CN113337507B - Construction method and application of deaf mouse model with Otof 1273(C > T) gene site-directed mutagenesis - Google Patents
Construction method and application of deaf mouse model with Otof 1273(C > T) gene site-directed mutagenesis Download PDFInfo
- Publication number
- CN113337507B CN113337507B CN202110894148.4A CN202110894148A CN113337507B CN 113337507 B CN113337507 B CN 113337507B CN 202110894148 A CN202110894148 A CN 202110894148A CN 113337507 B CN113337507 B CN 113337507B
- Authority
- CN
- China
- Prior art keywords
- otof
- mouse
- deafness
- gene
- sgrna
- 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
Links
Images
Classifications
-
- 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
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
- A61K38/465—Hydrolases (3) acting on ester bonds (3.1), e.g. lipases, ribonucleases
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
- A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/16—Otologicals
-
- 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
- C12N15/89—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microinjection
-
- 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
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/22—Ribonucleases RNAses, DNAses
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
-
- 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
-
- 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]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The invention discloses a construction method and application of a deaf mouse model with Otof 1273(C > T) gene site-directed mutagenesis, wherein the deaf mouse model constructed by the construction method can be stably inherited, and the construction method has the advantages of high mutation efficiency, short screening period and test period, low cost and the like, provides a good visual animal model for further discussing the pathogenesis of hereditary deafness, screening auditory protective drugs at high flux, exploring the method and feasibility of gene therapy and screening candidate drugs for preventing and/or treating hereditary deafness, and has important clinical guiding significance for preventing and treating deafness.
Description
Technical Field
The invention belongs to the technical field of animal model construction, particularly relates to a construction method of a deaf mouse model, and more particularly relates to a construction method of a deaf mouse model with Otof 1273(C > T) gene site-directed mutagenesis and application thereof.
Background
Hearing loss or Hearing impairment (HL) refers to the partial loss of the ability of one or both ears to hear sound; deafness (Deafness) is a disease in which auditory nerves and various levels of centers in the conduction and conduction paths of the sound transmission, the sound sensation and the hearing thereof in the auditory system are diseased, which causes auditory dysfunction and produces different degrees of hearing loss, and is also a very severe hearing loss or hearing impairment. Global Disease Burden studies have explored the time of prevalence indicators for patients with deafness, and the results suggest that deafness is the fourth leading cause of the Global population (GBD 2015 DISEASE AND INJURY INCIDENCE AND PREVALENCE COLLABORATORS. Global, regional, and national origin, prediction, and yearlive with diagnosis for 310 diseases and injuries, 1990:. a systematic analysis for the Global Burden of the Disease Study [ J ]. Lancet, 2016, 388: 1545-. According to the latest data of the world health organization, about 4.66 hundred million people causing disabled hearing loss worldwide account for more than 5% of the total number of people worldwide, wherein 3-4 hundred million people are children, and the number of people causing disabled hearing loss reaches 6.3 hundred million by 2030 and possibly exceeds 9 hundred million by 2050 without effective intervention measures. Wherein, hereditary deafness accounts for about 50% of all deaf people. Hereditary hearing loss refers to further auditory dysfunction caused by ear dysplasia or metabolic disorder due to parent pathogenic genes or newly-occurring deafness gene variation. Hereditary hearing loss is classified into Synthetic Hearing Loss (SHL) and nonsynthetic hearing loss (NSHL) according to whether the disease is accompanied by extraaural tissue abnormality, pathological changes or systemic diseases, and the SHL and NSHL account for 30% and 70%, respectively.
Deafness has serious influence on the normal life of human beings, hearing is one of the most developed senses of human beings, plays an important role in daily communication, and deafness patients usually have certain uncomfortable symptoms such as isolated feeling and feeling of loss. Therefore, establishing a stable and heritable gene site-directed mutagenesis deafness mouse model, and further providing a research basis for further research on the injury mechanism of deafness becomes a technical problem to be solved by those skilled in the art. With the development and improvement of high-efficiency gene editing technologies such as CRISPR/Cas9, Zinc Finger Nucleases (ZFNs), Transcription Activator-like Effector Nucleases (TALENs) and the like, the construction of an animal model of gene knockout or gene site-directed mutation provides a more effective way for researching pathogenesis of hereditary hearing loss on the basis of etiology and molecular pathology. At present, more than 400 genetic deafness animal models are reported, including three types of genetic engineering animal models, ENU chemical mutagenesis animal models and spontaneous mutation animal models. Genetic engineering deafness animal models are the most, and in addition, high-throughput random mutagenesis generated by mutagenizing animal genomes by utilizing ENU is also an effective way for preparing a new deafness animal model and discovering a new deafness gene. However, the method for constructing the deaf mouse model commonly used in the prior art has the defects of poor genetic stability, high cost, low mutation efficiency, long screening period and the like of the constructed mouse model. Until now, no relevant report on the construction method of the deafness mouse model with Otof 1273(C > T) gene site-directed mutation based on the CRISPR/Cas9 technology is seen.
