Method for establishing a dog model of central diabetes insipidus

Abstract

The present invention relates to a method for establishing a central diabetes insipidus (CDI) model dog, the method comprising obtaining an AVP gene knockout canine fertilized egg or canine somatic cell by using gene editing technology. The present invention also relates to an AVP gene knockout CDI disease model dog obtained by the method and its cells, tissues and organs, making it feasible to use dogs as CDI disease animal model research.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering, and specifically relates to a method for preparing a central diabetes insipidus (CDI) disease model dog using gene editing technology, and in particular to a method for preparing an AVP gene-edited CDI disease model dog using gene editing technology. A method for establishing a CDI disease model dog. Background Art

[0002] Central diabetes insipidus (CDI) is a rare disease, characterized by clinical patients excreting large amounts of dilute urine (polyuria), consuming excessive amounts of water (polydipsia), and elevated serum sodium concentrations (hypernatremia). The onset of CDI is mostly between 8 and 12 years old, and in adults it is mostly between 25 and 40 years old. The main cause is a group of syndromes caused by various factors affecting the insufficient secretion of AVP (arginine-vasopressin, also known as antidiuretic hormone, ADH). AVP is synthesized as a hormone precursor in the magnocellular neurons of the supraoptic (SON) and paraventricular (PVN) hypothalamus together with its carrier protein, posterior pituitary hormone transporter protein II (NPII, neurophysin II), and then stored in the posterior pituitary and secreted into the circulatory system. It binds to AVP receptor 2 in the kidney and promotes the transduction of aquaporin (AQP2) that mediates antidiuretic effects. Insufficient production and secretion of AVP will lead to CDI, while the kidneys are insensitive to the antidiuretic effect, resulting in nephrogenic diabetes insipidus (NDI).

[0003] At present, studies have shown that mice and rats carrying spontaneous or engineered mutations in the AVP-NPII gene will show a phenotype similar to that of CDI patients (polydipsia and polyuria). Clinically, a high proportion of CDI patients have mental problems, such as depression and anxiety. In addition to its antidiuretic function, AVP-NPII plays an important role in mediating social behavior in the brain through AVP receptor 1.

[0004] The AVP knockout mouse model has confirmed that mutations in the AVP-NPII gene will result in the inability to secrete and express AVP protein normally, causing mice to have a phenotype similar to that of CDI patients. AVP-NPII knockout mice show symptoms of polyuria and polydipsia at 2 months of age. Studies of the supraoptic (SON) and paraventricular (PVN) hypothalamus revealed a progressive loss of neurons that induce the chaperone protein BiP and neurons that produce AVP. In addition, no AVP gene product was detected in neuronal projections. In summary, it has been demonstrated that mutations in the AVP-NPII gene will cause CDI in mice to reproduce many characteristics of the human disease.

[0005] Although rodents are the most commonly used and widely used models in biological and medical research, their application in translational research is somewhat limited, probably because their organ size, physiology, brain proteome, pathological trajectories, and drug responses are less similar to humans than larger animals such as dogs, especially in terms of behavior and mood. Studies have shown that AVP receptors are expressed in specific brain regions in rodents, and that CDI patients show a higher proportion of psychiatric phenotypes, such as depression and anxiety. Since dogs have a higher similarity with humans in brain anatomy and behavior, canine models are particularly suitable for studying aspects of psychiatry.

[0006] Therefore, an appropriate canine model is needed to better simulate AVP gene-related CDI diseases for clinical translation and related drug development research. Summary of the invention

[0007] In order to solve the problems existing in the prior art, the present invention provides a method for establishing a central diabetes insipidus (CDI) disease model dog with AVP gene knockout by gene editing technology. The present invention also relates to the established AVP gene knockout CDI disease dog model, and its cells and tissues.

[0008] In a first aspect, the present invention provides a method for establishing a central diabetes insipidus (CDI) disease model dog, the method comprising the following steps: using gene editing technology to obtain canine fertilized eggs or canine somatic cells with AVP (arginine antidiuretic hormone) gene mutations.

[0009] In some embodiments, the gene editing technology is selected from BE3 single-base editing technology, CRISPR, TALEN and ZFN, preferably CRISPR / Cas9.

