CN114868705B - Construction method of retinitis pigmentosa mouse model - Google Patents

Construction method of retinitis pigmentosa mouse model Download PDF

Info

Publication number
CN114868705B
CN114868705B CN202210443478.6A CN202210443478A CN114868705B CN 114868705 B CN114868705 B CN 114868705B CN 202210443478 A CN202210443478 A CN 202210443478A CN 114868705 B CN114868705 B CN 114868705B
Authority
CN
China
Prior art keywords
mouse
gene
rho
mice
sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202210443478.6A
Other languages
Chinese (zh)
Other versions
CN114868705A (en
Inventor
谷峰
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Wenzhou Medical University
Original Assignee
Wenzhou Medical University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Wenzhou Medical University filed Critical Wenzhou Medical University
Priority to CN202210443478.6A priority Critical patent/CN114868705B/en
Publication of CN114868705A publication Critical patent/CN114868705A/en
Application granted granted Critical
Publication of CN114868705B publication Critical patent/CN114868705B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
    • A01K67/027—New breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0278—Humanized animals, e.g. knockin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/11—Protein-serine/threonine kinases (2.7.11)
    • C12Y207/11014—Rhodopsin kinase (2.7.11.14)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/072—Animals genetically altered by homologous recombination maintaining or altering function, i.e. knock in
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0306—Animal model for genetic diseases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]

Abstract

The invention relates to a retinal pigment degeneration mouse disease model, which is based on RHO genes of retinal pigment degeneration patients, 75nt DNA fragments of the patients are knocked into corresponding sites in the genome of the mice by using CRISPR/Cas9, and the fragments contain pathogenic mutation, so that a T17M gene knocked-in mouse disease model is constructed, wherein the pathogenic mutation is named as c.C50T from the nucleic acid level, and the pathogenic mutation is named as p.T17M from the protein level. The retinitis pigmentosa mouse disease model has patient-specific mutant fragments, and provides powerful support for the research and development of corresponding medicaments and gene therapy methods.