Disclosure of Invention
In view of the above, in order to make up for the defects existing in the current field, the invention aims to provide a construction method for constructing a deafness mouse model with Otof 1273(C > T) gene site-directed mutation based on a CRISPR/Cas9 technology, wherein the deafness mouse model constructed by the construction method can be stably inherited, and clinical test materials are provided for the pathogenesis research of hereditary hearing loss and the exploration of a treatment method of hereditary hearing loss.
The above object of the present invention is achieved by the following technical solutions:
in a first aspect of the invention, there is provided a mouse Otof mutant gene causing deafness.
Further, the mutant gene has a base 1273 in the sequence of the Otof gene changed from C to T as compared with that of the Otof gene of a wild-type mouse.
The Otof in the invention is otoferlin Gene of a mouse (Mus musculus), the Ensembl version database number is ENSMUSG00000062372, and the Gene ID in the NCBI database is 83762.
In a second aspect of the invention, a construct is provided.
Further, the construct comprises a mutant gene according to the first aspect of the invention;
preferably, the construct comprises a plasmid, phage, virus, artificial chromosome, cosmid;
more preferably, the construct is a plasmid.
In a third aspect of the invention, a recombinant cell is provided.
Further, the recombinant cell is obtained by transforming a recipient cell with the construct of the second aspect of the present invention;
preferably, the recipient cell comprises an escherichia coli cell, a mammalian cell, a yeast cell, an insect cell, a plant cell;
more preferably, the recipient cell is a mammalian cell.
A fourth aspect of the invention provides a sgRNA for Otof gene editing.
Further, the sequence of the sgRNA is shown in SEQ ID NO. 1.
In a fifth aspect of the present invention, there is provided a Donor oligo for Otof gene editing.
Further, the sequence of the Donor oligo is shown in SEQ ID NO. 2.
A sixth aspect of the invention provides a CRISPR/Cas9 system for Otof gene editing.
Further, the system comprises Cas9 mRNA, sgRNA according to the fourth aspect of the invention, and Donor oligo according to the fifth aspect of the invention.
The seventh aspect of the invention provides a construction method of a deafness mouse model with site-directed mutagenesis of an Otof 1273(C > T) gene;
further, the method comprises changing an Otof gene of a normal mouse to change the 1273 th base of the Otof gene sequence from C to T;
preferably, the method comprises the steps of:
(1) designing an sgRNA sequence for efficiently recognizing an Otof gene;
(2) in vitro transcribing Cas9 mRNA and sgRNA, and synthesizing a Donor oligo in vitro;
(3) configuring a mixture comprising Cas9 mRNA, sgRNA, Donor oligo;
(4) injecting the mixture in the step (3) into mouse fertilized eggs, transplanting the fertilized eggs into a surrogate mouse body, performing pregnancy, producing the mouse and identifying the genotype to obtain an Otof 1273(C > T) gene site-directed mutation deafness mouse model;
more preferably, the sequence of the sgRNA described in step (1) is shown in SEQ ID No. 1;
more preferably, the sequence of the Donor oligo in step (2) is shown in SEQ ID NO. 2;
more preferably, the amount of Cas9 mRNA in the mixture described in step (3) is 100-150 ng;
more preferably, the amount of sgRNA in the mixture described in step (3) is 30-90 ng;
more preferably, the amount of the Donor oligo in the mixture in step (3) is 100-150 ng;
most preferably, the amount of Cas9 mRNA in the mixture described in step (3) is 125 ng;
most preferably, the amount of sgRNA in the mixture described in step (3) is 62.5 ng;
most preferably, the amount of the Donor oligo in the mixture in step (3) is 125 ng;
most preferably, the mixture described in step (3) further comprises a solvent;
most preferably, the solvent is a buffer;
most preferably, the volume of the mixture is 1-10 μ L;
most preferably, the volume of the mixture is 5 μ Ι _;
more preferably, the fertilized egg of step (4) is derived from a C57BL/6J mouse;
more preferably, the injection in step (4) comprises microinjection, ultrasonic wave induction, electroporation, sonoporation, photoporation, magnetic transfer, thermal shock, calcium phosphate, liposome and polymer, nanoparticle, and viral transformation;
most preferably, the injection in step (4) is by microinjection;
more preferably, the duration of the pregnancy in step (4) is 19 to 21 days;
most preferably, the duration of the pregnancy in step (4) is 19 to 20 days;
preferably, the method further comprises superovulation of a fertilized egg donor mouse;
more preferably, the superovulation of the fertilized egg donor mouse includes the steps of:
(1) pregnant mare serum gonadotropin treatment donor female mice;
(2) injecting human chorionic gonadotropin 40-50 h later, mating with male mice, and collecting fertilized eggs the next day;
more preferably, the mouse described in step (1) is a C57BL/6J mouse;
more preferably, human chorionic gonadotropin is injected after 47 h in step (2).