[0010] In some embodiments, the method comprises the steps of:

[0011] (1) Determine the target site based on the sequence of exon 1 and exon 2 of the canine AVP-NPII gene;

[0012] (2) synthesizing the sgRNA sequence according to the targeting site determined in step (1), and then connecting the synthesized sequence to the backbone vector to construct an sgRNA targeting vector;

[0013] (3) Obtaining in vitro transcription products of sgRNA and CRISPR / Cas9 respectively through in vitro transcription;

[0014] (4) Introducing the sgRNA obtained in step (3) and the in vitro transcription product of CRISPR / Cas9 into canine fertilized eggs or canine somatic cells to obtain canine fertilized eggs or canine somatic cells with AVP gene knockout.

[0015] In some embodiments, two targeting sites are determined based on the sequences of exon 1 and exon 2 of the canine AVP-NPII gene. Preferably, the sgRNA sequence of the targeting site and its complementary sequence include the following sequences:

[0016] S1 site: GGCGGACGATGTAGGTCTTGACG (SEQ ID NO: 2), complementary sequence is CGTCAAGACCTACACGTCCGCC (SEQ ID NO: 3);

[0017] S2 site: TTCGGGCCCAGCATCTGCTGCGG (SEQ ID NO: 4), and the complementary sequence is CCGCAGCAGATGCTGGGCCCGAA (SEQ ID NO: 5).

[0018] In some embodiments, the canine somatic cells are from the following tissues or organs: fetal tissue, skin, muscle, ear, mammary gland, fallopian tube, ovary, blood, urine, fat, bone marrow, blood vessels and luminal endothelium.

[0019] In some embodiments, the canine somatic cell is selected from the group consisting of fetal fibroblasts, skin cells, epithelial cells, ear cells, fibroblasts, endothelial cells, muscle cells, mammary cells, fallopian tube cells, ovarian cells, cumulus cells, neural cells, and osteoblasts.

[0020] In some embodiments, the method further comprises transplanting the obtained AVP gene knockout canine fertilized egg into the oviduct of a recipient female dog, thereby preparing a CDI disease model dog.

[0021] In some embodiments, the method further comprises transplanting the nucleus of the obtained AVP gene knockout canine somatic cell into a canine enucleated oocyte, and then transplanting the canine enucleated oocyte after nuclear transplantation into the fallopian tube of a recipient female dog, thereby preparing a CDI disease model dog.

[0022] In some embodiments, the backbone vector used may include gene vectors such as lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, and non-viral vectors in addition to eukaryotic vectors.

[0023] In some embodiments, the present invention utilizes gene editing technology to select a targeting site sequence according to the exons of the canine AVP-NPII gene sequence, and constructs an sgRNA targeting vector and a CRISPR / Cas9 expression vector according to the targeting site sequence. After the vector is verified to be effective, it is transcribed into mRNA in vitro, and then the mRNA is injected into a canine fertilized egg by cytoplasmic injection. The canine fertilized egg is then transplanted into one oviduct of a female dog whose bilateral oviducts have been flushed with embryos, thereby preparing an AVP gene knockout CDI disease model dog.

[0024] In a second aspect, the present invention provides a targeting vector for canine AVP gene knockout, wherein the targeting vector is composed of an sgRNA sequence designed for a targeting site sequence determined for exon 1 and exon 2 of the canine AVP-NPII gene and a backbone vector.

[0025] In some embodiments, the sgRNA sequence and its complementary sequence include the following sequence:

[0026] S1 site: GGCGGACGATGTAGGTCTTGACG (SEQ ID NO: 2), complementary sequence is CGTCAAGACCTACACGTCCGCC (SEQ ID NO: 3);

[0027] S2 site: TTCGGGCCCAGCATCTGCTGCGG (SEQ ID NO: 4), and the complementary sequence is CCGCAGCAGATGCTGGGCCCGAA (SEQ ID NO: 5).

[0028] In a third aspect, the present invention provides canine somatic cells, tissues or organs of a CDI disease model dog obtained by the method of the first aspect.

