The procedure for genotype identification in step (2) is as follows: surviving mouse embryonic stem cells are lysed using a monoclonal identification kit to obtain crude DNA. The crude DNA is then amplified by PCR using the designed primers, and the amplified product is sequenced using sequencing primers.

6. The method for constructing a DFNB1-type deafness mouse model with a frameshift mutation in the GJB2 gene according to claim 1, characterized in that, The tetraploid blastocyst in step (3) is obtained by electrofusion.

7. The method for constructing a DFNB1-type deafness mouse model with a frameshift mutation in the GJB2 gene according to claim 1, characterized in that, The screening process in step (4) is based on sequencing primers designed with the c.299_300delAT point mutation sequence for detection and screening.

8. A DFNB1-type deaf mouse model with a frameshift mutation in the GJB2 gene obtained by the construction method according to any one of claims 1-7.

9. The DFNB1-type deafness mouse model with GJB2 gene frameshift mutation according to claim 8, characterized in that, The model's hearing is close to that of clinical patients carrying the same type of mutation.

10. The application of the DFNB1-type deafness mouse model with GJB2 gene frameshift mutation as described in claim 8 in studying the hearing phenotype, inner ear pathology, or pathogenesis of congenital deafness, characterized in that... The congenital deafness is caused by a mutation at the c.299_300delAT site in the GJB2 gene.

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