A method for constructing and applying an autosomal dominant polycystic kidney disease mouse model.

Abstract

This invention relates to a method for constructing a mouse model of autosomal dominant polycystic kidney disease and its applications, particularly to a method for preparing a non-human animal model carrying a nonsense mutation in the Pkd1 gene c.616_618GAG>TAG based on CRISPR / Cas9 gene editing technology. The method involves co-injecting mouse zygotes with gRNA targeting exon 5 of the Pkd1 gene, homologous recombinant donor oligonucleotides containing the c.616_618GAG>TAG mutation, and Cas9 nuclease. Mutant mice are obtained via embryo transfer. The mutation site is verified by PCR combined with sequencing, and a stable mutant line is established through breeding. This invention also includes the application of this model animal as a research tool for autosomal dominant polycystic kidney disease, particularly in the areas of pathogenesis analysis, drug screening, and therapeutic target validation. This model can stably simulate the typical clinical manifestations and pathological features of human autosomal dominant polycystic kidney disease, providing an important tool for basic research and translational medicine of this disease.

Description

Technical Field

[0001] This invention belongs to the technical field of creating gene-mutated animal models using gene editing technology, specifically relating to the construction method and application of a mouse model of autosomal dominant polycystic kidney disease caused by Pkd1 gene mutation. Background Technology

[0002] The CRISPR / Cas (Clustered Regularly Interspaced Short Palindromic Repeats / CRISPR-associated) system is the acquired immune system of prokaryotes, which defends against invasion by integrating foreign DNA fragments and guiding Cas endonucleases to cleave the foreign sequences. Among them, the Type II system (CRISPR / Cas9), due to its simplicity and high editing efficiency, has been widely used in genome modification of model organisms since its successful application in mammalian cell gene editing in 2013. It uses sgRNA to guide the Cas9 protein to cleave the DNA double strand at a specific site, triggering cellular repair mechanisms (random insertion / deletion or template-dependent repair) to achieve permanent genome modification, providing a powerful tool for disease model construction.

[0003] Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common inherited kidney diseases, with an incidence of approximately 1 / 400-1 / 1000 in the population. Its main characteristic is the presence of numerous progressively enlarging fluid-filled cysts in both kidneys, accompanied by renal parenchymal destruction, eventually progressing to end-stage renal disease (ESRD). In addition, patients may also present with extrarenal manifestations such as hepatic cysts, pancreatic cysts, and intracranial aneurysms. ADPKD has a long and "silent" progression: most people are asymptomatic in the early stages, and by the time they seek medical attention, their kidney function is already severely impaired. The core issue is a gene mutation that causes cysts to form in the renal tubules. These cysts gradually enlarge and multiply, compressing normal kidney tissue and eventually leading to kidney failure. The entire process can be divided into the following stages: Early stage: Asymptomatic, due to the kidney's compensatory capacity; Budding stage: Onset occurs in adolescence or childhood, with small, few cysts that can only be detected by imaging. The kidney can maintain function with over 50% normal tissue, so there is no discomfort, and it is easily missed during routine checkups; Progression stage: Mostly between 30 and 50 years of age, with cysts growing rapidly, compressing kidney tissue and causing renal parenchyma atrophy. However, the kidneys continue to "work overtime," and some people only experience occasional lower back pain or mild hypertension, which can be misdiagnosed as fatigue, leading to missed opportunities for intervention; Symptom onset stage: A concentrated outbreak occurs after age 50, with cysts filling the kidneys, causing the compensatory function to collapse. Significant lower back pain, hematuria, urinary tract infections, or symptoms of declining kidney function such as fatigue, edema, and nausea will appear. By the time patients seek medical attention, they are often in the middle to late stages of chronic kidney disease, or even approaching uremia. ADPKD can be asymptomatic for decades, during which the condition can quietly worsen.