Further, the microinjection method is a method in which a glass microinjection needle with a very fine tip (0.1 to 0.5 μm) is used to directly inject an exogenous gene fragment into a prokaryotic embryo or a cultured cell, and then the exogenous gene is inserted into the chromosome of the host by recombination (replication), Deletion (Deletion), replication (Duplication), Translocation (Translocation) or the like that may occur in the host genome sequence.
An eighth aspect of the present invention provides a pharmaceutical composition for the treatment and/or prevention of deafness.
Further, the pharmaceutical composition comprises an agent that changes the 1273 base of the Otof gene sequence from T to C;
preferably, the agent is an agent for site-directed mutagenesis of a gene.
The ninth aspect of the invention provides a kit for screening an Otof 1273(C > T) gene mutant deaf mouse.
Further, the kit comprises primers for specifically detecting mutations of the Otof 1273(C > T) gene;
preferably, the sequence of the primer is shown in SEQ ID NO. 5-6.
A tenth aspect of the invention provides the use of any one of the following aspects:
(1) the mutant gene of the first aspect of the invention is applied to the construction of an Otof 1273(C > T) gene site-directed mutant deafness mouse model;
(2) the application of the mutant gene of the first aspect of the invention in preparing products for diagnosing and/or treating mouse deafness;
(3) the application of the construct of the second aspect of the invention in constructing an Otof 1273(C > T) gene site-directed mutation deafness mouse model;
(4) the recombinant cell of the third aspect of the invention is applied to the construction of an Otof 1273(C > T) gene site-directed mutation deafness mouse model;
(5) the sgRNA of the fourth aspect of the invention is applied to the construction of an Otof 1273(C > T) gene site-directed mutation deafness mouse model;
(6) the application of the Donor oligo in the fifth aspect of the invention in constructing the deafness mouse model of the Otof 1273(C > T) gene site-directed mutagenesis;
(7) the system of the sixth aspect of the invention is applied to the construction of an Otof 1273(C > T) gene site-directed mutation deafness mouse model;
(8) the application of the primers shown in SEQ ID NO.5 and SEQ ID NO.6 in the preparation of a kit for screening an Otof 1273(C > T) gene mutant deaf mouse;
(9) the application of the deafness mouse model of the Otof 1273(C > T) gene site-directed mutation constructed by the method of the seventh aspect of the invention in screening candidate drugs for preventing and/or treating deafness;
(10) the kit of the eighth aspect of the invention is applied to screening deaf mice with Otof 1273(C > T) gene mutation;
(11) use of an agent for changing the 1273 base of the Otof gene sequence from T to C in the preparation of a pharmaceutical composition for treating and/or preventing deafness.
In order to further explain the present invention, terms involved in the present invention are explained as follows. The explanations in this section should not be construed as limitations of the present invention, but interpreted to allow those skilled in the art to better understand the contents of the present invention, and the terms are explained as follows:
the term "construct" as used herein is used in conjunction with "recombinant constructs", "expression constructs" and "chimeric constructs". A recombinant construct refers to a nucleic acid fragment comprising a mutant gene in which base 1273 of the otto gene sequence is changed from C to T, such as an artificial combination of a regulatory sequence and a coding sequence that do not occur together under natural conditions; chimeric constructs are meant to comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source but arranged in a manner different from that found in nature, and such constructs may be used alone or in combination with a vector, the choice of which, if used, depends on methods well known to those skilled in the art to be used to transform a host cell, e.g., a plasmid vector may be used. The skilled artisan is well aware of the genetic elements that must be present on a vector to successfully transform, select and propagate a host cell comprising a nucleic acid fragment of a mutant gene of the invention. The construct may comprise any combination of deoxyribonucleotides, ribonucleotides, and/or modified nucleotides, the construct may be transcribed to form RNA, wherein the RNA may be capable of forming a double-stranded RNA and/or hairpin structure, the construct may be expressed in a cell, or isolated, or made synthetically, the construct may further comprise a promoter or other sequence that facilitates manipulation or expression of the construct.
The term "recombinant cell" as used herein refers to a cell that contains or into which a construct (nucleic acid construct) has been introduced and supports replication and/or expression of the construct. The cell may be a prokaryotic cell, such as E.coli, or a eukaryotic cell, such as a fungal, yeast, insect, amphibian, nematode, mammalian cell, or a monocotyledonous, dicotyledonous plant cell, and in particular embodiments of the invention, the cell is preferably a mammalian cell.
The term "recipient cell" as used herein includes, but is not limited to, prokaryotic cells including, but not limited to, escherichia coli, mycoplasma, chlamydia, archaea, actinomycetes, rickettsia, spirochetes, lactobacillus, bacillus thuringiensis; the eukaryotic cells include, but are not limited to, yeast cells, insect cells, plant cells, animal cells (e.g., mammalian cells including mouse cells, rat cells, human cells, monkey cells, etc.), and non-mammalian cells including avian cells, etc., and in particular embodiments of the invention, the recipient cells are preferably mammalian cells.