[0029] In some embodiments, the canine somatic cell, tissue or organ comprises the nucleotide sequence shown in SEQ ID NO:10.

[0030] In some embodiments, the canine somatic cell, tissue or organ comprises the amino acid sequence shown in SEQ ID NO:11.

[0031] In a fourth aspect, the present invention provides a primer pair, the sequence of the primer pair is as follows:

[0032] Forward primer: CACGTCGTCTCCAAGGGAAT (SEQ ID NO: 6); and

[0033] Reverse primer: CACCCACCGTCGTTGCAGCA (SEQ ID NO: 7).

[0034] In a fifth aspect, the present invention provides a kit, wherein the sequences of the primer pairs are as follows:

[0035] Forward primer: CACGTCGTCTCCAAGGGAAT (SEQ ID NO: 6); and

[0036] Reverse primer: CACCCACCGTCGTTGCAGCA (SEQ ID NO: 7).

[0037] In a sixth aspect, the present invention provides use of the primer pair described in the fourth aspect or the kit described in the fifth aspect in detecting a CDI disease model dog having a genomic sequence comprising a sequence fragment shown in SEQ ID NO:10.

[0038] AVP (arginine-vasopressin, also known as antidiuretic hormone, ADH) is synthesized as a hormone precursor in the magnocellular neurons of the supraoptic (SON) and paraventricular (PVN) of the hypothalamus together with its carrier protein NPII (neurophysin II), and then stored in the posterior pituitary and secreted into the circulatory system. It binds to AVP receptor 2 in the kidneys and promotes the transduction of aquaporin (AQP2) that mediates antidiuretic effects. If AVP is not produced and secreted enough, it will lead to central diabetes insipidus (CDI), and the kidneys are insensitive to the antidiuretic effect, leading to nephrogenic diabetes insipidus (NDI).

[0039] The domestic dog (Canis familiaris) is an attractive model organism for studying human diseases. Unlike mice and rats, its organ size, anatomy, physiology, pathological trajectory, and spatiotemporal brain proteome are more similar to humans than rodents. Domestic dogs also have richer social behaviors similar to humans than rodents. The present invention generates a canine model of CDI by mutating the AVP gene through CRISPR / Cas9-mediated gene editing, which achieves effective editing of the AVP gene in the genome, enriches the canine disease model biological sample library, and is the first large animal model generated for CDI.

[0040] The present invention shows from the findings of molecular, biochemical and pharmacological analysis and magnetic resonance imaging (MRI) that the mutant dogs produced show clinically typical CDI. Using domestic dogs to study CDI is of great significance for clinical analysis and medication. At the same time, CDI patients show a higher proportion of psychiatric phenotypes. Since dogs have a higher similarity with humans in brain anatomy and behavior, the canine model is particularly suitable for studying the psychiatric aspects of CDI patients. In addition, a group can be established from female mutants through conventional mating breeding with wild types, somatic cell cloning, or a combination of both techniques to better explore molecular and neuronal pathogenesis and develop CDI therapies. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 A schematic diagram of the AVP-NPII gene targeting site sequence obtained for dogs of the present application is shown.

[0042] Figure 2 The gene sequencing results of the gene-edited dog No. 220216 of the present application are shown.

[0043] Figure 3 The amino acid sequence comparison results of the gene-edited dog No. 220216 of the present application and the wild-type dog are shown.

[0044] Figure 4 The comparative results of water deprivation and desmopressin reaction tests of the gene-edited dogs No. 220216 of the present application and wild-type dogs are shown.

[0045] Figure 5 The serum AVP test results of the gene-edited dog No. 220216 of the present application and the wild-type dog are shown.

[0046] Figure 6 The MRI analysis comparison results of the gene-edited dog No. 220216 of the present application and the wild-type dog are shown. DETAILED DESCRIPTION

[0047] In order to make the purpose, technical scheme and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments and drawings. The specific embodiments described herein are only used to explain the present invention and are not intended to constitute any limitation to the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concepts of the present disclosure. Such structures and technologies are also described in many publications.