[0004] The pathogenesis of ADPKD is closely related to mutations in the PKD1 or PKD2 genes, with PKD1 mutations accounting for approximately 85%-90%. Both follow autosomal dominant inheritance, meaning that carrying just one mutated pathogenic allele (heterozygote) is sufficient to cause the disease; both alleles do not need to be mutated (homozygote). Clinically, almost all ADPKD patients are heterozygous; homozygotes are extremely rare. Homozygous ADPKD patients are usually fatal during fetal or infancy and rarely survive to adulthood. The period between 30 and 50 years of age is the most common stage for the appearance of symptoms in PKD1 mutation-related ADPKD and is also the main period for clinical diagnosis. During this stage, patients' renal cysts gradually increase in number and size. Some patients still have normal renal function at this time, but with age, renal function gradually declines. If not intervened in time, it may progress to renal failure around age 60.

[0005] The PKD1 gene encodes polycystic protein 1 (PC1), a transmembrane protein mainly expressed in renal tubular epithelial cells, which participates in the regulation of cell proliferation, differentiation, apoptosis, and cyst formation. Under normal circumstances, PC1 and PC2 (the product of the PKD2 gene) form a complex that maintains the polarity of renal tubular epithelial cells and the stability of renal tubular structure. When nonsense mutations occur in PKD1, it can lead to truncation or loss of function of the PC1 protein, disrupting the stability of the complex, causing abnormal cell proliferation, disordered apoptosis, and increased fluid secretion, ultimately leading to cyst formation.

[0006] Currently, three different types of Pkd1 mouse models have been constructed abroad. In 2002, Anil Kumar Bhunia used gene knockout technology to construct a mouse model in which most of the region of exon 2 and the entire region of exon 3 of the Pkd1 gene were replaced by the lacZ gene. In 2004, Klaus B Piontek used Cre-loxP site-specific recombination technology to construct a mouse model with conditional deletion of exons 2-4 of the Pkd1 gene. In 2012, Katharina Hopp used gene knock-in technology to construct a mouse model with the c.9805_9807AGA>TGC mutation in the Pkd1 gene. However, given the different mutation locations in these three models, the severity of the disease exhibited by the corresponding mice also varies.

[0007] Existing Pkd1 mouse models are all conditional gene knockout homozygous models, exhibiting rapid cyst progression that does not accurately reflect human disease progression, making it difficult to comprehensively reflect the disease process. This is especially true for ADPKD caused by nonsense mutations in the PKD1 gene, where a stable model simulating the "progressive enlargement of cysts" is lacking, limiting research into disease mechanisms and targeted therapy strategies. Therefore, this invention utilizes CRISPR / Cas9 technology to construct a Pkd1 gene mutant mouse model to simulate the pathogenesis of human ADPKD. This will greatly aid in understanding the mechanism of this gene in disease development and progression, and is of great significance for elucidating disease mechanisms and developing novel gene therapy methods. Summary of the Invention

[0008] The first objective of this invention is to provide a method for constructing an autosomal dominant polycystic kidney disease mouse model, which utilizes CRISPR / Cas9 technology to construct a heterozygous mouse model of the Pkd1 gene c.616_618GAG>TAG nonsense mutation. This mouse model develops renal cysts in middle and old age, which well simulates the pathogenesis of human ADPKD.

[0009] The second objective of this invention is to provide an application of an autosomal dominant polycystic kidney disease mouse model.

[0010] The first objective of this invention is achieved by the following technical solution.

[0011] Mice have a relatively short lifespan (usually 2-3 years). Young mice (1-3 months old) grow rapidly; 1 month old corresponds to approximately 15 years old in humans, and 3 months old to approximately 28 years old. After reaching adulthood (from 6 months old), the aging process slows down relatively; 6 months old corresponds to approximately 40 years old in humans. By 12 months of age, the kidneys, metabolism, and other physiological functions of mice have entered the late adult stage, transitioning towards old age. Their organ maturity and aging degree roughly match the physiological state of humans aged 50-60, consistent with the age-corresponding requirements of animal models in research scenarios for diseases such as ADPKD. Therefore, it is anticipated to construct a heterozygous mouse model with a nonsense mutation of the Pkd1 gene c.616_618GAG>TAG to simulate the pathogenesis of ADPKD. Verification has shown that this model conforms to the clinical phenotype and pathological characteristics of autosomal dominant polycystic kidney disease, which will contribute to research in areas such as the pathogenesis and therapeutic targets of autosomal dominant polycystic kidney disease, and the development of new drugs.