The term "kit for screening deaf mice having Otof 1273(C > T) gene mutation" as used herein refers to a kit comprising liquid or powdered primers specifically recognizing Otof 1273(C > T) gene mutation, said kit may further comprise other reagents required for PCR, such as buffer, dNTP, polymerase; the kit can also comprise reagents and consumables required by recovering PCR products, such as a sol solution, a collecting tube, a washing solution and the like, and in addition, the kit also comprises an instruction book which describes how to detect a sample to be detected and how to judge whether the Otof 1273(C > T) gene mutation is contained according to the detection result. The kit and the instruction for screening the deaf mouse with the Otof 1273(C > T) gene mutation are used for detection by taking the DNA of a sample to be detected as a template, the operation is simple and convenient, and a large number of samples can be quickly identified.
The term "prevention and/or treatment", as used herein, includes "prevention" and "treatment", wherein prevention refers to the prevention or inhibition, in whole or in part, of the symptoms of a disease or the frequency with which such symptoms occur, or the reduction of the risk of acquiring a given symptom of a disease. In a specific embodiment of the invention, the disease is deafness. Prevention includes inhibition and/or prevention of symptoms associated with deafness, reduction in severity of symptoms associated with deafness, or amelioration of signs and symptoms associated with deafness, prevention includes inhibition, prevention, or reduction in severity of symptoms associated with deafness, which term includes the effect that occurs before a patient begins to suffer from deafness or an associated disorder, i.e., delaying the onset of symptoms associated with deafness, and/or inhibiting or reducing severity of symptoms associated with deafness; treatment refers to reducing or eliminating the severity of symptoms of deafness disease, the frequency of occurrence of such symptoms, or both, and the term includes the effect that occurs when a patient suffers from deafness or an associated condition, i.e., reducing the severity of one or more symptoms or effects of deafness-associated symptoms.
The invention has the advantages and beneficial effects that:
(1) the invention provides a construction method of a deafness mouse model with Otof 1273(C > T) gene site-directed mutation based on CRISPR/Cas9 technology, which is carried out in an Otof gene site-directed mutation mode, so that the mouse generates genome level change, diseases of the mouse after birth occur and develop under natural rules, the diseases of the mouse model are closer to the occurrence and development of real diseases, and research results have reference value;
(2) the method for constructing the deaf mouse model commonly used in the prior art has the defects of poor genetic stability, high cost, long experimental period, low mutation efficiency, long screening period and the like of the constructed mouse model, and the deaf mouse model with the fixed-point mutation of the Otof 1273(C > T) gene constructed by the construction method provided by the invention has the advantages of stable inheritance, short screening period and experimental period, high mutation efficiency and the like;
(3) the deafness mouse model of the Otof 1273(C > T) gene site-directed mutation constructed by the construction method provides a good visual animal model for deeply discussing the pathogenic mechanism of hereditary deafness, screening auditory protective drugs with high flux, exploring the method and feasibility of gene therapy and screening candidate drugs for preventing and/or treating hereditary deafness.
Drawings
Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:
FIG. 1 shows a schematic diagram of the base sequences of sgRNA and donor oligo;
FIG. 2 shows an experimental flow chart for constructing a deafness mouse model with site-directed mutation of Otof 1273(C > T) gene based on CRISPR/Cas9 technology;
FIG. 3 shows the results of high fidelity enzymatic PCR amplification of a DNA fragment (about 120bp) encoding the sequence of sgRNA 1;
FIG. 4 shows a graph of the results of DNA recovery of the T7 promoter/sgRNA PCR product using the DNA Clean & concentrator (TM) -5 kit;
FIG. 5 is a drawing showing the results of agarose gel electrophoresis;
FIG. 6 shows a result diagram of a deafness mouse model with site-directed mutation of Otof 1273(C > T) gene constructed based on CRISPR/Cas9 technology, wherein a triangle indicates a positive fountain mouse (initial mouse);
FIG. 7 is a view showing the results of electrophoresis obtained when PCR products are detected by agarose gel electrophoresis;
fig. 8 shows a graph of the results obtained by sequencing the PCR products of positive mice, in which, graph a: 1# Otof 1273(C > T) gene site-directed mutation positive mice, panel B: 8# Otof 1273(C > T) gene site-directed mutation positive mouse;
FIG. 9 shows the result of hearing test of the constructed deaf mouse model by ABRs hearing test method, wherein the straight line is the hearing test result of the deaf mouse with fixed point mutation of the Otof 1273(C > T) gene constructed based on CRISPR/Cas9 technology, and the broken line is the hearing test result of the wild mouse.
Detailed Description
The present invention is further illustrated below with reference to specific examples, which are intended to be illustrative only and are not to be construed as limiting the invention. As will be understood by those of ordinary skill in the art: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents. The following examples are examples of experimental methods not indicating specific conditions, and the detection is usually carried out according to conventional conditions or according to the conditions recommended by the manufacturers.
Example 1 construction of Otof 1273(C > T) Gene site-directed mutagenesis deafness mouse model based on CRISPR/Cas9 technology
1. Experimental Material
Q5 Hot Start high fidelity DNA polymerase (NEB, M0493); hieffTMPCR reaction mix (Yeasen, 10102); DNA purification kit (Zymo Research, D4014); zymocleanTMGel DNA recovery kit (Zymo Research, D4007); mMESSAGE mMACHINETMT7 high-throughput capped RNA transcription kit (Invitrogen, AM 1345); MEGAshortscriptTMT7 transcription kit (Invitrogen, AM 1354); MEGAclear ™ transcription purification kit (Invitrogen, AM 1908); pEASY-T1 series cloning vectors (Transgen, CT 111); animal tissue/cell genomic DNA extraction kit (Solarbio, D1700); PX330 plasmid (Addgene, plasmid # 42230); trans2K Plus DNA Marker (Transgen, BM 111); pregnant Mare Serum Gonadotropin (PMSG) (Prospec, HOR-272); human chorionic gonadotropin (hCG) (Prospec, HOR-250); m2 medium (Sigma-Aldrich, M7167-100 ML); EmbryoMax modified M16 medium (1X) (Merck, MR-016-D); mineral oil (Sigma-Aldrich, M8410); hyaluronidase (Sigma-Aldrich, H3506); the pseudopregnant female mouse is ICR mouse (Weitonghua)) (ii) a The fertilized egg for injection is in C57BL/6J (Wittingle) background.
2. Design of sgRNA sequence and donor oligo sequence
The sequence of the sgRNA and the sequence of the donor oligo (see FIG. 1) were designed as follows:
(1) sgRNA sequence:
5’-GTGAAAATTTACCGAGCAGA-3’ (SEQ ID NO.1)；
(2) donor oligo sequence:
5’-TGAACGCCTTCTTCACGTTGGCCATGAGGCTTGTGTTCATCCGGGGCAGTCCCTCTGCTCAGTAAATTTTCACATAGAACCGTGCCCACTGCCGTTCGGGGGGCACGCCCTCGGGGAGCAG-3’ (SEQ ID NO.2)。
3. preparation of sgRNA in vitro transcription template
(1) Synthesis of oligodeoxynucleotide (DNA oligo, 5 '→ 3')
sgRNA F：TAATACGACTCACTATAGGTGAAAATTTACCGAGCAGAGTTTAAGAGCTATGCTGGAAA (SEQ ID NO.3)；
sgRNA R：AAAAGCACCGACTCGGTGCC (SEQ ID NO.4)。
(2) High fidelity enzymatic PCR amplified DNA fragments (about 120bp) encoding sgRNA sequences, each sgRNA in 3 tubes of the same system (see table 1 and fig. 3).
TABLE 1 composition of the system
(3) PCR reaction procedure
The PCR reaction was carried out according to the PCR reaction procedure described in Table 2.
TABLE 2 PCR reaction procedure
(4) DNA from the T7 promoter/sgRNA PCR product was recovered using the DNA Clean & concentrator (TM) -5 kit (see FIG. 4), and the DNA concentrations were recovered: 255 ng/. mu.L; 260/280: 1.8; 260/230: 1.9.
4. sgRNA in vitro transcription and recovery
(1) Using MEGASHORTScript T7 Transcription kit, an external ligand Transcription reaction system, the composition of which is shown in Table 3.
TABLE 3 reaction System composition
Mixing, and reacting at 37 deg.C for 2 h.
(2) Add 1. mu.L of TURBO DNase, mix well, react at 37 ℃ for 15 min.
(3) sgRNA was recovered using MEGAclear ™ Transcription clear-Up Kit.
(4) Mix well, 1.5 μ L (RNA40) was taken to assay, 260/280 was normal at around 2.0, and the remaining sgrnas were placed on ice. Recovery of gRNA concentration: 2089 ng/. mu.L; 260/280: 2.4; 260/230: 2.5.
(5) a1. mu.L aliquot of the gel was run on a 2% agarose gel, normally with a bright band around 100 bp (see FIG. 5).
(6) And if the concentration and the gum running result are normal, subpackaging, and quickly freezing in a refrigerator at minus 80 ℃ to obtain the sgRNA.
5. Otof sgRNA, Cas9 mRNA and Donor oligo mixture
The mixture used for two-cell embryo injection was 25 ng/. mu.L Cas9 mRNA, 12.5 ng/. mu.L Otof sgRNA, 25 ng/. mu.L donor oligo. Typically, 5. mu.L of the mixture is prepared for injection, and the composition of the mixture is shown in Table 4.