[0048] Example

[0049] 1. Construction of transgenic targeting vector

[0050] According to the sequence information of canine AVP-NPII gene (AVP-NPII; NM_001197149.1) in Genbank, the targeting site was determined based on exons 1 and 2 of the canine AVP-NPII gene. The sequence of the targeting site is shown in the following SEQ ID NO: 1:

[0051]

[0052] One sgRNA was designed for exon 1 and one for exon 2 (see Figure 1 ), the sgRNA sequence is specifically:

[0053] S1 site: GGCGGACGATGTAGGTCTTGACG (SEQ ID NO: 2), complementary sequence is CGTCAAGACCTACACGTCCGCC (SEQ ID NO: 3);

[0054] S2 site: TTCGGGCCCAGCATCTGCTGCGG (SEQ ID NO: 4), and the complementary sequence is CCGCAGCAGATGCTGGGCCCGAA (SEQ ID NO: 5).

[0055] The vector plasmid px330 was digested to linearize, 1% agarose gel electrophoresis was performed, and the gel was cut and recovered to determine the concentration. According to the system with a molar ratio of 1:3 between the linearized vector and the AVP-NPII gene targeting sgRNA, T4 DNA ligase was used to connect overnight at 16°C. The ligation product was transformed into an ampicillin-resistant LB plate for screening, colony PCR was used to identify positive clones, and the plasmid was extracted using a plasmid extraction kit after inoculation. 10 μL of the recombinant plasmid was taken and submitted to Shanghai Shenggong Biotechnology Co., Ltd. for sequencing. Snapgene was then used to analyze and compare the sequencing results, and the plasmid with the correct sequencing comparison was saved for later use.

[0056] 2. In vitro transcription

[0057] First, the CRISPR / Cas9 plasmid was linearized, and the reaction system was: 30μg plasmid, 5μL restriction endonuclease AflII; 10μL 10×Buffer and ddH2O, with a total volume of 100μL. Then, 100μL phenol: imitation: isoamyl alcohol (25:24:1) was added to purify the linearized plasmid DNA, and centrifuged at 12000g for 5min; 50μl of supernatant was pipetted into a 1.5ml centrifuge tube without RNase, and 1 / 10 volume of sodium acetate and 3 volumes of anhydrous ethanol were added to precipitate the plasmid DNA, and centrifuged at 12000g for 5min; the supernatant was discarded, and the residual supernatant was discarded as much as possible, and 150μL 70% ethanol was added to wash the plasmid, and the centrifuged at 12000g for 5min; it was dried in air for 3-5min, and the DNA was dissolved with 15μL RNase-free ddH2O, and the concentration was determined.

[0058] In vitro transcription mRNA kit method (Thermo Scientific):

[0059] The in vitro transcription system is: 1μg linearized plasmid DNA, 10μL 2×NTP / CAP, 2μL 10×Buffer, 2μL RNA synthase and ddH2O, with a total volume of 20μL. After mixing, incubate at 37℃ for 1 hour; add 1μL TURBO DNase to digest the plasmid template and incubate at 37℃ for 30min. Then mix 20μL in vitro transcription product, 20μL 10×Reaction Buffer, 10μL ATP (10mM), 2.5μL RNase inhibitor, 2μL Poly(A) polymerase and nuclease-free ddH2O to prepare a total volume of 100μL in vitro transcribed mRNA plus polyA system, and incubate at 37℃ for 1 hour. After incubation, add 350 μL of binding buffer to the reaction system and mix by pipetting; then add 250 μL of anhydrous ethanol and mix evenly; then transfer the sample to the mRNA purification column and centrifuge at 10,000 g at room temperature for 1 min; discard the filtrate, reinstall the column, rinse the column with 500 μL of eluent, and centrifuge at 10,000 g at room temperature for 1 min; repeat the rinse once, discard the filtrate, and centrifuge the empty column for 1 min to elute impurities such as proteins; then place the column in a new centrifuge tube, add 50 μL of RNA eluent to the center of the column, cover the lid, incubate at 65°C for 10 min, and centrifuge at 10,000 g at room temperature for 1 min; detect the RNA quality and concentration.

[0060] CRISPR sgRNA and Cas9 mRNA were mixed to a final concentration of 50 ng / μL for sgRNA and 200 ng / μL for Cas9, and stored at -80°C for cytoplasmic injection.