[0012] A method for constructing an autosomal dominant polycystic kidney disease mouse model involves using CRISPR / Cas9 technology to mutate the GAG ​​codon at position 616-618 of exon 5 of the mouse Pkd1 gene to the TAG codon, resulting in glutamate changing to a stop codon (p.E206X), corresponding to the p.Q206X mutation (CAG→TAG, i.e., glutamine changing to a stop codon) in the human PKD1 gene NM_001009944.3 transcript.

[0013] It includes the following steps:

[0014] S1. Design a gRNA plasmid targeting exon 5 of the mouse Pkd1 gene. The gRNA is named gRNA-Pkd1-A1, with the sequence 5'-CTGCAGGCCTCAGTCTCCAGTGG-3' (SEQ ID NO.2), and PAM is TGG. The sequence of exon 5 of the mouse Pkd1 gene is shown in SEQ ID NO.1.

[0015] S2. Obtain the gRNA designed in S1 using in vitro transcription technology;

[0016] S3. GRNA targeting the mouse Pkd1 gene, homologous recombinant donor oligonucleotides containing the c.616_618GAG>TAG mutation, and Cas9 nuclease were co-injected into mouse zygotes, and the zygotes were transplanted into recipient mother mice to develop into mice.

[0017] Preferably, the method further includes a step of identifying the mouse genotype using primers. F1 generation mice are identified by polymerase chain reaction (PCR) and sequence analysis, and then bred with wild-type mice to test for pedigree transmission and the generation of F1 generation Pkd1:c.616_618GAG>TAG mice.

[0018] Preferably, the sequence of the forward primer used for identification is 5'-AAAGGCATACATGGGAAGATAGTG-3' (SEQ ID NO.3), and the sequence of the reverse primer is 5'-AGGAAGTAATATGGAAGTCTGCTGG-3' (SEQ ID NO.4). The primer sequences used for identifying mouse genotypes are designed based on the corresponding region of the Pkd1 gene and can specifically amplify genomic fragments containing the c.616_618GAG>TAG mutation site.

[0019] Preferably, it also includes at least one of reverse transcription PCR evaluation, histopathological examination evaluation, and Western blotting evaluation of the mouse model.

[0020] Preferably, the pathological histological examination evaluation includes HE staining and / or polycystic protein 1 immunohistochemical staining.

[0021] The second objective of this invention is achieved by the following technical solution.

[0022] A method for constructing an autosomal dominant polycystic kidney disease mouse model was proposed, and the Pkd1:c.616_618GAG>TAG mice obtained were used as a research model mouse for autosomal dominant polycystic kidney disease.

[0023] Beneficial Effects: This invention discloses a method for constructing a non-human animal model carrying the c.616_618GAG>TAG nonsense mutation in the Pkd1 gene based on CRISPR / Cas9 gene editing technology. This method involves co-injecting gRNA targeting exon 5 of the Pkd1 gene, homologous recombinant donor oligonucleotides containing the c.616_618GAG>TAG mutation, and Cas9 nuclease into mouse fertilized eggs. Mutant mice are obtained via embryo transfer. The mutation site is verified by PCR combined with sequencing, and a stable mutant line is established through one generation of breeding. Experimental verification shows that this model can stably simulate the typical clinical manifestations (such as multiple renal cysts and progressive renal function decline) and pathological features of human ADPKD from asymptomatic childhood to renal function decline in middle and old age. This will contribute to research in areas such as the pathogenesis and therapeutic targets of autosomal dominant polycystic kidney disease, and the development of new drugs. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 A strategy diagram for constructing Pkd1 gene c.616_618GAG>TAG mutant mice.

[0026] Figure 2 The results show the PCR products and sequencing maps of the PCR products from F1 generation positive mice with the c.616_618GAG>TAG mutation in the Pkd1 gene. Among them, (a) is a schematic diagram of the editing target site of the gene knock-in allele and the location of the PCR primer pair; (b) is a gel electrophoresis image of the PCR products of F1 generation positive mice, where lanes 14, 15, 16 and 20 are PCR products of F1 generation positive mice, lane WT is wild-type mice, and lane M is the marker molecular weight standard; (c) is the sequencing map of the PCR products of F1 generation positive mice.