Description

Construction method of retinitis pigmentosa mouse model
Technical Field
The invention relates to the field of genetic engineering, and relates to a mouse model for retinitis pigmentosa and a construction method thereof.
Background
Retinitis pigmentosa (retinitis pigmentosa, RP) is a hereditary eye disease in which progressive apoptosis of retinal photoreceptor cells occurs due to genetic mutations. The disease is mainly manifested by progressive vision loss, visual field constriction, night blindness, fundus pigmentation, and Electroretinogram (ERG) abnormality. It has been counted that the population incidence of retinitis pigmentosa is about 1/4000 worldwide, and has become a major blinding disease. The genetic patterns are classified into three types, namely, autosomal dominant inheritance (autosomal dominant RP, ADRP), autosomal recessive inheritance (autosomal recessive RP, ARRP) and sex chromosome linked recessive inheritance (X-linked RP, XLRP). In autosomal dominant inheritance RP (autosomal dominant retinitis pigmentosa, ADRP), rhodopsin (RHO) gene mutation rates are high, accounting for approximately 25% -30% of the incidence of ADRP. The RHO gene is located at 3q22.1 and consists of 5 exons, encoding 348 amino acids. Mutation of the RHO gene causes dysfunctions of rhodopsin protein, and finally causes apoptosis of photoreceptor cells. These mutation sites are therefore also important targets in gene therapy. The construction of ADRP disease models caused by RHO gene mutation is of great significance.
Because of the difference between the mouse genome and the human genome, the currently reported retinitis pigmentosa mouse model has a great difference from the genes of the patient, so that the existing methods for gene editing developed based on the retinitis pigmentosa mouse model cannot be directly used for clinical treatment of the patient, and the methods seriously affect the gene editing for the treatment of retinitis pigmentosa diseases.
Disclosure of Invention
In view of the above, the present invention aims to develop a mouse model of retinal pigment degeneration, which has patient-specific mutant fragments at the corresponding genomic sites, and provides a powerful support for the development of corresponding drugs and gene therapy methods.
In order to achieve the above objects, the present invention has developed a model of a disease of a mouse with retinal pigment degeneration, based on the RHO gene of a patient with retinal pigment degeneration, using CRISPR/Cas9 to knock a 75nt DNA fragment of the patient into a corresponding site in the genome of the mouse, the fragment containing a pathogenic mutation designated c.c50t from the nucleic acid level and p.t17m from the protein level, thereby constructing a model of a disease of a mouse with T17M gene knock-in.
Preferably, the mutant Rho gene has a protein sequence shown in SEQ ID NO. 1.
Preferably, the T17M gene knock-in mouse Rho portion has the gene sequence shown in SEQ ID NO. 3.
Preferably, the construction method of the retinal pigment degeneration mouse model, which constructs a gene knock-in mouse by CRISPR/Cas9, comprises the following steps,
(1) Designing two specific sgRNA sequences;
(2) Introducing missense mutation RHO, p.T17M on the mouse Rho gene exon 1, designing a repair template for gene knock-in, wherein the sequence of the repair template is shown as SEQ ID NO.2;
(3) Injecting in vitro transcribed Cas9 mRNA, sgRNA and a repair template into a C57BL/6J mouse fertilized egg by adopting a microinjection mode, and carrying out gene cutting in the mouse fertilized egg by a CRISPR/Cas9 gene editing system to induce homologous recombination repair, wherein the fertilized egg is developed into an embryo;
(4) The embryo after gene editing is immediately transferred into the uterus of pseudopregnant female mice, and the gene knock-in mice are obtained after production.
Preferably, the two specific sgRNA sequences include sgRNA1 and sgRNA2, which are respectively targeted to specific genomic sites, and the drawn line sequence is PAM, and the sequences of the sgRNA1 and the sgRNA2 are as follows:
sgRNA1:CGGCTCTCGAGGCTGCCCCACGG；
sgRNA2:CTTCTCCAACGTCACAGGCGTGG。
preferably, the CRISPR/Cas9 gene editing system binds to the target site of the genome, cas9 exerts a cleavage activity, generating a DNA double strand break, thereby inducing DNA damage repair, the cell repair DNA by homologous recombination, and the repair template is site-directed knocked into the mouse Rho gene.
Preferably, a 195nt repair template is designed comprising 75nt of the patient DNA sequence and the pathogenic mutation RHO, p.T17M, which comprises BstXI cleavage site 5'-CCANNNNNNTGG-3' by comparison with the wild-type mouse RHO gene sequence, whereas the wild-type mouse RHO gene sequence is absent, so that the cleavage site can be used for genotyping.
Preferably, the repair template sequence is shown as SEQ ID NO.2;
the T17M gene knock-in mouse Rho part gene sequence is shown in SEQ ID NO. 3.