[0061] 3. Cytoplasmic injection, embryo transfer and identification

[0062] Cas9 mRNA and two sgRNAs were mixed in a ratio of 2:1 and injected into the cytoplasm of canine fertilized eggs. A total of 10 embryo transfers were performed, with 63 embryos transferred, 10 transplant recipients, 23 puppies born, and 1 gene-edited positive dog (see Table 1 for details). The specific operation includes: a total of 10 naturally estrous beagle female dogs were used as fertilized egg donors and embryo transplant recipients for experiments. Blood was collected from all female dogs to test the progesterone concentration in serum. When the progesterone concentration reached 4-7ng / mL, it could be determined as the ovulation period. Natural mating was performed 48h after ovulation, and then fertilized embryos were flushed out. A total of 63 fertilized eggs were obtained from 10 female dogs. After collecting the fertilized eggs, the cumulus granulosa cells were removed using TCM199 medium containing 0.1% hyaluronidase, and then placed in a microdroplet of HEPES-buffered TCM199 medium (HM, GIBCO11150), and then placed on an inverted microscope equipped with a micromanipulator. A mixture containing the mRNA of the sgRNA prepared above and the mRNA of Cas9 in a ratio of 4:1 by volume was drawn up with a microinjection needle and then injected into the cytoplasm of the fertilized egg. The oviduct was flushed with 10 mL of HEPES-buffered TCM199 medium (HM, GIBCO11150) containing 10% fetal bovine serum, and the oviduct flushing fluid flowed out from the injection needle ligated at the fimbria of the oviduct and collected in a 10 mL centrifuge tube. After the cytoplasmic injection was completed, the embryo was placed in an embryo transfer tube, and the embryo in the embryo transfer tube was injected from the fimbria into the oviduct on the side with less bleeding during the embryo flushing. A total of 23 puppies were finally born.

[0063] Table 1. Cytoplasmic injection and embryo transfer results

[0064]

[0065] After the puppies were born, ear tissue (E) and tail tissue (T) were collected for identification. After the tissue blocks were cut into pieces in a centrifuge tube, proteinase K was added to the centrifuge tube and lysed in a water bath at 56°C for 1-3 hours. Then 700μL of Genomic Lysis Buffer was pipetted and added to the lysis system, mixed evenly by inversion, and centrifuged at 10,000g for 1 minute. The supernatant was pipetted to the purification column, centrifuged at 10,000g for 1 minute at room temperature, and centrifuged for 1 minute. A new collection tube was replaced, 200μL of DNA Pre-Wash Buffer was added to the centrifuge column, centrifuged at 10,000g for 1 minute at room temperature, and the waste liquid was discarded. 400μL of g-DNA Wash Buffer was added to the centrifuge column, centrifuged at 10,000g for 1 minute at room temperature, and the waste liquid was discarded. The purification column and collection tube were centrifuged again at 10,000g for 2 minutes. Replace the purification column into a new 1.5 mL centrifuge tube, add 50 μL of Elution Buffer to elute the DNA, and place at room temperature for 2 minutes. Centrifuge at 12000 rpm for 1 minute to obtain the canine genomic DNA solution.

[0066] PCR and PCR product sequencing were performed using canine genomic DNA as a template, where the genotype identification primer pair was:

[0067] Forward primer: 5'-CACGTCGTCTCCAAGGGAAT-3' (SEQ ID NO: 6)

[0068] Reverse primer: 5'-CACCCACCGTCGTTGCAGCA-3' (SEQ ID NO: 7)

[0069] Among them, the PCR identification system (30μL) is: forward primer 1μL, reverse primer 1μL, KODone enzyme 15μL, ddH2O12μL, template 1μL; PCR reaction conditions are: 98℃3min, (98℃10s, 60℃10s, 68℃10s) 30 cycles; 68℃5min; stored at 4℃.

[0070] After gel excision and purification of the PCR product, it was connected to the T vector and transformed into Escherichia coli. Fifteen colonies were selected from each plate for sequencing. The sequencing results were compared with the wild-type sequence, and the monoclonal gene mutation was analyzed to obtain the gene-edited dog individual numbered 220216.