[0027] Figure 3The sequencing results of Pkd1 gene RT-PCR products from multiple tissues of Pkd1:c.616_618GAG>TAG mice (hereinafter referred to as mutant mice) and wild-type mice are shown in (a) as a schematic diagram of primer localization; (b) as a gel electrophoresis diagram of total RNA, where lanes 1-3 are blood samples from mutant mice, lanes 5-7 are heart samples from mutant mice, lanes 9-11 are kidney samples from mutant mice, lanes 13-15 are brain samples from mutant mice, lanes 17-19 are lung samples from mutant mice, lane 4 is blood samples from wild-type mice, lane 8 is heart samples from wild-type mice, lane 12 is kidney samples from wild-type mice, lane 16 is brain samples from wild-type mice, lane 20 is lung samples from wild-type mice, and lane M is a marker molecular weight standard; (c) as the sequencing results of Pkd1 gene RT-PCR products from multiple tissues of mutant mice and wild-type mice.

[0028] Figure 4 Histopathological images of kidney tissue from wild-type and Pkd1:c.616_618GAG>TAG mice.

[0029] Figure 5 Immunohistochemical results of PC1 protein in the kidneys of wild-type and Pkd1:c.616_618GAG>TAG mice.

[0030] Figure 6 The image shows the Western blot results of PC1 protein in the kidneys of wild-type and Pkd1:c.616_618GAG>TAG mice; where numbers 1, 2, and 9 represent mutant mice, and numbers 3, 4, 5, and 6 represent wild-type mice. Detailed Implementation

[0031] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Throughout this specification, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. In case of any conflict, this specification shall prevail.

[0032] Unless otherwise specified, all materials, reagents, instruments and equipment used in this invention can be obtained by purchasing them from the market or by existing methods.

[0033] Example 1 - Construction of Pkd1: c.616_618GAG>TAG mice

[0034] Using CRISPR / Cas9 gene editing technology, a mouse model with the Pkd1 gene c.616_618GAG>TAG mutation was constructed based on C57BL / 6J mice. The design scheme of the gene knock-in mouse model is as follows.

[0035] 1. Determine the basic information of the knockout gene: The target gene for mutation is named Pkd1 (GenBank accession number: NM_013630.2; Ensembl number: ENSMUSG00000032855), and the corresponding transcript for the mutation scheme is Pkd1. 201 (Ensembl No.: ENSMUST00000035565), mutation site: Substitution c. 616_618GAG>TAG of MousePkd1.

[0036] 2. Construct the Pkd1:c.616_618GAG>TAG mouse strategy, such as... Figure 1 As shown: A gRNA plasmid with the c.616_618GAG>TAG mutation was designed targeting exon 5 of the mouse Pkd1 gene (sequence shown in SEQ ID NO.1). The gRNA sequence name is gRNA-Pkd1-A1, and the gRNA sequence is 5'-CTGCAGGCCTCAGTCTCCAGTGG-3' (SEQ ID NO.2). PAM is TGG (located at the 3' end of the gRNA). Design principle: Using CRISPR / Cas9 technology, the GAG ​​codon at position 616-618 of exon 5 of the mouse Pkd1 gene was mutated to the TAG codon, resulting in glutamate changing to a stop codon (p.E206X), corresponding to the p.Q206X mutation (CAG→TAG, i.e., glutamine changing to a stop codon) in the human PKD1 gene NM_001009944.3 transcript.

[0037] 3. gRNA was synthesized using in vitro transcription, and homologous recombinant donor oligonucleotides were constructed simultaneously. gRNA targeting the Pkd1 gene (50 ng / μL), homologous recombinant donor oligonucleotides containing the c.616_618GAG>TAG mutation (5 ng / μL), and Cas9 nuclease (80 ng / μL) were co-injected into mouse zygotes using micromanipulation techniques. The microinjected zygotes were then transferred to the uterus of pseudopregnant recipient mice to develop into F0 generation mice. F0 generation positive mice were mated with wild-type mice to obtain F1 generation mice.

[0038] Example 2 - Genotyping of F1 generation mice

[0039] 1. PCR amplification

[0040] Design specific primers to amplify the genomic fragment containing the c.616_618GAG>TAG mutation site. Use mouse tail genomic DNA as a template, such as... Figure 2 As shown in (a), PCR amplification was performed using forward primer F1 (sequence 5'-AAAGGCATACATGGGAAGATAGTG-3', SEQ ID No. 3) and reverse primer R1 (sequence 5'-AGGAAGTAATATGGAAGTCTGCTGG-3', SEQ ID No. 4). The PCR amplification reaction system is shown in Table 1, and the reaction procedure is shown in Table 2.

[0041] Table 1 PCR amplification reaction system

[0042]

[0043] Table 2 PCR amplification reaction procedure

[0044]

[0045] 2. DNA electrophoresis identification

[0046] The PCR amplification products were added to a 1.5% agarose gel and electrophoresed at a constant voltage of 120V for 30 min. The results were observed as follows. Figure 2 As shown in (b). The PCR amplification products were sequenced by Sanger sequencing (sequencing primers are shown in SEQ ID No. 3), and the results are as follows. Figure 2 (c) shows that mice numbered 14, 15, 16 and 20 were confirmed to be mutant mice with a mutation in the GAG ​​codon at position 616-618 of the Pkd1 gene to the TAG codon. The wild-type mouse numbered WT was confirmed to have no mutation in the GAG ​​codon at position 616-618 of the Pkd1 gene.

[0047] Example 3 - Pkd1: c.616_618GAG>TAG mouse multi-tissue Pkd1 gene RT-PCR

[0048] 1. Total RNA extraction from samples (Trizol method)

[0049] (1) Blood, heart, kidney, brain and lung samples from three Pkd1:c.616_618GAG>TAG mutant mice and one wild-type mouse were treated with Trizol. Each sample was treated with 1 mL of Trizol and then transferred to 1.5 mL EP tubes.

[0050] (2) Add 500 μL of phenol chloride to a 1.5ml EP tube, shake to mix, and let stand for 5 min;

[0051] (3) Centrifuge at 12000 rpm for 10 min at 4℃, and carefully aspirate the supernatant into a new 1.5 mL EP tube;

[0052] (4) Add 700 μL of isopropanol to the separated supernatant and mix thoroughly;

[0053] (5) Centrifuge at 12000 rpm for 10 min at 4℃ and carefully discard the supernatant;

[0054] (6) Wash the precipitate once with 75% ethanol and air dry at room temperature;

[0055] (7) Dissolve the RNA precipitate in 50 μL of DEPC water;

[0056] (8) Quality was determined by agarose gel electrophoresis.

[0057] 2. Total RNA quality testing

[0058] (1) Preparation: Before measurement, zero the nucleic acid concentration meter with DEPC water for dissolving RNA, add the sample to the detection base of the nucleic acid concentration meter, and start the absorbance detection using the software on the computer.

[0059] (2) Concentration determination: A reading of 1 at 260 nm indicates 40 ng RNA / μL; the formula for calculating the sample RNA concentration is: A260 × 40 ng / μL; the average RNA concentrations measured in each tissue in this example are: blood 63.2 ng / µL, heart 252.2 ng / µL, kidney 442.0 ng / µL, brain 445.7 ng / µL and lung 313.5 ng / µL.

[0060] (3) Purity detection: The ratio of A260 / A280 of RNA solution is a method for detecting RNA purity. The ratio range of 1.8 to 2.1 is preferred. In this example, the average RNA purity in each tissue was measured as follows: blood 1.97, heart 2.01, kidney 2.03, brain 2.01 and lung 2.02.

[0061] 3. Reverse transcription to synthesize cDNA

[0062] Reverse transcription was performed using Takara's Primescript RT reagent kit with gDNAEraser. A primer diagram is shown below. Figure 3 As shown in (a).

[0063] Prepare the RT-PCR reaction system according to Table 3. The sequence of the forward primer mPkd1-PCR-F1 is 5'-ACATCAGGTGCATGTGGTACA-3' (SEQ ID No. 5), and the sequence of the reverse primer mPkd1-PCR-R1 is 5'-GGCAGAGTTGAGGGACAGTG-3' (SEQ ID No. 6). After mixing the system, briefly incubate it and place it on the PCR instrument. Perform the reaction according to the RT-PCR reaction procedure in Table 4.

[0064] Table 3 RT-PCR reaction system

[0065]

[0066] Table 4 RT-PCR reaction procedure

[0067]

[0068] The RT-PCR products were added to a 1.5% agarose gel and electrophoresed at a constant voltage of 120V for 30 min. The results are as follows. Figure 3 As shown in (b). The RT-PCR product was sequenced by Sanger sequencing (sequencing primers are shown in SEQ ID No. 5), and the results are as follows. Figure 3 As shown in (c).

[0069] The mutation of the GAG ​​codon at position 616-618 of the Pkd1 gene in mutant mice numbered 1-3 (blood), 5-7 (heart), 9-11 (kidney), 13-15 (brain), and 17-19 (lung) was confirmed to be a TAG codon. The mutation of the GAG ​​codon at position 616-618 of the Pkd1 gene in wild-type mice numbered 4 (blood), 8 (heart), 12 (kidney), 16 (brain), and 20 (lung) was confirmed to be absent.

[0070] Example 4 - Histopathological examination of Pkd1:c.616_618GAG>TAG mice

[0071] 1. Paraffin sections.

[0072] Wild-type mice (C57BL / 6J, hereinafter the same) and Pkd1:c.616_618GAG>TAG mice were sectioned in paraffin. The paraffin sectioning procedure is as follows:

[0073] (1) After taking the sample, fix it quickly, and then dehydrate it with 80%, 90%, 95% and 100% ethanol for 2 hours in sequence, and use xylene to make it transparent;

[0074] (2) Allows paraffin to penetrate the tissue, replacing the clearing agent present in the tissue;

[0075] (3) The tissue after being impregnated with wax is placed in melted solid paraffin. After the paraffin solidifies, the tissue is embedded in it.

[0076] (4) Cut off the wax block and slice it. Use ophthalmic forceps to pick up the slice and gently lay it flat on the surface of water at 45°C. Use the surface tension and temperature of the water to naturally flatten the slightly wrinkled slice.

[0077] (5) After the slide is fully flattened on the constant temperature water surface, pick up the slide and pour off the excess water on the slide. Place it in a 65℃ constant temperature oven or in the oven of the slide drying temperature controller and bake for 30 minutes to remove the paraffin that has melted the interstitial space.

[0078] 2. The morphological changes of mouse kidney tissue were observed using HE staining.

[0079] The HE staining procedure is as follows:

[0080] (1) Dewaxing paraffin sections to water: The sections were placed in xylene I for 10 min and xylene II for 10 min to dewax, and then placed in anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 95% ethanol for 5 min, 90% ethanol for 5 min, 80% ethanol for 5 min and 70% ethanol for 5 min, and then washed with distilled water to complete the gradient hydration.

[0081] (2) Hematoxylin staining of cell nuclei: After washing, the sections were stained with Harris hematoxylin for 8 min, washed with tap water, then differentiated with 1% hydrochloric acid alcohol for a few seconds, rinsed with tap water, soaked in 0.6% ammonia water to turn blue, and rinsed with running water.

[0082] (3) Eosin staining of cytoplasm: Immerse the sections in eosin staining solution for 3 min;

[0083] (4) Dehydration and mounting: The sections are 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 in turn to dehydrate. Then, they are placed in xylene I for 5 min and xylene II for 5 min in turn to dehydrate and become transparent. The sections are removed from the xylene and dried slightly, and then mounted with neutral resin.

[0084] Microscopic observation of HE-stained sections revealed that the kidneys of Pkd1:c.616_618GAG>TAG mice exhibited normal morphology and function within the first 12 months of life (asymptomatic period). Kidney sections from 3-month-old Pkd1:c.616_618GAG>TAG mice showed no difference from those from wild-type mice. Kidney cysts gradually appeared from 12 months onwards (onset period). The HE-stained sections from 50-week-old wild-type and Pkd1:c.616_618GAG>TAG mice showed the following results: Figure 4 As shown: Compared with wild-type mice, Pkd1:c.616_618GAG>TAG mice showed two small cysts containing eosinophilic material in the renal medullary flexure (yellow arrows); local tubular and glomerular atrophy (black arrows), with a small cyst containing eosinophilic material in the atrophic lesion.

[0085] This indicates that the Pkd1:c.616_618GAG>TAG mice constructed in this invention exhibit age-dependent pathogenesis, consistent with the pathogenesis of ADPKD in the middle and elderly in humans; and that cyst formation shows a gradual growth from non-existent to present, highly consistent with the pathological characteristics of human ADPKD, reproducing the clinical characteristic that "structural damage in human ADPKD precedes functional damage." It can be used to study: (1) molecular events in the initial stage of cyst formation (early intervention targets); (2) novel treatment strategies to delay or block cyst growth; (3) screening and verification of early biomarkers for ADPKD.

[0086] 3. The localization and expression of PC1 protein in mouse kidney tissue were detected by immunohistochemical SP method.

[0087] The immunohistochemical (IHC) experimental procedure is as follows:

[0088] (1) Dewaxing paraffin sections to water: Place the paraffin sections into an automatic staining and mounting machine and dewax them according to the standard operating procedure; then place them 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, and 70% alcohol for 5 min in sequence, and then wash them with distilled water 3 times (5 min each time).

[0089] (2) Antigen retrieval: After washing, the sections are immersed in citrate buffer or EDTA 9.0 antigen retrieval solution and microwaved at high temperature for 10 min, then allowed to cool naturally to room temperature; wash three times with 0.1% Triton TBS (3-5 min each time).

[0090] (3) Blocking endogenous peroxidase: Add 3% H2O2 aqueous solution and incubate at room temperature for 5 min; wash 3 times with 0.1% Triton TBS (5 min each time);

[0091] (4) Blocking non-specific binding: Add animal serum of the same origin as the secondary antibody, incubate at room temperature for 30 min, and then discard the blocking solution without washing.

[0092] (5) Primary antibody incubation: Add diluted PKD1 / PKC mu primary antibody and incubate at room temperature for 60 min; wash 3 times with 0.1% Triton TBS (5 min each time);

[0093] (6) Secondary antibody incubation: Add MicroStacker polyHRP-labeled goat anti-rabbit polymer secondary antibody and incubate at room temperature for 30 min; wash 3 times with 0.1% Triton TBS (5 min each time).

[0094] (7) Enzyme-linked incubation: Add streptavidin-HRP complex and incubate at room temperature for 20 min; wash 3 times with 0.1% Triton TBS (5 min each time);

[0095] (8) DAB color development: Freshly prepared DAB color development solution was added to the surface of the slide and observed under a microscope for 3-5 minutes. The color development was stopped by adding distilled water in time.

[0096] (9) Counterstaining: hematoxylin counterstaining for 30 s, hydrochloric acid alcohol differentiation for 5 s, and rinsing with tap water to return to blue for 10 min;

[0097] (10) Dehydration and transparency: Place in 75% ethanol for 5 s, 95% ethanol for 5 s, anhydrous ethanol I for 1 min, and anhydrous ethanol II for 5 min in sequence, and then place in xylene 3 times (5 min each time);

[0098] (11) Mounting: Mount with neutral resin, observe and acquire images under an optical microscope.

[0099] Table 5. Multiple protein signaling regions (ROIs) in PC1 protein immunohistochemistry

[0100]

[0101] The results are as follows Figure 5 As shown in Table 5, the positive area of ​​PC1 protein in wild-type mice was 0.00500%, representing the basic expression level of PC1 protein under normal physiological conditions. Compared with wild-type mice, the positive area of ​​PC1 protein in Pkd1:c.616_618GAG>TAG mice was 0.00046%, which was only about 1 / 10 of that in the wild-type control group. This indicates that the c.616_618GAG>TAG mutation in the Pkd1 gene significantly reduced the expression level of PC1 protein.

[0102] The above histopathological results indicate that the Pkd1:c.616_618GAG>TAG mice constructed in this invention exhibit histopathological features of polycystic kidney disease.

[0103] Example 5 - Western blot (WB) experiment on Pkd1:c.616_618GAG>TAG mice

[0104] The expression level of PC1 protein in mouse kidney tissue was detected by Western blotting, and the experimental steps are shown below.

[0105] (1) Sample preparation: Take about 50 mg of fresh kidney tissue, add pre-cooled RIPA lysis buffer containing 1% PMSF and phosphatase inhibitor (total volume 100 μL), homogenize thoroughly on ice, and let stand at 4℃ for 30 min for lysis.