TABLE 4 composition of the mixture
Centrifuging at 4 deg.C and 10000 rpm for 10 min, and storing at-80 deg.C.
6. Prokaryotic injection of embryos
Injecting the mixture of the Otof sgRNA, the Cas9 mRNA and the Donor oligo into the prokaryotic embryo by using a micromanipulator, and injecting about 90 prokaryotic embryos;
the prokaryotic embryo is obtained by superovulation of a fertilized egg donor mouse, and comprises the following steps: treating donor female C57BL/6J mouse with Pregnant Mare Serum Gonadotropin (PMSG), injecting Human Chorionic Gonadotropin (HCG) after 47 h, mating with male mouse, and collecting fertilized egg the next day to obtain prokaryotic embryo;
an experimental flow chart of constructing an Otof 1273(C > T) gene site-directed mutation deafness mouse model based on CRISPR/Cas9 technology is shown in figure 2.
7. Embryo transplantation and genotype identification of first-established mice
After injecting the prepared mixture of Otof sgRNA, Cas9 mRNA and Donor oligo into prokaryotic embryos and transplanting into oviducts of 3 pseudopregnant female mice, the mice are generally bred for 19-20 days. 8 newborn mice were born (see FIG. 6), wherein the triangle among the 8 newborn mice indicates a positive fountain mouse (naive mice). After the mouse is born for 6 days, the toes of the mouse are separated, the toes can be cut for numbering and marking, and the small tissue of the tail tip is cut for extracting the genome DNA. In addition, in the present embodiment, the applicant designs a plurality of sgrnas for the Otof gene, the sgrnas used in the present embodiment are significantly superior to other sgrnas, and can perform the Otof 1273(C > T) site-specific mutation on the mouse Otof gene with high efficiency, specificity, stability, difficulty in off-target, and no interference with other genes, thereby greatly improving the efficiency of site-specific mutation on the mouse Otof gene, while other sgrnas have the disadvantages of easy off-target, instability, poor specificity, and the like.
Example 2 genotyping of deafness mouse model with site-directed mutagenesis of Otof 1273(C > T) Gene constructed based on CRISPR/Cas9 technique
1. Primer information
The sequences of the forward and reverse primers were as follows:
Primer F：5’-TGTTTTCCAGCTGGGGGTTT-3’ (SEQ ID NO.5)；
Primer R：5’-TGCAAGCATTCACTTGCTTTGT-3’ (SEQ ID NO.6)。
the theoretical length of the wild type (wt) product is about 457 bp.
2. PCR reaction system
The PCR reaction system is shown in Table 5.
TABLE 5 PCR reaction System
3. PCR reaction conditions
The PCR reaction conditions are shown in Table 6.
TABLE 6 PCR reaction conditions
4. Results of the experiment
The resulting electrophoretogram is shown in FIG. 7, wherein M is DNA Marker (from bottom to top: 100 bp, 200 bp, 500 bp, 750 bp, 1000 bp, 2000 bp), and 1-8 is rat tail DNA; the sequencing result of the PCR product of the positive mouse shows that the sequencing peak diagram of the PCR product of the DNA of the rat tail of the fixed point mutation fountain mouse (a first-building mouse) of the Otof 1273(C > T) gene shows the replacement of T basic group at the position (an arrow) of the Otof 1273 (see a figure 8A and a figure 8B), which proves the success of the point mutation, and 2 positive mice (1#, 8#) (female and male respectively) of the fixed point mutation of the Otof 1273(C > T) gene are constructed in the invention.
Example 3 phenotypic identification of Otof 1273(C > T) Gene site-directed mutagenesis deafness mouse model constructed based on CRISPR/Cas9 technology
1. Experimental methods
In this embodiment, an Auditory brainstem response detection method (ABRs) is used to detect hearing of the constructed deafness mouse model, and the detailed experimental steps are as follows:
(1) using TDT audiometry equipment (Tucker Davis Technologies, Alachua, FL, USA) and Biosig audiometry software to detect ABR threshold and latency of each wave of the mice;
(2) ABR detection is carried out in a shielding room with good sound insulation, the mouse is anesthetized before audiometry, the mouse is anesthetized systemically by adopting ketamine (intraperitoneal injection, 100 mg/kg) and xylazine (intraperitoneal injection, 10 mg/kg), and the anesthetized mouse is placed on a heat-insulating pad at 37 ℃ to maintain the body temperature of the mouse;
(3) after anesthesia succeeds, a recording electrode (+) of the ABR is inserted into a subcutaneous part at the midpoint of a connecting line of the anterior border of auricles on two sides of a mouse, a reference electrode (-) of the ABR is inserted into a subcutaneous part behind the ear of a tested ear, a grounding electrode (-) of the ABR is inserted into a subcutaneous part behind the ear of a tested opposite side, and a sound-giving earphone is placed at a position which is about 0.5 cm away from an opening of an external auditory canal;
(4) sound giving setting for auditory brainstem response detection: short sound (Click) and short pure Tone (Tone-burst) are adopted as the stimulation sound of the ABR; parameter setting of auditory brainstem response detection: the band-pass filtering is 300-3000 Hz, the stacking times are 1024, and the scanning time is 10 ms; ABR dosing frequencies of 4, 8, 12, 16, 32, 40 kHz and clicks (100 μ s), respectively, ABR detection dosing and recording was done by System 3 hardware (Tucker Davis Technologies, TDT, Alachua, USA) and SigGen/BioSig software (Tucker Davis Technologies, TDT);
(5) the intensity of the applied stimulation sound is gradually decreased from 90 dB SPL at a distance of 10 dB SPL, and when a repeatable ABR waveform cannot be detected, the sound intensity is increased by 5 dB SPL on the basis of the applied sound intensity. The intensity of the stimulating sound when the repeated ABR waveform is recorded is the ABR threshold of the mouse;
(6) the amplitudes and latencies of the I-wave and v-wave of ABR are calculated at 90 dB for the acoustic intensity level at each frequency.