[0071] The wild-type nucleic acid sequence of exon 1 and part of exon 2 of the AVP-NPII gene contains the sequence shown in SEQ ID NO:8, which encodes the amino acid shown in SEQ ID NO:9. After sequencing and sequence information alignment, the gene-edited dog individual No. 220216 has the sequence shown in SEQ ID NO:10, which encodes the amino acid shown in SEQ ID NO:11. The mutant contains a mutation at site 1 of exon 1 (3bp deletion in No. 220216), and a mutation at site 2 of exon 2 (3bp deletion in No. 220216), which result in a total of 2 amino acid (aa) deletions in the gene-edited dog No. 220216. The gene sequencing results of the gene-edited dog No. 220216 are shown as follows. Figure 2 As shown, the amino acid sequence comparison results of the gene-edited dog No. 220216 and the wild-type dog are as follows Figure 3 shown.

[0072] Wild-type nucleic acid sequence:

[0073]

[0074] The wild-type amino acid sequence is:

[0075] MPDTMLLPACFLGLLAFTSACYFQNCPRGGKRAMSDLELRQCLPCGPGGKGRCFGPSICCGDELGCFVGTAEALRCQEENYLPSPCQSGRTPCGSGGRCAAAGICCNDESCVTEPECREGAGLHRRARASDRSNATQLDGPTGALLLRLVQLAGAPEPAEPAPPGVY*(SEQ ID NO: 9)

[0076] Nucleotide sequence of mutant No. 220216:

[0077]

[0078] The amino acid sequence of mutant No. 220216 is:

[0079] MPDTMLLPACFLGLLAFTSAYFQNCPRGGKRAMSDLELRQCLPCGPGGKGRCFGPSICGDELGCFVGTAEALRCQEENYLPSPCQSGRTPCGSGGRCAAAGICCNDESCVTEPECREGAGLHRRARASDRSNATQLDGPTGALLLRLVQLAGAPEPAEPAPPGVY*(SEQ ID NO: 11)

[0080] 4. Phenotypic verification of AVP gene-edited dogs

[0081] (1) Water deprivation and desmopressin response test

[0082] Four-month-old AVP mutant dogs (no. 220216) and wild-type dogs were tested for water deprivation and desmopressin responses. At the beginning of the test, a urinary catheter was inserted to empty the bladder and collect urine at specific time points. After 7 hours of water deprivation, 4 μg of desmopressin was injected intramuscularly. Urine osmolality was measured 1 and 2 hours after injection (i.e. 8 and 9 hours after the start of the test), while plasma osmolality and sodium concentration were measured 6, 7, 8, and 9 hours later (results are shown in Tables 2 and Figure 4 ), the results showed that the AVP mutant dog (no. 220216) showed a typical clinical phenotype of central diabetes insipidus

[0083] Table 2 Osmotic pressure after missing and desmopressin test

[0084]

[0085] The mutant dogs weighed 5.0 kg and the wild-type weighed 7.6 kg (7.2, 7.6 and 8.1 kg), and the mutants were leaner than the wild-type controls.

[0086] 3 ml of whole blood from AVP mutant dogs and wild-type dogs were collected using anticoagulation tubes at 6h, 7h, and 8h after dehydration and desmopressin treatment, respectively, and centrifuged at 1600xg and 4℃ for 15min. The serum was separated into EP tubes, and the samples were stored in a -80℃ refrigerator for subsequent experiments. The AVP concentration was measured using the arg 8-vasopressin ELISA kit. According to the product manual and published methods, a volume of 700μl of serum was extracted with acetone and petroleum. Extraction is necessary to eliminate interfering substances in the sample and to concentrate AVP for accurate measurement within the analytical range. The extracted samples were freeze-dried and reconstituted in 120μl of detection buffer, and 100μl was used for enzyme-linked immunosorbent assay (ELISA). The optical density of the reaction was detected using a Thermo Microplate Reader (Thermo Fisher Scientific, Waltham, USA), and the wavelength was set at 450nm. The results are shown in the table below. Figure 5 As shown, the results showed that the AVP content in the wild-type control serum was very high, while AVP was almost undetectable in the mutant serum.