Preferably, the step of identifying the mice comprises:
after the pseudopregnant female mice farrowing, the mice are F0 generation mice;
taking the tail and the toe of an F0 generation mouse, extracting the whole genome, carrying out PCR amplification and sequencing, hybridizing an F0 generation positive mouse with a wild type mouse to obtain an F1 generation mouse, taking the tail and the toe of the mouse, extracting the whole genome, and carrying out enzyme digestion verification or gene sequencing identification by using BstXI after PCR amplification.
In the invention, the structure and the function of the retina of the T17M gene knock-in mouse are obviously abnormal, which is the same as the characterization of clinical patients. Therefore, the invention provides strong support for the development of clinical therapeutic drugs and gene therapy methods for retinitis pigmentosa.
Drawings
FIG. 1 is a diagram of the construction of a mouse model of retinitis pigmentosa;
FIG. 2 is a graph showing the identification result of T17M knock-in mice;
FIG. 3 shows the results of T17M knock-in mouse retinal structure (OCT and immunohistochemistry) identification;
FIG. 4 shows the results of T17M knock-in mouse retinal function (ERG) assay;
FIG. 5 is a diagram of T17M knock-in mouse fundus and angiogram;
Detailed Description
In order to further describe the technical means and effects adopted by the present invention for achieving the intended purpose, the following detailed description will refer to the specific implementation, structure, characteristics and effects according to the present invention with reference to the accompanying drawings and preferred embodiments.
Referring to FIGS. 1 to 5, the transgenic mouse model for retinitis pigmentosa, which is disclosed by the invention, is based on the RHO gene of a patient suffering from retinitis pigmentosa, and a 75DNA fragment of the patient is knocked into the genome of the mouse by using CRISPR/Cas9, wherein the fragment contains RHO and p.T17M pathogenic mutation, so that a T17M gene knocked-in mouse disease model is constructed. Wherein T17M is a mutation site on the RHO gene.
The protein sequence corresponding to the Rho gene of the T17M gene knock-in mouse model is shown in SEQ ID NO. 1:
MNGTEGPNFYVPFSNAMGVVRSPFEQPQYYLAEPWQFSMLAAYMFLLIVLGFPIN
FLTLYVTVQHKKLRTPLNYILLNLAVADLFMVFGGFTTTLYTSLHGYFVFGPTGCNLEG
FFATLGGEIALWSLVVLAIERYVVVCKPMSNFRFGENHAIMGVVFTWIMALACAAPPLV
GWSRYIPEGMQCSCGIDYYTLKPEVNNESFVIYMFVVHFTIPMIVIFFCYGQLVFTVKE
AAAQQQESATTQKAEKEVTRMVIIMVIFFLICWLPYASVAFYIFTHQGSNFGPIFMTLP
AFFAKSSSIYNPVIYIMLNKQFRNCMLTTLCCGKNPLGDDDASATASKTETSQVAPA*.
mutant amino acids are underlined.
The construction method of the transgenic mouse model for retinal pigment degeneration comprises the following steps:
(1) Two specific sgRNA sequences were designed.
Two specific sgRNA sequences, including sgRNA1 and sgRNA2, target genomic specific sites, respectively, the sequences of sgRNA1 and sgRNA2 of CRISPR/Cas9 are as follows:
sgRNA1:CGGCTCTCGAGGCTGCCCCACGG；
sgRNA2:CTTCTCCAACGTCACAGGCGTGG。
(2) Introducing missense mutation (RHO, p.T17M) into mouse Rho gene exon 1, designing repair template sequence for gene knock-in,
the repair template sequence is shown as SEQ ID NO. 2:
5'-GGGAGCCGTCAGTGGCTGAGCTCGCCAAGCAGCCTTGGTCTCTGTCTACGAAGAGCCCGTGGGTCA GCCACAAGGGCCACAGCCATGAATGGCACAGAAGGCCCTAACTTCTACGTGCCCTTCTCCAATGCG ATGGGCGTGGTGCGGAGCCCCTTCGAGCAGCCGCAGTACTACCTGGCGGAACCATGGCAGTTC-3' a missense mutation (RHO, p.T17M) was introduced in exon 1 of the mouse Rho gene, the mutation site is underlined, and by comparison analysis with the wild-type mouse Rho gene sequence, it was found that the repair template contained BstXI cleavage site 5'-CCANNNNNNTGG-3', which was not present in the wild-type mouse Rho gene sequence, and therefore the cleavage site was useful for genotyping.
(3) The T7 promoter sequence was used as an in vitro transcription template by PCR amplification prior to insertion into the coding region of Cas9 (SpCas 9 utilized, coding sequence from https:// www.addgene.org/42230 /) and the sgRNA sequence. And injecting the in vitro transcribed Cas9 mRNA, sgRNA and repair template into the C57BL/6J fertilized egg by adopting a microinjection mode, and microinjection of the CRISPR/Cas9 system into the mouse fertilized egg for gene cleavage to induce homologous recombination repair.
The CRISPR/Cas9 gene editing system is combined with a target site of a genome, the Cas9 plays a cutting activity, and DNA double-strand breaks are generated, so that DNA damage repair is induced, cells repair DNA through homologous recombination, and a repair template is knocked into a mouse Rho gene at a fixed point.
(4) The fertilized eggs after gene editing develop into embryos, and are transferred into the uterus of pseudopregnant female mice, and the gene knock-in mice are obtained after production.
The partial sequence of the T17M gene knock-in mouse Rho gene is shown in SEQ ID NO. 3:
gcgttagtatgatatctcgcggatgctgaatcagcctctggcttagggagagaaggtcactttataagggtctggggggggtcagtgcctggagttgcgctgtgggagccgtcagtggctgagctcgccaagcagccttggtctctgtctacgaagagcccgtGGGTCAGCCACAAGGGCCACAGCCATGAATGGCACAGAAGGCCCTAACTTCTACGTGCCCTTCTCCAATGCGATgggcgtggtgcggagccccttcgagcagccgcagtactacctggcggaaccatggcagttctccatgctggcagcgtacatgttcctgctcatcgtgctgggcttccccatcaacttcctcacgctctacgtcaccgtacagcacaagaagctgcgcacacccctcaactacatcctgctcaacttggccgtggctgacctcttcatggtcttcggaggattc the DNA sequence (uppercase) is a 75nt DNA fragment from humans and the mutation site (RHO, p.T17M) is underlined.
In the present invention, the steps of breeding mice and identifying offspring include:
after the pseudopregnant female mice farrowing, the mice are F0 generation mice; tail and toe of the mouse are taken, and BstXI enzyme digestion is used for verification or gene sequencing identification is carried out after PCR amplification. The F0 generation positive mice are hybridized with wild mice to obtain F1 generation mice, the tails and toes of the F1 generation mice are taken, the whole genome is extracted, PCR amplification is carried out, bstXI enzyme digestion verification is carried out or gene sequencing identification is carried out. The F1 mice continue to breed to obtain more disease mice.
As shown by referring to FIG. 2, a repair template for respectively targeting a target sequence and 195nt of sgRNA1 and sgRNA2 is designed, wherein the target sequences of the sgRNA1 and the sgRNA2 are shown as A in FIG. 2. The repair template comprises 75nt human DNA sequence and a pathogenic mutation (RHO, p.t17m). The fertilized mouse eggs were edited and developed into embryos. The embryo after gene editing is then transplanted into the uterus of pseudopregnant female mice, and the gene knock-in mice are obtained after production (see FIG. 1). In designing the repair template, since the template contained 75nt of human DNA sequence, it was compared with the wild-type mouse Rho gene sequence, and found that the repair template contained BstXI cleavage site (5 '-CCANNNNNNTGG-3'), so that the gene knock-in mouse Rho gene sequence contained the cleavage site, whereas the wild-type (WT) mouse Rho gene sequence did not. After amplification by PCR and BstXI cleavage, agarose gel electrophoresis showed that two bands were present, the gene knocked-in mice, and that only one band was representative of the wild type mice, thus identifying (FIG. 2B). Genotyping was also performed by Sanger sequencing (C of fig. 2).
As shown in FIG. 3, the structural change of the retina of the gene knockout mouse was further observed. OCT (Optical Coherence Tomography ) showed clear structure of wild-type (WT) mouse retina, whereas gene knock-in mouse retina photoreceptor cell layers were significantly thinner, especially the outer nuclear layer (A, B of fig. 3). Further hematoxylin-eosin staining as in FIG. 3C, gene knock-in mice showed retinal structural abnormalities, most notably thinning of the outer nuclear layer to almost vanish, compared to WT mice.
From the above, it is clear from the description of FIG. 4 of the present invention that the T17M gene knock-in mouse retina structure change is remarkable, and the electrophysiological function of the mouse retina was evaluated. Electroretinograms are the total electrophysiological response of the retina recorded from the corresponding site when the retina is stimulated by light. When the dark-adapted electroretinogram detects rod cell responses (A, E), the b-wave amplitude is significantly reduced compared to WT (n=10, p < 0.0001); during dark adaptation mixing reaction, a normal wild-type mouse sequentially has a smaller negative a wave and a larger positive b wave, the a wave and b wave amplitude (B, D, E) of the T17M gene knock-in mouse are obviously reduced, and the difference is obvious (n=10, P < 0.0001); when examining cone function (C, D, E), there was no significant difference in the a-wave (n=10, p > 0.05) versus WT, the b-wave amplitude was significantly reduced (n=10, p < 0.01), suggesting that there was massive rod apoptosis in T17M knocked-in mouse retina, but cone cells also retained some functions. The apparent decrease in function of T17M gene knocked-in mice retina is consistent with OCT-detected epiretinal nuclear layer thinning.
As shown by reference to fig. 5 of the present invention, it was observed that the T17M gene knock-in mouse retinal fundus showed significant retinal pigment degeneration, retinal artery tapering, and beaded-like changes by fundus retinal detection and angiography (as indicated by arrows in fig. 5).
In conclusion, obvious abnormality appears in the structure and function of the retina of the T17M gene knock-in mouse, which is the same as the characterization of clinical patients, and the successful construction of the T17M gene knock-in mouse disease model is demonstrated.
The present invention is not limited to the above embodiments, but is capable of modification and variation in detail, and other modifications and variations can be made by those skilled in the art without departing from the scope of the present invention.