2. Results of the experiment
The hearing test result of the ABR of deafness mice constructed by Otof 1273(C > T) gene site-directed mutation based on the CRISPR/Cas9 technology shows that under the stimulation of 90 dB sound intensity, no effective waveform is led out from frequencies of 4K, 8K, 16K and 32K of adult mutation mice; the wild type mice (control group) have waveforms at all frequencies, the hearing level is normal (see figure 9), and the mice with site-directed mutagenesis of the Otof 1273(C > T) gene constructed by the invention are proved to be deaf mice.
The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.
Sequence listing
<110> affiliated Beijing friendship hospital of capital medical university
Construction method and application of deafness mouse model with site-directed mutation of <120> Otof 1273(C > T) gene
<141> 2021-08-04
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
gtgaaaattt accgagcaga 20
<210> 2
<211> 121
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
tgaacgcctt cttcacgttg gccatgaggc ttgtgttcat ccggggcagt ccctctgctc 60
agtaaatttt cacatagaac cgtgcccact gccgttcggg gggcacgccc tcggggagca 120
g 121
<210> 3
<211> 59
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
taatacgact cactataggt gaaaatttac cgagcagagt ttaagagcta tgctggaaa 59
<210> 4
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
aaaagcaccg actcggtgcc 20
<210> 5
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
tgttttccag ctgggggttt 20
<210> 6
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
tgcaagcatt cacttgcttt gt 22
Claims (5)
1. A method for constructing a deafness mouse model with site-directed mutation of an Otof 1273(C > T) gene, which is characterized by comprising the following steps:
(1) designing an sgRNA sequence for efficiently recognizing an Otof gene;
(2) in vitro transcribing Cas9 mRNA and sgRNA, and synthesizing a Donor oligo in vitro;
(3) configuring a mixture comprising Cas9 mRNA, sgRNA, Donor oligo;
(4) injecting the mixture obtained in the step (3) into mouse fertilized eggs, transplanting the fertilized eggs into a surrogate mouse body, performing pregnancy, producing the mouse and identifying the genotype to obtain an Otof 1273(C > T) gene site-directed mutation deafness mouse model;
the sequence of the sgRNA in the step (1) is shown in SEQ ID NO. 1;
the sequence of the Donor oligo in step (2) is shown in SEQ ID NO. 2.
2. The method of claim 1, wherein the amount of Cas9 mRNA used in the mixture in step (3) is 125 ng;
the amount of sgRNA in the mixture in step (3) was 62.5 ng;
the amount of the Donor oligo in the mixture in the step (3) is 125 ng;
the volume of the mixture in the step (3) is 5 mu L;
the fertilized egg in the step (4) is derived from a C57BL/6J mouse;
the injection mode in the step (4) is a microinjection method;
the time of the pregnancy in the step (4) is 19 to 21 days.
3. The method of constructing according to claim 1 or 2, further comprising superovulation of a fertilized egg donor mouse;
the superovulation of the fertilized egg donor mouse comprises the following steps:
(1) pregnant mare serum gonadotropin treatment donor female mice;
(2) injecting human chorionic gonadotropin 40-50 h later, mating with male mice, and collecting fertilized eggs the next day;
the mice in the step (1) are C57BL/6J mice.
4. The application of a CRISPR/Cas9 system in constructing an Otof 1273(C > T) gene site-directed mutation deafness mouse model;
the system comprises Cas9 mRNA, sgRNA with a sequence shown in SEQ ID NO.1 and Donor oligo with a sequence shown in SEQ ID NO. 2.