[0087] (2) MRI analysis

[0088] One AVP-edited dog and one wild-type beagle dog aged 4 months were scanned using a Siemens Prismafit 3.0 T MR scanner equipped with a homemade 4-channel Tx / RxRF coil at the Beijing Brain Research MRI Center, Institute of Biophysics, Chinese Academy of Sciences. After 6 hours of water deprivation, each dog was pre-anesthetized by intramuscular injection of atropine (0.25 mg / kg) and anesthesia was maintained with inhaled isoflurane in oxygen (up to 5% for initial induction and 2-2.5% for maintenance) using a precision MRI-compatible vaporizer. Then, 3D-T1-weighted images were acquired using a magnetization prepared rapid acquisition gradient echo (MPRAGE) sequence (FOV = 128x128mm2, 0.5mm iso, TR / TE / TI = 2370 / 3.68 / 1030ms, 200 flip angle = 8), and the results are shown in Figure 2. Figure 6 As shown, the results showed that the bright signal caused by AVP storage was clearly visible in the posterior pituitary of the wild type, while the bright signal was lost in the posterior pituitary of the mutant due to the lack of AVP.

[0089] according to Figure 4 , Figure 5 and Figure 6 The water deprivation and desmopressin reaction tests and MRI comparison results further verified that the AVP gene knockout CDI disease model dog was obtained in the present invention.

[0090] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. A method for establishing a central diabetes insipidus (CDI) disease model dog, the method comprising the following steps: (1) Determine two target sites based on the sequences of exon 1 and exon 2 of the canine AVP-NPII gene; (2) Synthesizing the sgRNA sequence according to the targeting site determined in step (1), and then connecting the synthesized sequence to the backbone vector to construct the sgRNA targeting vector; (3) Obtaining in vitro transcription products of sgRNA and CRISPR / Cas9 respectively through in vitro transcription; (4) introducing the sgRNA obtained in step (3) and the in vitro transcription product of CRISPR / Cas9 into canine fertilized eggs or canine somatic cells to obtain canine fertilized eggs or canine somatic cells with AVP gene knockout; The method further comprises: The obtained AVP gene knockout canine fertilized eggs are transplanted into the oviduct of a recipient female dog, thereby preparing a CDI disease model dog; or the obtained AVP gene knockout canine somatic cell nuclei are transplanted into canine enucleated oocytes, and then the canine enucleated oocytes after nuclear transplantation are transplanted into the oviduct of a recipient female dog, thereby preparing a CDI disease model dog; Among them, the sgRNA sequence of the targeting site and its complementary sequence include the following sequences: S1 site: GGCGGACGATGTAGGTCTTGACG (SEQ ID NO: 2), complementary sequence is CGTCAAGACCTACACGTCCGCC (SEQ ID NO: 3); S2 site: TTCGGGCCCAGCATCTGCTGCGG (SEQ ID NO: 4), complementary sequence is CCGCAGCAGATGCTGGGCCCGAA (SEQ ID NO: 5).

2. The method according to claim 1, characterized in that The canine somatic cells are derived from the following tissues or organs: fetal tissue, skin, muscle, ear, mammary gland, fallopian tube, ovary, blood, urine, fat, bone marrow, blood vessel and luminal endothelium.

3. A targeting vector for canine AVP gene knockout, the targeting vector comprising an sgRNA sequence designed for the targeting site sequence determined for exon 1 and exon 2 of the canine AVP-NPII gene and a backbone vector, in, The sgRNA and its complementary sequence include the following sequences: S1 site: GGCGGACGATGTAGGTCTTGACG (SEQ ID NO: 2), complementary sequence is CGTCAAGACCTACACGTCCGCC (SEQ ID NO: 3); S2 site: TTCGGGCCCAGCATCTGCTGCGG (SEQ ID NO: 4), complementary sequence is CCGCAGCAGATGCTGGGCCCGAA (SEQ ID NO: 5).

4. Canine somatic cells of a CDI disease model dog obtained by the method according to claim 1 or 2.

5. The canine somatic cell according to claim 4, characterized in that The canine somatic cell comprises the sequence shown in SEQ ID NO: 10.

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