Construction method of alport syndrome mouse nmd escape model and application thereof

Abstract

The application discloses a kind of Alport syndrome mouse NMD escape model construction method and its application, based on CRISPR / Cas9 gene editing technology preparation carries Col4a5 gene c.4432delG frame mutation's non-human animal model.The method is by to mouse zygote co-injection targeted Col4a5 gene 49th exon gRNA, contains c.4432delG mutation's homologous recombination donor oligonucleotide and Cas9 nuclease, obtains F0 generation mutant mouse by embryo transplantation;By PCR in combination with sequencing verification mutation site, again by two generations of reproduction establishes stable genetic mutant line.It is verified that the model meets the pathological characteristics of Alport syndrome, can stably simulate the typical clinical manifestations and pathological characteristics of human X-linked Alport syndrome, and can be used as an important tool for Alport syndrome research.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of bioengineering, and particularly relates to a method for constructing an Alport syndrome mouse NMD escape model and application thereof. BACKGROUND

[0002] Alport syndrome is a genetic collagen disease caused by mutations in the genes COL4An (n=3, 4, 5) that encode the alpha-3, alpha-4 and alpha-5 chains of type IV collagen, resulting in basement membrane lesions. It most commonly involves the kidneys, followed by the eyes and ears. Due to the above mutations, the collagen structure of the organs is abnormal, which further manifests as hereditary glomerular disease, including hematuria, proteinuria and progressive decline in renal function, often accompanied by sensorineural hearing loss and ocular abnormalities. Male patients with X-linked dominant inheritance and patients with autosomal recessive inheritance progress relatively rapidly, often developing into end-stage renal disease at around 20 years old, with severely impaired renal function, and requiring kidney replacement therapy to maintain life activities. The progression of female patients with X-linked dominant inheritance and patients with autosomal dominant inheritance is relatively slow, and most of them will show renal failure after 60 years old, and the renal function can be maintained in a relatively stable state for a long period of time, but eventually the decline of renal function cannot be avoided.

[0003] Nonsense-mediated mRNA decay (NMD) is a ubiquitous mRNA quality control mechanism in eukaryotes, which plays a key role in maintaining the precise regulation of gene expression. Under normal physiological conditions, premature termination codons (PTCs) generated by alternative splicing, transcription errors or programmed gene rearrangements will trigger the NMD pathway, causing the mRNA containing PTCs to be specifically recognized and degraded, thereby avoiding the production of harmful truncated proteins and ensuring normal physiological functions of cells. However, under pathological conditions, gene mutations, especially nonsense and frameshift mutations, also produce PTCs and activate NMD, leading to mRNA degradation, ultimately reducing the expression of target proteins and triggering a series of diseases. Among these diseases, the phenomenon of NMD escape gradually enters the researchers' field of vision. NMD escape refers to the fact that some PTCs do not trigger NMD, and NMD escape leads to the degradation of mRNA containing premature termination codons, which in turn produces truncated proteins and triggers diseases. The occurrence of this phenomenon makes the pathogenesis of diseases more complex, and also brings new challenges and research directions for the treatment of related diseases.

[0004] The gene frequency of Alport syndrome is 1 / 10 000-1 / 5000. The common genetic patterns are X-linked inheritance (80%-85%), autosomal recessive inheritance (15%) and autosomal dominant inheritance (5%) in turn. The patients with X-linked inheritance (XLAS) are almost caused by mutations in COL4A5 gene, which is located in the X chromosome q22 fragment and encodes the α5 chain of type IV collagen. In XLAS patients, the most diagnostic significance is the α5 chain staining of glomerular basement membrane. Due to mutations in COL4A5 gene, the synthesis of α5 chain is abnormal or missing, and in most cases, the α5 chain staining of glomerular basement membrane is negative or significantly weakened. This is one of the important pathological features of XLAS and has key diagnostic value in immunofluorescence detection of kidney biopsy. The mutation types of COL4A5 gene are diverse, including missense mutation, deletion mutation and insertion mutation. Different mutations may have different effects on the synthesis, structure and function of α5 chain. Most of the mutations producing PTC result in complete absence of α5 chain due to triggering NMD pathway, so that the staining is negative; while a few mutations producing PTC result in abnormal function of α5 chain due to NMD escape, but there is still partial expression, so the staining may be weakly positive or patchy positive, rather than completely negative.

[0005] At present, three different types of XLAS mouse models have been constructed at home and abroad: In 2004, Michelle N. Rheault constructed an Alport syndrome mutant mouse model with G5X mutation by targeting the Col4a5 gene of mice; in 2019, Kentarou Hashiami constructed an Alport syndrome mutant mouse model with Col4a R471X mutation by using CRISPR-associated technology; in 2020, Li Xiaojun constructed an Alport syndrome mutant mouse model with Del-ATGG mutation in Col4a5 gene by using CRISPR / Cas9 technology. However, the mutation positions of the three models are different, and the severity of the corresponding mice also varies, which makes it difficult to unify the existing models to simulate the typical pathological features of human XLAS, such as progressive renal failure and glomerular basement membrane layering, and cannot cover all types of gene mutations related to the disease, limiting the in-depth study of gene function and disease mechanism. Moreover, in terms of nonsense mutation-mediated mRNA degradation pathway, the existing models cannot accurately reflect the development process of the disease under NMD escape, and cannot fully reveal the pathogenesis of Alport syndrome.

[0006] In view of the potential important role of NMD escape in the development of the disease, it is of great scientific and clinical significance to construct an Alport syndrome mouse model that can occur NMD escape. SUMMARY

[0007] The first object of the present application is to provide a method for constructing an Alport syndrome mouse NMD escape model.

[0008] The second object of the present application is to provide an application of the Alport syndrome mouse NMD escape model.

[0009] The first object of the present application is implemented by the following technical solutions:

[0010] The method for constructing an Alport syndrome mouse NMD escape model deletes a single base G at the c.4432 site on the 49th exon of the mouse Col4a5 gene (the sequence of which is shown in SEQ ID NO. 1) by using CRISPR / Cas9 technology.

[0011] The method comprises the following steps: S1, designing a gRNA plasmid for expressing a mouse-derived Col4a5 gene 49th exon, the gRNA sequence being 5'-CAGAAGCACCACAATGCCCACGG-3' (SEQ ID NO. 2), and the PAM being CGG; S2, obtaining the gRNA designed in S1 by using an in vitro transcription technology; S3, co-injecting the gRNA targeting the mouse Col4a5 gene, a donor oligonucleotide containing the c.4432delG mutation, and a Cas9 nuclease into a mouse zygote, and transplanting the zygote into a recipient female mouse to develop the mouse.

[0012] Preferably, the method further comprises a step of identifying the mouse genotype by using a primer pair; the forward primer used in the identification is 5'-CCCAGAGCTTGCTTAATCTTGTAA-3' (SEQ ID NO. 3), and the reverse primer is 5'-GTGACTGAAGTTATTTGGCTCTGA-3' (SEQ ID NO. 4).

[0013] Preferably, the method further comprises one or more of the following: a histopathological examination evaluation of the mouse model, a systematic evaluation of urine biochemical indicators, and a real-time fluorescent quantitative PCR evaluation. The consistency of the constructed model with human diseases is verified by genotype identification and multi-dimensional characterization evaluation.

[0014] Preferably, the pathological histological examination evaluation includes at least one of HE staining, PAS staining, Masson staining, kidney transmission electron microscopy, type IV collagen alpha 5 chain staining. HE staining can be used to observe glomerular capillary sclerosis, mesangial widening and other lesions; PAS staining can be used to detect basement membrane fibrosis; Masson staining can be used to evaluate collagen fiber deposition; kidney transmission electron microscopy can be used to observe the status of glomerular basement membrane; type IV collagen alpha 5 chain staining can be used to evaluate the expression of type IV collagen alpha 5 chain.

[0015] Preferably, the urine biochemical index system evaluation index includes at least one of urine creatinine, urine albumin, urine microalbumin / creatinine ratio.

[0016] Preferably, the real-time fluorescent quantitative PCR evaluation step includes: sample total RNA extraction, total RNA quality detection, reverse transcription of qualified RNA to synthesize cDNA, real-time fluorescent quantitative detection of the cDNA obtained by reverse transcription.

[0017] The second object of the application is implemented by the following technical scheme:

[0018] The use of Col4a5: c.4432delG mice obtained by the construction method of an Alport syndrome mouse NMD escape model as an Alport syndrome research model mouse.

[0019] Beneficial effects: The application provides a construction method of an Alport syndrome mouse NMD escape model, selects a Col4a5 gene 49th exon c.4432delG frame shift mutation, simulates a common mutation type of human XLAS, can trigger an NMD escape mechanism, and causes a type IV collagen alpha 5 chain complete functional fragment to be missing. The constructed c.4432delG mutant mouse model is verified by experiments to meet the pathological characteristics of Alport syndrome, can stably simulate the typical clinical manifestations and pathological characteristics of human X-linked Alport syndrome, such as glomerular basement membrane stratification, foot process fusion, and proteinuria. The established model can reflect the occurrence and development process of the disease under the condition of NMD escape, and is helpful for in-depth analysis of the pathogenesis of Alport syndrome, especially the interaction mechanism between podocyte damage and basement membrane abnormalities. Can be used as an important tool for Alport syndrome research, and is applied to the fields of pathogenesis analysis, drug screening, and treatment target verification. BRIEF DESCRIPTION OF DRAWINGS

[0020] 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. Obviously, the accompanying drawings in the following description only only some embodiments of the present application, and for those skilled in the art, other drawings can be obtained without creative work based on these drawings.

[0021] Figure 1 The strategy diagram for constructing Col4a5 gene c.4432delG mutant mice; the knock-in mutation site is located in the 49th exon of the mouse Col4a5 gene;

[0022] Figure 2 PCR product gel electrophoresis of F1 generation deletion positive mice and sequencing map of PCR product; (a) is the PCR product gel electrophoresis of F1 generation deletion positive mice, wherein, the numbers 5, 6, 12-16, 18, 19, 25 are mutant mice, and the numbers 1-4, 7-11, 17, 20-24 are wild type mice; (b) is the sequencing map of PCR product of F1 generation deletion positive mice;

[0023] Figure 3 Kidney histopathology of wild type and Col4a5:c.4432delG mice;

[0024] Figure 4 Kidney electron microscopy results of wild type and Col4a5:c.4432delG mice;

[0025] Figure 5 Collagen type IV alpha 5 chain staining of wild type and Col4a5:c.4432delG mice;

[0026] Figure 6 Biochemical indicators of urine of wild type and Col4a5:c.4432delG mice;

[0027] Figure 7 Gel electrophoresis of total RNA extracted from wild type and Col4a5:c.4432delG mice; wherein, the numbers 1-7 are the test groups (Col4a5:c.4432delG mice), and the numbers 8-10 are the control groups (wild type mice);

[0028] Figure 8 Melting curve of each PCR amplification product in Example 5;

[0029] Figure 9 RNA relative expression of collagen type IV alpha 1-alpha 6 chain RT-qPCR of wild type and Col4a5:c.4432delG mice. DETAILED DESCRIPTION

[0030] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative work fall within the protection scope of the present application. In the entire specification, unless otherwise defined, all the technical and scientific terms used herein have the same meaning as that generally understood by a person of ordinary skill in the art to which the present application belongs. The present specification is preferred in case of conflict.

[0031] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present application can be obtained by market purchase or can be obtained by existing methods.

[0032] The general idea of the present application is as follows:

[0033] The CRISPR / Cas9 system, as an acquired immune system of prokaryotes, has been widely used in the field of gene editing due to its simple operation, high editing efficiency and wide species adaptability. This technology guides Cas9 nuclease to target gene for site-specific cleavage through gRNA, triggers cell DNA repair mechanism (such as non-homologous end joining or homologous recombination), and realizes permanent modification of genome. However, in the construction of Alport syndrome model, the existing technology has not fully utilized the advantage of precise mutation in the middle region of the gene to construct a model closer to the characteristics of human diseases. Based on the CRISPR / Cas9 gene editing technology, the present application induces c.4432delG frameshift mutation on exon 49 of mouse Col4a5 gene to construct a mouse model that can stably simulate human X-linked Alport syndrome (XLAS). Through gRNA design, in vitro transcription, microinjection, genotype identification and phenotype verification, the construction and function verification of the mutant model are realized, which provides a tool for disease mechanism research and drug development.

[0034] Example 1 - Construction of Col4a5: c.4432delG mouse

[0035] A mouse model of Col4a5 gene terminal region c.4432delG mutation is constructed, and the design scheme of the gene knock-in mouse model is as follows.

[0036] (1) Basic information of the mutant gene: the name of the gene to be mutated is Col4a5 (GenBank accession number: NM_001163155.1; Ensembl number: ENSMUSG00000031274), the mutant scheme corresponds to the transcript Col4a5-202 (Ensembl number: ENSMUST00000112931), and the mutation site is Deletion G(4432) of MouseCol4a5.

[0037] (2) Construction of Col4a5: c.4432delG mouse strategy, as shown in Figure 1 : design gRNA targeting the 49th exon of the mouse Col4a5 gene (sequence as shown in SEQ ID NO. 1), the gRNA sequence is named gRNA-Col4a5-A1, the gRNA sequence is 5'-CAGAAGCACCACAATGCCCACGG-3' (SEQ ID NO. 2), and the PAM sequence is CGG (located at the 3' end of gRNA); the sequence targets the vicinity of the c.4432 site to ensure precise cutting of the Cas9 nuclease to the target gene. Design principle: through the complementary binding of gRNA and target gene, guide Cas9 protein to produce DNA double-strand break (DSB) at 3 bp upstream of PAM, trigger cell repair mechanism. Through gRNA-guided Cas9 nuclease precise cutting of the target gene, combined with homologous recombination donor oligonucleotide to realize site-directed mutagenesis, the mutation efficiency is significantly higher than that of traditional gene editing technology.