5. Use of the deafness mouse model of site-directed mutation of the Otof 1273(C > T) gene constructed according to the construction method of claim 1 in screening candidate drugs for preventing and/or treating deafness.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110894148.4A CN113337507B (en) | 2021-08-05 | 2021-08-05 | Construction method and application of deaf mouse model with Otof 1273(C > T) gene site-directed mutagenesis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110894148.4A CN113337507B (en) | 2021-08-05 | 2021-08-05 | Construction method and application of deaf mouse model with Otof 1273(C > T) gene site-directed mutagenesis |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113337507A CN113337507A (en) | 2021-09-03 |
| CN113337507B true CN113337507B (en) | 2021-11-12 |
Family
ID=77480841
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110894148.4A Active CN113337507B (en) | 2021-08-05 | 2021-08-05 | Construction method and application of deaf mouse model with Otof 1273(C > T) gene site-directed mutagenesis |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113337507B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA3168055A1 (en) | 2020-02-21 | 2021-08-26 | Emmanuel John Simons | Compositions and methods for treating non-age-associated hearing impairment in a human subject |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110225975A (en) * | 2016-08-23 | 2019-09-10 | 阿库斯股份有限公司 | For treating the composition and method of non-age-dependent hearing impairment in people experimenter |
| CN110468154A (en) * | 2019-07-05 | 2019-11-19 | 南京医科大学 | A kind of reconstructed eggs and its construction method of deafness swine model, deaf swine model, construction method and its application |
| CN112020561A (en) * | 2018-02-22 | 2020-12-01 | 阿库斯股份有限公司 | Compositions and methods for treating non-age-related hearing impairment in a human subject |
-
2021
- 2021-08-05 CN CN202110894148.4A patent/CN113337507B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110225975A (en) * | 2016-08-23 | 2019-09-10 | 阿库斯股份有限公司 | For treating the composition and method of non-age-dependent hearing impairment in people experimenter |
| CN112020561A (en) * | 2018-02-22 | 2020-12-01 | 阿库斯股份有限公司 | Compositions and methods for treating non-age-related hearing impairment in a human subject |
| CN110468154A (en) * | 2019-07-05 | 2019-11-19 | 南京医科大学 | A kind of reconstructed eggs and its construction method of deafness swine model, deaf swine model, construction method and its application |
Non-Patent Citations (8)
Also Published As
| Publication number | Publication date |
|---|---|
| CN113337507A (en) | 2021-09-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| WO2018177351A1 (en) | Method for preparing non-chimeric gene knockout animal based on crispr/cas9 technology | |
| Yao et al. | Homology-mediated end joining-based targeted integration using CRISPR/Cas9 | |
| Shao et al. | CRISPR/Cas-mediated genome editing in the rat via direct injection of one-cell embryos | |
| Harel et al. | Efficient genome engineering approaches for the short-lived African turquoise killifish | |
| Wu et al. | Engineering CRISPR/Cpf1 with tRNA promotes genome editing capability in mammalian systems | |
| CN110551759B (en) | Composition and method for improving recombination efficiency of transgenic cells | |
| CN108753772A (en) | The construction method of the human neuroblastomacells of CAPNS1 genes is knocked out based on CRISPR/Cas technologies | |
| CN108707628A (en) | The preparation method of zebra fish notch2 gene mutation bodies | |
| Zhou et al. | Programmable base editing of the sheep genome revealed no genome-wide off-target mutations | |
| Lone et al. | CRISPR/Cas9 system: a bacterial tailor for genomic engineering | |
| CN106047930A (en) | Method for preparing flox rats for PS1 gene conditional knockout | |
| CN104404036A (en) | Conditional gene knockout method based on CRISPR/Cas9 technology | |
| CN105683379A (en) | Delivery, engineering and optimization of systems, methods and compositions for targeting and modeling diseases and disorders of post mitotic cells | |
| CN110484549B (en) | Genome targeted modification method | |
| CN113337507B (en) | Construction method and application of deaf mouse model with Otof 1273(C > T) gene site-directed mutagenesis | |
| CN110249051A (en) | Enhance the method and composition that functional myelin generates | |
| CN110257435A (en) | A kind of construction method of PROM1-KO mouse model and its application | |
| CN110305896A (en) | A kind of construction method of zebra fish kidney progenitors label transgenosis system | |
| CN104611368A (en) | Carrier incapable of generating frameshift mutation after recombination as well as method and application for gene fixe-point knock-in in Xenopus laevis genome | |
| CN113088521A (en) | Construction method of Ahnak2 gene knockout animal model based on CRISPR/Cas9 technology | |
| Lu et al. | Base-edited cynomolgus monkeys mimic core symptoms of STXBP1 encephalopathy | |
| CN114480497B (en) | Construction and application method of ep400 gene knockout zebra fish heart failure model | |
| CN111705063B (en) | ASGR1 mutant gene and application thereof in preparation of mammal liver injury sensitive model | |
| CN113234756A (en) | Construction method of LAMA3 gene knockout animal model based on CRISPR/Cas9 technology | |
| Brakebusch | Generation and analysis of genetically modified mice |
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 |