Claims (5)

1. A construction method of a mouse model for retinitis pigmentosa is characterized by comprising the following steps: using CRISPR/Cas9
The construction of the knock-in mice includes the steps of,
(1) Designing two specific sgRNA sequences;
two specific sgRNA sequences include sgRNA1 and sgRNA2, targeting the forward and reverse sequences, respectively, at specific sites in the genome, the sequences of the sgRNA1 and the sgRNA2 are as follows:
sgRNA1:CGGCTCTCGAGGCTGCCCCACGG；
sgRNA2:CTTCTCCAACGTCACAGGCGTGG；
(2) Introducing missense mutation RHO, p.T17M on the mouse Rho gene exon 1, designing a repair template for gene knock-in, wherein the sequence of the repair template is shown as SEQ ID NO.2;
(3) Injecting in vitro transcribed Cas9 mRNA, sgRNA and a repair template into a C57BL/6J mouse fertilized egg by adopting a microinjection mode, and carrying out gene cutting in the mouse fertilized egg by a CRISPR/Cas9 gene editing system to induce homologous recombination repair, wherein the fertilized egg is developed into an embryo;
(4) The embryo after gene editing is immediately transferred into the uterus of pseudopregnant female mice, and the gene knock-in mice are obtained after production.
2. The method for constructing a mouse model of retinal pigment degeneration according to claim 1, characterized in that: the CRISPR/Cas9 gene editing system is combined with a target site of a genome, the Cas9 plays a cutting activity, and DNA double-strand breaks are generated, so that DNA damage repair is induced, cells repair DNA through homologous recombination, and a repair template is knocked into a mouse Rho gene at a fixed point.
3. The method for constructing a mouse model of retinal pigment degeneration according to claim 1 or 2, characterized in that: 195nt repair template was designed comprising 75nt of the patient DNA sequence and the pathogenic mutation RHO, p.t17m, which comprises the BstXI cleavage site 5'-CCANNNNNNTGG-3' by comparison with the wild-type mouse RHO gene sequence, whereas the wild-type mouse RHO gene sequence is absent, so that the cleavage site can be used for genotyping.
4. The method for constructing a mouse model of retinitis pigmentosa as claimed in claim 3, which is characterized by
The characteristics are that:
the repair template sequence is shown as SEQ ID NO.2;
the T17M gene knock-in mouse Rho part gene sequence is shown in SEQ ID NO. 3.
5. The method of claim 1, wherein the step of identifying the mouse comprises,
after the pseudopregnant female mice farrowing, the mice are F0 generation mice;
taking the tail and the toe of an F0 generation mouse, extracting the whole genome, carrying out PCR amplification and sequencing, hybridizing an F0 generation positive mouse with a wild type mouse to obtain an F1 generation mouse, taking the tail and the toe of the mouse, and carrying out enzyme digestion verification or gene sequencing identification by using BstXI after PCR amplification.
CN202210443478.6A 2022-04-26 2022-04-26 Construction method of retinitis pigmentosa mouse model Active CN114868705B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202210443478.6A CN114868705B (en) 2022-04-26 2022-04-26 Construction method of retinitis pigmentosa mouse model

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210443478.6A CN114868705B (en) 2022-04-26 2022-04-26 Construction method of retinitis pigmentosa mouse model

Publications (2)

Publication Number Publication Date
CN114868705A CN114868705A (en) 2022-08-09
CN114868705B true CN114868705B (en) 2024-02-06

Family

ID=82670995

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210443478.6A Active CN114868705B (en) 2022-04-26 2022-04-26 Construction method of retinitis pigmentosa mouse model

Country Status (1)

Country Link
CN (1) CN114868705B (en)