[0038] (3) Synthesize gRNA using in vitro transcription technology (such as T7 RNA polymerase), and construct homologous recombination donor oligonucleotide (Donor oligo) at the same time, the nucleotide sequence is shown in SEQ ID NO. 5. Mix gRNA targeting Col4a5 gene (concentration 100 ng / μL), Donor oligo containing c.4432delG mutation (concentration 3 ng / μL), and Cas9 nuclease (concentration about 50 ng / μL) to obtain a mixed solution. Inject the mixed solution into the cytoplasm or nucleus of the fertilized egg of C57BL / 6J mouse by microsurgical technique, and transplant the injected fertilized egg into the uterus of a pseudopregnant female mouse to develop into F0 generation mice. Cross the F0 generation positive mice with wild type mice to obtain F1 generation mice.

[0039] Example 2-Genotype identification and model establishment of F1 generation mice

[0040] (1) PCR amplification

[0041] Design specific primers to amplify the genomic fragment containing the c.4432delG mutation site. Use F1 generation mouse tail genomic DNA as template, PCR amplification is carried out by forward primer F1: 5'-CCCAGAGCTTGCTTAATCTTGTAA-3' (SEQ ID No. 3) and reverse primer R1: 5'-GTGACTGAAGTTATTTGGCTCTGA-3' (SEQ ID No. 4). The PCR amplification reaction system (total amount 50 μL): mouse tail genomic DNA is 2 μL, forward primer (10 μM) is 2 μL, reverse primer (10 μM) is 2 μL, dNTPs (2.5 mM) is 6 μL, 5X LongAmp Taq reaction buffer is 10 μL, LongAmp Taq DNA polymerase is 2 μL, double distilled water is 26 μL; the reaction program is shown in Table 1.

[0042] Table 1 PCR amplification reaction program

[0043]

[0044] (2) DNA electrophoresis identification

[0045] The PCR product is added to 1.5% agarose gel, electrophoresis at 120V for 30 min, and the results are shown in Figure 2 . The positive band size of MT mutant is 417 bp, and that of WT wild type is 418 bp. The positive reaction confirms the deletion of G (4432) in Exon49 of Col4a5 gene, and the genotype is determined by direct sequencing method (the sequencing primer is shown in SEQ ID No. 2) of PCR product. The sequencing confirms that the single base G is deleted at site c.4432 (frameshift mutation).

[0046] (3) Establishment of stable genetic line

[0047] F1 generation mice are interbred, and F2 generation mice are screened by PCR and sequencing to establish a stable genetic Col4a5:c.4432delG mutant mouse model (hereinafter referred to as Col4a5:c.4432delG mouse), which ensures the repeatability and long-term research value of the model.

[0048] Example 3 - Pathological histological examination of Col4a5:c.4432delG mice

[0049] 1. Kidney tissue staining observation

[0050] The wild type mice (C57BL / 6J, same below) and Col4a5:c.4432delG mice were respectively paraffin sectioned, and the morphological differences of the kidney tissues of the two mice were observed after staining: the glomerular capillary sclerosis, mesangial widening and other lesions were observed by HE staining, the basement membrane fibrosis was detected by PAS staining, and the collagen fiber deposition was evaluated by Masson staining.

[0051] (1) Paraffin section steps

[0052] ①After taking the material, it is quickly fixed, and then dehydrated with 80%, 90%, 95%, and 100% ethanol for 2 h, and transparentized with xylene;

[0053] ②The paraffin is immersed into the tissue to replace the transparent agent (xylene) contained in the tissue, and then the wax-impregnated tissue is placed in the melted solid paraffin, and after the paraffin solidifies, the tissue is embedded in it;

[0054] ③The wax block is cut into sections, and the sections are gently laid on the water surface at 45°C with ophthalmic forceps, and the slightly wrinkled sections are naturally flattened by the water tension and temperature;

[0055] ④After flattening, the sections are taken to the middle section of the glass slide, the remaining water on the glass slide is poured off, and the sections are placed in a 65°C constant temperature oven or a section baking temperature controller oven for 30 min to remove the paraffin in the dissolved tissue gap.

[0056] (2) HE staining steps

[0057] ①Paraffin section deparaffinization to water: the sections are sequentially placed in xylene I for 10 min, xylene II for 10 min for deparaffinization, and then sequentially placed in anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 95% alcohol for 5 min, 90% alcohol for 5 min, 80% alcohol for 5 min, 70% alcohol for 5 min, and then washed with distilled water to complete gradient hydration;

[0058] ②Staining: the washed sections are placed in Harris hematoxylin for 8 min, washed with tap water, differentiated with 1% hydrochloric acid alcohol for a few seconds, washed with tap water, soaked in 0.6% ammonia water to return blue, and then the sections are placed in eosin staining solution for 3 min; complete hematoxylin staining of cell nuclei and eosin staining of cytoplasm;

[0059] ③Dehydration and mounting: the sections are sequentially placed in 95% alcohol I for 5 min, 95% alcohol II for 5 min, anhydrous ethanol I for 5 min, and anhydrous ethanol II for 5 min for dehydration, and then sequentially placed in xylene I for 5 min and xylene II for 5 min for transparency, and the sections are taken out of the xylene and slightly dried, and then mounted with neutral balsam.

[0060] (3) PAS staining steps

[0061] ① Paraffin sections were dewaxed to water, washed with distilled water, and washed with 70% alcohol;

[0062] ② Oxidation was performed by immersing in a high iodine acid alcohol solution for 10 min (solution temperature 20℃);

[0063] ③ After washing with 70% alcohol, immerse in a reducing solution for 1 min (solution temperature 20℃), then wash with 70% alcohol, and then immerse in a colorless safranine solution for 1.5 h, and place in a 37℃ incubator; the reducing solution is prepared by preparing 1 g of sodium thiosulfate, 30 mL of 95% alcohol, and 20 mL of distilled water; add 0.5 ml of 2 mol / L HCl, wait until completely dissolved, then add 1 g of potassium iodide; if there is a small amount of sulfur precipitate, it does not affect the staining, and can be stored in a 4℃ refrigerator;

[0064] ④ Wash with running water for 10 min, and then re-stain the cell nucleus with Mayer's hematoxylin reagent for 5 min, and then differentiate with 1% hydrochloric acid alcohol; after washing with running water, dehydrate, transparentize, and mount.

[0065] (4) Masson staining steps

[0066] ① Paraffin sections were dewaxed to water, and then stained with Weigert iron hematoxylin for 10 min; differentiate with an acidic ethanol differentiation solution, and then wash with distilled water for 1 min;

[0067] ② Re-blue with Masson blue solution, and then wash with distilled water for 1 min; stain with ponceau red staining solution for 10 min;

[0068] ③ Prepare a weak acid working solution in a ratio of distilled water: weak acid solution = 2:1; wash with the weak acid working solution for 1 min; wash with phosphomolybdic acid solution for 2 min, and then wash with the prepared weak acid working solution for 1 min;

[0069] ④ Directly immerse in aniline blue staining solution for 2 min, and then wash with the prepared weak acid working solution for 1 min;

[0070] ⑤ Rapidly dehydrate with 95% ethanol; dehydrate with anhydrous ethanol three times, each time for 10 s;

[0071] ⑥ Transparentize with xylene three times, each time for 2 min; mount with neutral balsam.

[0072] (5) Results

[0073] Observe the HE staining, Masson staining, and PAS staining sections under a microscope, and the results are as follows Figure 3HE staining showed glomerular basement membrane injury and tubular cell necrosis in Col4a5:c.4432delG mice compared with wild-type C57BL / 6J mice; Masson staining showed thickening of the glomerular basement membrane and severe interstitial fibrosis; PAS staining showed a large increase in mesangial matrix.

[0074] 2. Kidney transmission electron microscopy

[0075] (1) Transmission electron microscopy section steps

[0076] ① Take the fixed sample: quickly cut the kidney tissue of wild-type mice and Col4a5:c.4432delG mice, and the sample size is 0.5-1 mm 3 , immerse the tissue in 4% glutaraldehyde within 1 min for fixation for 4 h or more, and store at 4°C; rinse with 1 M phosphate buffer for 3 times, 15 min each time;

[0077] ② Post-fixation: immerse in 1% osmium tetroxide for 2 h, and store at 4°C refrigerator; rinse with 1 M phosphate buffer for 3 times, 15 min each time;

[0078] ③ Room temperature dehydration: sequentially use 30% acetone, 50% acetone, 70% acetone, 80% acetone, and 90% acetone for gradient dehydration, 15 min each step; use 100% acetone for dehydration 3 times, 30 min each time;

[0079] ④ Infiltration and embedding: infiltrate the embedding agent at room temperature for 3 hours to gradually infiltrate the embedding agent into the tissue cells; 100% acetone: embedding solution = 1:1, incubate at 37°C for 60 min; pure embedding solution is incubated at 37°C for 5 h; use the embedding agent to embed the sample in the embedding plate;

[0080] ⑤ Polymerization: place the embedding plate in the oven, heat at 37°C for 24 h, and heat at 60°C for 48 h;

[0081] ⑥ Ultrathin sectioning: use an ultramicrotome to cut the sample into ultrathin sections with a thickness of 50 nm;

[0082] ⑦ Electron staining: first, ultrathin sections are stained with lead citrate for 10 min, and then washed with carbon dioxide-free hydrogen peroxide for 3 times, and then stained with uranyl acetate for 30 min, and then washed with Milli-Q water for 3 times; after the ultrathin sections are dried, they can be observed under a transmission electron microscope.

[0083] (2) Results

[0084] The electron microscopy results are as follows Figure 4As shown, the basement membrane (BM) of the kidney of the wild-type mouse at 28 weeks was clear and had no obvious rupture, and most of it was uniform in thickness, and thickening was observed in individual areas. Under transmission electron microscopy, it was observed that the glomerular basement membrane of the kidney of the Col4a5: c.4432delG mouse was uneven in thickness, and the foot processes were fused or disappeared in large areas, and the interpodocyte space was widened.

[0085] 3. Immunostaining of mouse kidney collagen

[0086] (1) Staining steps

[0087] ① De-waxing: sequentially immerse in dimethylbenzene for 10 min x 2 times, 100% ethanol for 5 min x 2 times, 95% ethanol for 5 min x 2 times, 85% ethanol for 5 min x 2 times, 75% ethanol for 5 min x 2 times, and distilled water for 5 min x 1 time;

[0088] ② Repair: PBS buffer for 2 min x 3 times, and wipe off the PBS outside the specimen with filter paper;

[0089] ③ Blocking: drop blocking serum, and incubate in a wet box at 37°C for 60 min; wipe off the blocking solution;

[0090] ④ Incubation: drop the primary antibody of appropriate concentration, and incubate in a wet box at 4°C overnight; then wash off the primary antibody with PBS for 5 min x 5 times, wipe off the PBS outside the specimen with filter paper, drop the fluorescent secondary antibody, and incubate in a wet box at 37°C for 1 hour in the dark; then wash off the secondary antibody with PBS for 5 min x 3 times, and wipe off the PBS outside the specimen with filter paper;

[0091] ⑤ Mounting: drop DAPI, and incubate in the dark for 10 min; then perform nucleic acid staining on the specimen, wash off the DAPI with PBS for 5 min x 3 times, and wipe off the PBS outside the specimen with filter paper. Finally, mount with glycerol, and observe immediately under a fluorescence microscope.

[0092] (2) Results

[0093] The mutation types of COL4A5 gene are diverse, including missense mutations, deletion mutations, and insertion mutations. Different mutations may have different degrees of influence on the synthesis, structure, and function of the a5 chain. Most mutations that produce PTC result in complete absence of the a5 chain due to triggering of the NMD pathway, thereby making the staining negative. A small number of mutations that produce PTC result in abnormal function of the a5 chain due to NMD escape, but there is still partial expression, at which time the staining may be weakly positive or patchy positive, rather than completely negative.

[0094] For example, Figure 5As shown, the glomerular capillary loop and Bowman's capsule wall of the kidney tissue of wild-type mice were continuously and clearly stained with green fluorescence, indicating that the a5 chain of type IV collagen was normally expressed in these parts, and the staining was positive. The staining of the a5 chain of type IV collagen in the glomerular capillary loop and Bowman's capsule wall of the kidney tissue of Col4a5:c.4432delG mice was patchy positive. This indicates that the protein expression amount and spatial uniformity of the a5 chain of type IV collagen protein are interfered by the c.4432delG gene mutation, the a5 chain is abnormally expressed, and truncated proteins can be translated, which may have toxicity or dominant negative effect, leading to diseases.

[0095] In summary, according to the results of the histopathological test, the kidneys of Col4a5:c.4432delG mice showed histopathological features of Alport syndrome.

[0096] Example 4 - Detection of urine biochemical indicators of Col4a5:c.4432delG mice

[0097] 1. Experimental method

[0098] Col4a5:c.4432delG male / female mice and wild-type male / female mice of the same age were used as controls and were raised in a SPF-level animal laboratory, with the environmental temperature maintained at 22±2°C, the humidity at 50±10%, a 12-hour light / dark cycle, and free access to food and water. The kidney function of Col4a5:c.4432delG mice was detected every 4 weeks: the mice were individually raised in 24-hour metabolic cages to ensure no fecal contamination, and 24-hour urine samples were collected, with the urine volume recorded.

[0099] The collected urine samples were centrifuged at 3000 rpm for 20 min at 4°C, and the supernatant was taken; a kidney function detection kit was used to perform quantitative analysis of urine creatinine (U-CRE), urine albumin (U-ALB), and urine trace albumin / urine creatinine ratio (U-ACR) indicators on a full-automatic biochemical analyzer. The detection data were statistically analyzed with the same indicator data of wild-type mice.

[0100] 2. Results

[0101] The specific results are as follows Figure 6As shown, with the development of the disease process, the urine creatinine and urine albumin of Col4a5: c.4432delG mice showed an upward trend; the indicators of wild-type mice were relatively stable. And compared with wild-type mice, the urine microalbumin / urine creatinine ratio of Col4a5: c.4432delG mice was significantly increased. It is shown that the c.4432delG mutation can cause kidney damage in mice, manifested as increased urine albumin leakage, altered urine creatinine excretion, and significantly decreased kidney function. Combined with the pathogenic relevance of this gene mutation and Alport syndrome, it is further shown that the mouse model meets the pathological characteristics of Alport syndrome. In addition, although the kidney function of female mutant mice has changed, it is relatively flat, and the overall difference is not as significant as that of male mutant mice, which further confirms that male patients with Alport syndrome are more severe than female patients.

[0102] Example 5- Real-time quantitative PCR of Col4a5 mRNA in kidney tissue of Col4a5: c.4432delG mice

[0103] 1. Total RNA extraction (Trizol method)

[0104] (1) The kidney tissue samples of wild-type mice and Col4a5: c.4432delG mice were ground and pulverized after being added to liquid nitrogen;

[0105] (2) The ground samples were transferred to a 1.5 mL EP tube with 1 mL Trizol;

[0106] (3) 500 μL of phenol chloroform was added to the 1.5 mL EP tube, and after shaking and mixing, it was static for 5 min; centrifuged at 12000 rpm for 10 min at 4°C, and the supernatant was carefully transferred to a new 1.5 mL EP tube;

[0107] (4) 700 μL of isopropanol was added to the separated supernatant and mixed thoroughly; centrifuged at 12000 rpm for 10 min at 4°C, and the supernatant was carefully discarded;

[0108] (5) The precipitate was washed once with 75% ethanol and dried at room temperature;

[0109] (6) 50 μL of DEPC water was added to dissolve the RNA precipitate;

[0110] (7) Agarose gel electrophoresis detection, as shown in Figure 7 , the 28S, 18S bands are clear, the brightness ratio is appropriate, and the 5S band exists and the brightness is relatively low, indicating that the total RNA extracted is of good integrity.

[0111] 2. Total RNA quality detection

[0112] (1) Preparation: Before measurement, use DEPC water to dissolve RNA to adjust the zero of the nucleic acid concentration detector, add the sample to the nucleic acid concentration detector detection base, and use the software on the computer to start the light absorption value detection;

[0113] (2) Concentration determination: the reading value at 260 nm is 1, indicating 40 ng RNA / μL; the sample RNA concentration calculation formula is: A260 x 40 ng / μL; the concentration average value is 897.18 ng / μL;

[0114] (3) Purity detection: the A260 / A280 ratio of the RNA solution is a method for detecting the purity of RNA, and the ratio range is 1.8 to 2.1; in this embodiment, the purity average value (n=10) is 2.004.

[0115] 3. Reverse transcription to synthesize cDNA

[0116] The reverse transcription uses the reverse transcription kit superscript III of invitrogen.

[0117] (1) Prepare reaction system 1 (total amount 12 μL: RNA is 200 ng (10 μL), Oligo-dT is 1 μL, and Random is 1 μL), mix reaction system 1, centrifuge, react at 65°C for 5 min, and then place on ice after the reaction is completed;

[0118] (2) Add reaction system 1 to reaction system 2 (total amount 20 μL: reaction system 1 is 12 μL, dNTP (10 μM) is 1 μL, 0.1M DTT is 2 μL, 5x Buffer is 4 μL, and RT enzyme is 1 μL), mix, centrifuge, and then react in a 42°C water bath for 60 min;

[0119] (3) After taking out, react at 85°C for 10 min to inactivate the reverse transcriptase;

[0120] (4) After the reaction is completed, place the product at -20°C for use.

[0121] 4. Real-time fluorescence quantitative (Quantitative Real-time PCR) detection

[0122] (1) The cDNA obtained by reverse transcription is used as a template, and different primers are added into a Real time PCR reaction system (total amount 20 μL: cDNA is 2 μL, PCR mix is 10 μL, forward primer F is 1 μL, reverse primer R is 1 μL, and ddH2O is 6 μL), mix, centrifuge once, and then place on a fluorescence quantitative PCR instrument to react according to the conditions in Table 2. The correspondence between each amplification target and primer is shown in Table 3.

[0123] Table 2 Realtime PCR reaction conditions

[0124]

[0125] Table 3 Real-time fluorescent quantitative PCR primer design

[0126]

[0127] Melting curve analysis was performed for each PCR product to avoid non-specific amplification. The relative expression was calculated according to the detected threshold cycle (Ct value), and the calculation formula was: , wherein , ; the target gene was Col4a1-Col4a6, and the internal reference was Actin. The test group was Col4a5: c.4432delG mouse, and the control group was wild type mouse.

[0128] 5、Results

[0129] As shown in Figure 8 , no impurity peak was found in the melting curve of the internal reference and each target gene group, indicating that the amplification product was single and specific, and there was no non-specific amplification; the internal reference detection quality was qualified and could be used as a standardized reference for subsequent relative quantitative analysis of Col4a1-Col4a6 expression.

[0130] The corresponding Ct value was read through PCR amplification curve, and the mRNA expression level (relative expression) of Col4a5 and other type IV collagen chains (α1-α6) was calculated, and the results were as follows Figure 9As shown, compared with wild type mice (normal), the expression of Col4a5 mRNA in c.4432delG mutant mice (knockout) was significantly decreased. The relative expression of Col4a3 and Col4a4 in c.4432delG mutant mice was increased compared with wild type mice, which reflected the compensatory expression of α3 and α4 chains; because of the abnormal function (mutation) of Col4a5 gene, the body may up-regulate the expression of α3 and α4 chain genes to compensate for the function of Col4a5 and maintain the overall function of type IV collagen. The expression of Col4a1 and Col4a2 in mutant and normal mice was small, which indicated that the expression of these two chain genes did not change significantly due to Col4a5 mutation, and their function may not be involved in significant compensation; the expression of Col4a6 in c.4432delG mutant mice was slightly increased, which may exist weak compensation or other regulatory relationship, but the change range was not as significant as α3 and α4. Overall, these changes in gene expression reflect the regulatory network of type IV collagen gene family in Col4a5 mutation, and Col4a5 mutation triggers partial gene compensation to maintain the structure and function of type IV collagen related in kidney and other tissues, and provides gene expression level data for the study of type IV collagen function compensation mechanism and kidney disease molecular pathology.

[0131] In summary, combined with genotype identification (PCR combined with sequencing), urine biochemical indicators (urine creatinine U-CRE, urine albumin U-ALB, urine trace albumin / creatinine ratio U-ACR), pathological histology (HE / PAS / Masson staining, electron microscopy observation, IV collagen α5 chain immunohistochemistry) and molecular biology (RT-qPCR detection of collagen chain expression), the whole chain verification system ensures that the model is highly consistent with the pathological characteristics of human XLAS. The c.4432delG mutant mouse model constructed can be applied to the study of Alport syndrome pathogenesis (such as NMD escape mechanism, podocyte-basement membrane interaction), drug screening (anti-fibrosis drugs, podocyte protectors) and treatment target verification (IV collagen metabolism pathway) and the like.

[0132] The above merely describes preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for constructing an Alport syndrome mouse NMD escape model, characterized by, A single base G at site c.4432 in exon 49 of the mouse Col4a5 gene is deleted by CRISPR / Cas9 technology; It comprises the following steps: S1, designing a gRNA plasmid expressing against the 49th exon of the mouse Col4a5 gene, the gRNA sequence is 5'-CAGAAGCACCACAATGCCCACGG-3', and the PAM is CGG; the sequence of the 49th exon of the mouse Col4a5 gene is shown in SEQ ID NO. 1; S2, obtaining the gRNA designed in S1 by in vitro transcription technology; S3, co-injecting the gRNA targeting the mouse Col4a5 gene, the donor oligonucleotide containing the c.4432delG mutation, and the Cas9 nuclease into mouse zygotes, and transplanting the zygotes into recipient female mice to develop into mice.

2. The method for constructing an Alport syndrome mouse NMD escape model according to claim 1, characterized in that, It also includes the step of identifying the genotype of the mouse using primers; the forward primer used for identification is 5'-CCCAGAGCTTGCTTAATCTTGTAA-3', and the reverse primer is 5'-GTGACTGAAGTTATTTGGCTCTGA-3'.

3. The method for constructing an Alport syndrome mouse NMD escape model according to claim 1, characterized in that, It also includes one or more of the following: histopathological examination evaluation of the mouse model, systematic evaluation of urine biochemical indicators, and real-time fluorescent quantitative PCR evaluation.

4. The method for constructing an Alport syndrome mouse NMD escape model according to claim 3, characterized in that, The histopathological examination evaluation includes at least one of HE staining, PAS staining, Masson staining, kidney transmission electron microscopy examination, and type IV collagen α5 chain staining.

5. The method for constructing an Alport syndrome mouse NMD escape model according to claim 3, characterized in that, The indicators of the urine biochemical indicator system evaluation include at least one of urine creatinine, urine albumin, and urine microalbumin / creatinine ratio.

6. The method for constructing an Alport syndrome mouse NMD escape model according to claim 3, characterized in that, The steps of real-time fluorescent quantitative PCR evaluation include: total RNA extraction, total RNA quality detection, reverse transcription of qualified RNA to synthesize cDNA, and real-time fluorescent quantitative detection of the cDNA obtained by reverse transcription.

7. The use of the Col4a5:c.4432delG mouse obtained by the method of claim 1 as an Alport syndrome research model mouse.

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