[0106] (2) Protein extraction: Centrifuge at 15,000 rpm for 10 min at 4℃, and carefully aspirate the supernatant to obtain the total protein extract.

[0107] (3) Protein quantification: The BCA protein quantification kit was used to detect protein concentration and standardize sample concentration according to the instructions.

[0108] (4) Sample processing and loading: Mix the total protein sample with 5× protein loading buffer at a ratio of 4:1, centrifuge briefly to mix, and then heat in a 100℃ metal bath for 10 min to denature; after a brief centrifugation, load 20-40 μg of standardized protein sample.

[0109] (5) SDS-PAGE electrophoresis: Prepare 12% separating gel and 5% stacking gel (separating gel: 4.8 mL Tris-HCl pH8.8, 7.6 mL 40% PAGE gel stock solution, etc.; stacking gel: 475 μL Tris-HCl pH6.6, 700 μL 40% PAGE gel stock solution, etc.); electrophoresis at constant voltage in 1× electrophoresis buffer, stacking gel 90V, separating gel 120V, until bromophenol blue migrates to the bottom of the gel.

[0110] (6) Transfer: The PVDF membrane was activated with methanol for 30 s and then immersed in 1× transfer buffer with the gel and filter paper to equilibrate. The transfer sandwich was assembled in the order of "sponge-filter paper-gel-PVDF membrane-filter paper-sponge" (without air bubbles), and the membrane was transferred at a constant current of 220 mA for 1.5 h under ice bath conditions.

[0111] (7) Blocking: After the membrane is transferred, rinse lightly with TBST, block with rapid blocking solution at room temperature for 20 min, and wash the membrane 3 times with TBST (5 min each time).

[0112] (8) Primary antibody incubation: Add diluted PKD1 / PKC mu primary antibody and incubate overnight on a shaker at 4°C; wash the membrane 3 times with TBST the next day (5 min each time).

[0113] (9) Secondary antibody incubation: Add MicroStacker polyHRP enzyme-labeled goat anti-rabbit polymer secondary antibody, incubate on a shaker at room temperature for 1.5 h, and wash the membrane 3 times with TBST (5 min each time).

[0114] (10) ECL developer solution A and solution B are mixed in a 1:1 ratio and evenly dropped onto the surface of PVDF film. The chemiluminescence imaging system captures and analyzes the signal image.

[0115] The results are as follows Figure 6 As shown, Pkd1:c.616_618GAG>TAG mice can express the complete PC1 protein, which is consistent with the molecular mechanism of heterozygous nonsense mutation.

[0116] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for constructing a mouse model of autosomal dominant polycystic kidney disease, characterized in that, The GAG ​​codon at position 616-618 of exon 5 of the mouse Pkd1 gene was mutated to the TAG codon using CRISPR / Cas9 technology. It includes the following steps: S1. Design a gRNA plasmid targeting exon 5 of the mouse Pkd1 gene. The sequence of the gRNA is shown in SEQ ID NO.2, and the PAM is TGG. The sequence of exon 5 of the mouse Pkd1 gene is shown in SEQ ID NO.

1. S2. Obtain the gRNA designed in S1 using in vitro transcription technology; S3. GRNA targeting the mouse Pkd1 gene, homologous recombinant donor oligonucleotides containing the c.616_618GAG>TAG mutation, and Cas9 nuclease were co-injected into mouse zygotes, and the zygotes were transplanted into recipient mother mice to develop into mice. S4. Use primers to identify mouse genotypes; the sequence of the forward primer used for identification is shown in SEQ ID NO.3, and the sequence of the reverse primer is shown in SEQ ID NO.

4.

2. The method for constructing an autosomal dominant polycystic kidney disease mouse model according to claim 1, characterized in that, It also includes at least one of the following: reverse transcription PCR evaluation, pathological histological examination evaluation, and Western blotting evaluation of the mouse model.

3. The method for constructing an autosomal dominant polycystic kidney disease mouse model according to claim 2, characterized in that, The pathological and histological examination evaluation includes HE staining and / or polycystic protein 1 immunohistochemical staining.

4. The use of Pkd1:c.616_618GAG>TAG mice obtained by the method for constructing an autosomal dominant polycystic kidney disease mouse model according to any one of claims 1-3 as a research model mouse for autosomal dominant polycystic kidney disease.

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