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107287243A (en) * 2017-06-20 2017-10-24 温州医科大学 A kind of humanized's retinoschisis transgene mouse model and its construction method
CN110241202A (en) * 2019-06-25 2019-09-17 复旦大学附属眼耳鼻喉科医院 Retinal pigment degeneration mutational site and its application
CN111850044A (en) * 2020-07-16 2020-10-30 中国科学技术大学 Method for constructing rhesus monkey model for retinitis pigmentosa based on in-vivo gene knockout
CN111979241A (en) * 2020-07-23 2020-11-24 上海市第一人民医院 Method for preparing non-human mammal model of retinitis pigmentosa
CN111979272A (en) * 2020-08-24 2020-11-24 上海朗昇生物科技有限公司 Method for preparing non-human mammal model of retinopathy and application thereof
CN112715484A (en) * 2020-12-29 2021-04-30 四川省人民医院 Method for constructing retinal pigment degeneration disease model, application and breeding method
CN112980819A (en) * 2019-12-18 2021-06-18 上海市第一人民医院 Construction method and application of retinitis pigmentosa animal model

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107287243A (en) * 2017-06-20 2017-10-24 温州医科大学 A kind of humanized's retinoschisis transgene mouse model and its construction method
CN110241202A (en) * 2019-06-25 2019-09-17 复旦大学附属眼耳鼻喉科医院 Retinal pigment degeneration mutational site and its application
CN112980819A (en) * 2019-12-18 2021-06-18 上海市第一人民医院 Construction method and application of retinitis pigmentosa animal model
CN111850044A (en) * 2020-07-16 2020-10-30 中国科学技术大学 Method for constructing rhesus monkey model for retinitis pigmentosa based on in-vivo gene knockout
CN111979241A (en) * 2020-07-23 2020-11-24 上海市第一人民医院 Method for preparing non-human mammal model of retinitis pigmentosa
CN111979272A (en) * 2020-08-24 2020-11-24 上海朗昇生物科技有限公司 Method for preparing non-human mammal model of retinopathy and application thereof
CN112715484A (en) * 2020-12-29 2021-04-30 四川省人民医院 Method for constructing retinal pigment degeneration disease model, application and breeding method

Also Published As

Publication number Publication date
CN114868705A (en) 2022-08-09

Similar Documents

Publication Publication Date Title
Slijkerman et al. The pros and cons of vertebrate animal models for functional and therapeutic research on inherited retinal dystrophies
Hoshijima et al. Precise editing of the zebrafish genome made simple and efficient
Won et al. Mouse model resources for vision research
CN112715484B (en) Method for constructing retinal pigment degeneration disease model, application and breeding method
Hu et al. In vivo CRISPR/Cas9-mediated genome editing mitigates photoreceptor degeneration in a mouse model of X-linked retinitis pigmentosa
CN115968834A (en) Preparation method and application of RS1 point mutation mouse model
CN112899311B (en) Construction method and application of RS1-KO mouse model
CN110628814B (en) Method for increasing SMN protein expression based on gene editing technology and application of method in SMA treatment
CN111485003B (en) Construction method and application of retinal vascular disease model
CN116218905B (en) Construction and application of point mutation rat epilepsy model
CN114868705B (en) Construction method of retinitis pigmentosa mouse model
CN106399369B (en) Build the method and targeting vector and kit in the mouse model of hippocampus regiospecificity knockout IKK α genes
CN112410340A (en) IDOL transgenic spontaneous atherosclerosis mouse model and construction method thereof
JP2010104326A (en) Model animal for age-related macular degeneration, and method for preparing the same
CN114752623B (en) Construction method and application of animal model of retinal pigment degeneration disease
EP1619248B1 (en) Mouse with deficiency of glutamate transporter glast function
CN115851833B (en) Mutant transgenic mouse with repeated amplification of NOTCH2NLC gene GGC and construction method and application thereof
US20060265772A1 (en) Histamine hyperproductive animal
Yu et al. Myelin regulatory factor deficiency is associated with the retinal photoreceptor defects in mice
KR100517831B1 (en) Double Transgenic Alzheimer&#39;s mice expressing mutant PS2 gene and APPsw gene
CN111979271A (en) Method for preparing Bietti crystal-like atrophy non-human mammal model and application thereof
CN114304068A (en) Preparation and application of epilepsy animal model
CN115197963A (en) Method for establishing SOX10 gene targeting mouse model
CN117604034A (en) Method for preparing humanized TTR mice and application thereof
CN117568398A (en) Method for constructing arrhythmia animal model by PGC-1 alpha gene knockout mice and application thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant