CN115074386A - Preparation method and application of zebra fish hoxbb gene cluster deletion mutant - Google Patents

Preparation method and application of zebra fish hoxbb gene cluster deletion mutant Download PDF

Info

Publication number
CN115074386A
CN115074386A CN202210758285.XA CN202210758285A CN115074386A CN 115074386 A CN115074386 A CN 115074386A CN 202210758285 A CN202210758285 A CN 202210758285A CN 115074386 A CN115074386 A CN 115074386A
Authority
CN
China
Prior art keywords
hoxbb
gene cluster
hoxb8b
hoxb1b
zebra fish
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.)
Pending
Application number
CN202210758285.XA
Other languages
Chinese (zh)
Inventor
祖尧
王冰琦
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.)
Shanghai Ocean University
Original Assignee
Shanghai Ocean 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 Shanghai Ocean University filed Critical Shanghai Ocean University
Priority to CN202210758285.XA priority Critical patent/CN115074386A/en
Publication of CN115074386A publication Critical patent/CN115074386A/en
Pending legal-status Critical Current

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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0276—Knock-out vertebrates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • 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
    • C12N15/89—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
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/15—Humanized animals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; 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
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/40—Fish
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; 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
    • A01K2267/035—Animal model for multifactorial diseases
    • A01K2267/0375—Animal model for cardiovascular 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]
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
    • Y02A40/81—Aquaculture, e.g. of fish

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • Environmental Sciences (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Animal Behavior & Ethology (AREA)
  • Animal Husbandry (AREA)
  • Biodiversity & Conservation Biology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention discloses a preparation method and application of a zebra fish hoxbb gene cluster deletion mutant, which comprises the following steps: determining that knockout targets of the hoxbb gene cluster are respectively located on the 1 st exon of the hoxb1b gene and the 1 st exon of the hoxb8b gene to design gRNA sequences, performing PCR amplification by using a gRNA framework plasmid as a template, performing PCR product purification and in vitro transcription to obtain gRNAs, mixing the gRNAs of the hoxb1b and the hoxb8b with the Cas9mRNA, injecting the mixture into fertilized eggs of zebra fish, and culturing to obtain the stably inherited hoxbb gene cluster deletion mutant. The invention provides a method for knocking out a plurality of continuous genes on a genome together and obtaining a stable mutant, and meanwhile, the phenotype of the zebra fish hoxbb gene cluster deletion mutant lays a foundation for researching the biological function of the hoxbb gene cluster and diseases related to hoxbb gene cluster deletion.

Description

Preparation method and application of zebra fish hoxbb gene cluster deletion mutant
Technical Field
The invention belongs to the field of molecular biology, and particularly relates to a preparation method and application of a zebra fish hoxbb gene cluster deletion mutant.
Background
CRISPR/Cas is an adaptive immune defense formed during long-term evolution of bacteria and archaea, and can be used to fight invading viruses and foreign DNA. There are three types of CRISPR/Cas9 systems: type i, type ii and type iii, wherein the type ii CRISPR/Cas system requires only one Cas9 endonuclease to cleave the DNA double strand, i.e. the CRISPR/Cas9 system. As a novel genome editing technology, the CRISPR/Cas9 system has the advantages of simple experiment, easy operation, time saving and the like. Gene-directed engineering has been achieved in multiple species of mice, arabidopsis, drosophila, etc. using CRISPR/Cas9 technology. In the research field of zebra fish, the technology is also widely applied to precise and targeted gene editing, and becomes a high-efficiency and convenient gene editing tool.
The Hox gene is a large family of transcription factors containing homeoboxes, the full name of homeotic genes. They are arranged in clusters on chromosomes and are distributed on different chromosomes. The Hox gene family controls the construction of body morphology during early embryonic development. Mutation of the Hox gene often results in corresponding malformations in the corresponding parts of the body. The Hox gene was first found in Drosophila and contains only one gene cluster. Most vertebrates have undergone genome doubling during evolution, and the Hox gene has also undergone a doubling process during this process. Most of them, including the model animal zebrafish, undergo an additional genome doubling, eventually forming 7-8 gene clusters. The zebrafish contains 7 hox gene clusters, and 48 genes are total. For example, the Hoxb gene cluster in mice has two in zebrafish: the hoxba gene cluster and the hoxbb gene cluster.
It has been reported that the Hox gene plays a role not only in anteroposterior axis formation and somite development, but also in cardiac development of vertebrates. Among these, patients with heterozygous deletion of the HOXB gene cluster show abnormalities in multiple organs including atrial and ventricular septal defects. In recent years, some reports have been made about the Hoxb gene cluster, and it was discovered that Olga Medina-Martinez et al originally deleted Hoxb1-Hoxb 99 genes in mice: the heterozygote mice after the Hoxb1-Hoxb9 gene deletion do not have defect phenotypes, and the homozygote mutant mice have cardiovascular diseases such as edema and cervical hemorrhage. Subsequent papers report that the heart and aorta of Hoxb mutant mice are deficient in size and morphology, with subsequent phenotypes such as deficient sternal development. In mice, there have been some reports about a single Hoxb gene, but the mechanism of the study has not been clarified yet due to some limitations. How the regulation mechanism of the hoxbb gene cluster on the cardiac development is realized in the zebra fish is not clearly reported at present. The hoxb cluster in zebrafish consists of four hoxb genes (hoxb1b, hoxb5b, hoxb6b and hoxb8b) spanning approximately 25.5kb from 3 'to 5' of chromosome 12. To study the function of the hoxbb cluster during cardiac development and to mimic the del [17] (q21.3q23) disease associated with deletion of the HOXB cluster in humans (Amores a et al, 1998; Pearson, Lemons and McGinnis, 2005), we developed a large fragment deletion of the entire hoxbb cluster in zebrafish by using the CRISPR/Cas9 system, and further studied the pathogenesis of this disease model.
In addition, the large fragment knockout technology in the prior art is relatively difficult and low in efficiency. In 2010, the Jin-Soo Kim laboratory successfully realizes large fragment knockout on 293T cells of human by using zinc finger nuclease, and the knockout efficiency is between 2% and 5%. In 2013, the large fragment knockout is successfully realized in zebra fish by a spot A.Wolfe laboratory through a TALEN technology, and the knockout efficiency is different from 1% to 15%. Meanwhile, researches report that the CRISPR/Cas9 technology is used for deleting large fragments of the zebra fish, the size of the deleted fragments is in the range of 0.2 kb-120 kb, and the fragment deletion efficiency is in the range of 0-1/3. The method for efficiently deleting large fragments of the hoxbb gene and the application thereof are created by combining the CRISPR/Cas9 technology with the molecular biology technology, and the method is favorable for making up the blank of the gene cluster in the heart disease research technology.
Disclosure of Invention
The invention mainly aims to provide a preparation method of a zebra fish hoxbb gene cluster deletion mutant, which utilizes a zebra fish model organism to edit a hoxbb gene cluster through a CRISPR/Cas9 technology, realizes the targeted knockout of a target gene, obtains the zebra fish hoxbb gene cluster deletion mutant, and provides a mode method for jointly knocking out a plurality of continuous genes on a genome and obtaining a stable mutant.
The invention also aims to provide a zebra fish hoxbb gene cluster deletion mutant used as an animal model for researching the biological function of the hoxbb gene cluster and simulating del [17] (q21.3q23) diseases related to HOXB cluster deletion in human, and provides a theoretical basis for the exploration of a heart mechanism caused by the hoxbb cluster in human.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a preparation method of a zebra fish hoxbb gene cluster deletion mutant, which comprises the following steps:
s1, determining target positions at two ends of a hoxbb gene cluster, and designing target gRNA sequences before a hoxb8b start codon and after a hoxb1b stop codon respectively;
s2, designing and synthesizing an upstream primer T7-hoxb8b-sfd, an upstream primer T7-hoxb1b-sfd and a downstream primer tracrrev of two pairs of gRNAs;
s3, performing PCR amplification by using a gRNA framework plasmid as a template and using primers T7-hoxb8b-sfd, T7-hoxb1b-sfd and a downstream primer tracrrev;
s4, respectively carrying out in vitro transcription on the PCR products obtained in the step S3, and transforming to obtain hoxb8b-gRNA and hoxb1 b-gRNA;
s5, microinjecting the purified gRNA and Cas9mRNA into a zebra fish single-cell embryo, culturing for 24h, extracting DNA, respectively designing two pairs of detection primers on two sides of a hoxb8b target spot and two sides of a hoxb1b target spot, detecting by using a double-outer-side primer PCR method, and determining that a hoxbb gene cluster knocks out positive F 0 Culturing to adult fish;
s6, step S5 screening the heritable parents F 0 The stable genetic F1 is obtained by outcrossing and cultured until sexual maturity, at F 1 Obtained from intercrossed fish 1/4F 2 Homozygous mutant by pairing F 2 Sciaenops ocellatus tail detection and sequencing verification, at F 2 And generating to obtain stably inherited zebra fish hoxbb gene cluster deletion mutant.
Preferably, in step S1, the target gRNA sequence is
hoxb8b：GGGAGTTCTCGGAGGTAGAG(SEQ ID NO:1)；
hoxb1b：GGAATGTTGCATTACACACC(SEQ ID NO:2)。
Preferably, in step S2, the upstream leadThing F1(T7+Target site+
Figure BDA0003723334640000031
) That is, the sequence of the primer T7-hoxb8b-sfd is
Figure BDA0003723334640000032
The sequence of primer T7-hoxb1b-sfd is
Figure BDA0003723334640000033
The sequence of a downstream primer R1(trans reverse), i.e., a gRNA reverse primer, is
AAAAAAAGCACCGACTCGGTGCCAC(SEQ ID NO:5)。
Preferably, in step S4, the sequence of hoxb8b gRNA is
TAATACGACTCACTATAGGGAGTTCTCGGAGGTAGAGGTTTTAGAGCTAGAAATAGCGGACAGATTCATGTCCTGGACGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT(SEQ ID NO:6)。
The sequence of the hoxb1b gRNA is
TAATACGACTCACTATAGGAATGTTGCATTACACACCGTTTTAGAGCTAGAAATAGCGGACAGATTCATGTCCTGGACGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT(SEQ ID NO:7)。
Preferably, in step S5, the detection primers have the sequences respectively
The upstream primer hoxb8 b-F: CGCTCGTACTGTAGCCTACTT (SEQ ID NO: 8);
the downstream primer hoxb8 b-R: GACGGTCCCCGAGCAATAAA (SEQ ID NO: 9);
the upstream primer hoxb1 b-F: TATTCGGACCTGTGACCTGAT (SEQ ID NO: 10);
the downstream primer hoxb1 b-R: AGTATCATTATGCTGTGCCGGA (SEQ ID NO: 11).
Preferably, in step S5, in the method for detecting double-outer-primer PCR, PCR amplification fragments are 449bp and 344bp, respectively, the total length of the hoxbb gene cluster is 25.5kb, when the primers are amplified by using the hoxb8b F/hoxb1b R, a band of about 400bp is amplified in a successfully deleted genome, a target band cannot be amplified in a successfully deleted genome, and a single-side primer of hoxb8b F/R is used as a control.
Preferably, the zebra fish hoxbb -/- The mutant causes early-stage pericardial edema and heart cyclization angle abnormality of the zebra fish and homozygous death phenomenon, the mutant has obvious ventricular abnormality at 5dpf, and the elongation and the thinning of the ventricular length are shown through observation, photographing and data statistics.
The invention also provides application of the zebra fish hoxbb gene cluster deletion mutant as an animal model in researching biological functions of the hoxbb gene cluster and diseases related to hoxbb gene cluster deletion, and the zebra fish hoxbb gene cluster deletion mutant is obtained by any one of the preparation methods of the zebra fish hoxbb gene cluster deletion mutant.
Patients with a loss of heterozygote from the HOXB cluster develop multiple organ abnormalities, including abnormalities in the heart, lungs, brain, face, hands and feet, consistent with some clinical observations, mice with a loss of homozygote from the HOXB cluster have been found to have abnormalities in the cervical and thoracic spine, and defects in sternal morphology. Soshenikova et al subsequently demonstrated that deletion of the HoxA or HoxB cluster in mice did not severely affect cardiac morphogenesis, but that the HoxA/B complex mutants failed to develop into proper cardiac circulation, resulting in embryonic death, and that studies indicated that the HoxB cluster plays a role in normal cardiac genesis, but they did not identify specific genes critical to this cluster. Furthermore, these studies have not extended further into the underlying pathways downstream of HoxB, nor have they provided a mechanistic link to explain abnormal morphology. Existing studies do not elucidate which cardiac progenitors are affected by HoxB deficiency during cardiac development. In mice, there are experimental techniques to perform fragmentation knockouts, whereas in zebrafish there are fewer large fragment knockouts using CRISPR/Cas9 technology. In addition, the size of the fragment to be knocked out may affect the efficiency of the knock out. According to the existing results, the zebra fish hoxbb gene cluster causes abnormal early heart phenotype, hoxbb -/- The mutant zebra fish all have homozygous lethality, and about 11dpf is dead. In addition, hoxbb -/- The mutant zebrafish has a serious phenotype, and obvious phenotypes such as pericardial cavity enlargement, abnormal cardiac cyclization, atrioventricular blood backflow and the like are observed at 5 dpf. The invention realizes the specific knockout of the hoxbb gene cluster in the zebra fish by using the CRISPR/Cas9 technology, and the obtained zebra fish hoxbb gene cluster deletion mutant can be used for the research on the influence of the zebra fish hoxb gene cluster on the heart function and the gene regulation network thereof, and provides accurate thought and reference for the treatment and prevention of related heart diseases.
Compared with the prior art, the invention has the following beneficial effects:
(1) the invention utilizes zebra fish as a model organism, carries out gene editing on a genome through a CRISPR/Cas9 technology, firstly designs a series of knockout targets before a HOXB8b start codon and after a HOXB1b stop codon respectively, selects a target sequence with higher knockout efficiency from the knockout targets, carries out large-fragment deletion on a zebra fish HOXB gene cluster to obtain a HOXB gene cluster mutant, and provides a theoretical basis for researching the function of the HOXB cluster in the heart development process, simulating diseases (Park et JP al.1992, Rooryck et al.2008) of HOXB cluster deletion del [17] (q21.3q23) in a human, and researching the heart mechanism caused by the HOXB cluster in the human, clinical treatment (including new drug trial) and the like.
(2) The hoxbb gene cluster of the zebra fish is knocked out without accidentally damaging other genes, is used as a model animal for knocking out the hoxbb gene cluster, can eliminate human factor intervention once being knocked out, and has great significance for researching the function of the hoxbb gene cluster. Meanwhile, compared with the traditional single-site gene knockout, the large fragment knockout technology has higher relative difficulty and lower efficiency, the invention successfully knocks out the 25.5kb of the zebra fish hoxbb gene cluster by using the CRISPR/Cas9 technology and adopting a double-target knockout method, provides valuable reference and thought for fragmentation knockout, and is used for the subsequent deep research of the functional mechanism of the hoxbb gene cluster.
Drawings
FIG. 1 shows that the zebrafish hoxbb gene cluster is successfully deleted by using CRISPR/Cas9 technology, and the targeting sequences are respectively positioned on the front side of a hoxb8b start codon and on the rear side of a hoxb1b stop codon.
FIG. 2 shows zebrafish F 0 、F 1 、F 2 The generation double outer primer genotype detection result is shown in the figure, wherein the former is a double outer primer PCR amplification strip, and the latter control group is a single side primer PCR amplification strip.
FIG. 3 is the result of a peak plot for PCR product sequencing showing successful deletion of the hoxbb gene cluster.
FIG. 4 shows hoxbb -/- Compared with the control group, the zebrafish embryo has an abnormal phenotype of pericardial cavity enlargement at 5dpf, ". mark the position of the abnormal phenotype, and the scale is 1000 um.
FIG. 5 shows the abnormal heart development of zebrafish embryos after the deletion of the hoxbb gene cluster; a-a': wild Type (WT) and hoxbb under 20 Xlight -/- A phenotype of zebrafish 5 dpf; b: the proportion of hoxbb mutants consistent with mendelian inheritance was confirmed earlier by mendelian law; c. hoxb1b, hoxb5b, hoxb6b, hoxb8b and hoxb from the WT genome -/- A cPCR product at 5 dpf; d-d': from WT and hoxbb -/- Confocal microscopy of isolated hearts, dashed lines and arrows indicate abnormal morphology of mutant hearts and defects of cardiac trabeculae, scale 25 μm; e: survival curve result chart, result shows hoxbb -/- Zebrafish are homozygous lethal, approximately 11dpf (day post fertilization) and all die.
FIG. 6 shows the elongation result of abnormal cardiac ventricular development of zebra fish after the hoxbb gene cluster is deleted; a: wild type zebrafish and hoxbb at 5dpf -/- Carrying out in situ hybridization on the zebra fish vmhc gene; the expression of vmhc showed zebrafish hoxbb at 5dpf -/- Abnormal, elongated and thinned ventricles; b: WT and hoxbb -/- Histogram statistics of ventricular length, hoxbb -/- Ventricles longer than WT, P<0.01，n>3, the scale is 0.1 mm; *: p<0.05，**：P<0.01，***：P<0.001。
FIG. 7 shows hoxbb +/- Tg (flk: mCherry) analysis results, showing control and hoxbb -/- The brain blood vessel and the main blood vessel of the homozygous mutant have no obvious difference.
Detailed Description
The present invention will be described in detail with reference to examples. The following examples will aid those skilled in the art in further understanding the present invention, but are not intended to limit the invention in any manner. It should be noted that it would be apparent to those skilled in the art that several modifications and improvements can be made without departing from the inventive concept. All falling within the scope of the present invention.
In vitro synthetic Cas9mRNA plasmids used for the experiments were from the literature Liu D, Wang Z, Xiao a, Zhang Y, Li W, Zu Y, Yao S, Lin S, Zhang b. efficient gene targeting in zebrafish media by zebrafish-code-optimized Cas9 and evaluation of off-targeting effect.j gene genomics.2014 Jan 20; 41, (1) 43-6; the in vitro synthetic gRNA backbone plasmids used were from the literature: chang N, Sun C, Gao L, Zhu D, Xu X, Zhu X, Xiong JW, Xi JJ. genome editing with RNA-guided Cas9 nucleic in zebrafish embryo, Cell Res,2013,23(4): 465-.
1. Design of gRNA and two-sided primers
1) Principle of target site design
The first principle is that the gRNA knockout site can destroy important gene sites, so that the target gene loses function. Meanwhile, the method is strictly designed according to the design principle of gRNA, and the detection primer is simple and clear when the knockout result is detected.
2) Target site design
Firstly, Cas9 target spots are used for predicting website http:// zifit.attners.org/ZiFiT/CSquare 9 Nuclear.aspx, target spots are searched for a target gene hoxbb cluster, some target spots are selected and recorded for later use, subsequently, efficient target spots are selected by counting gene knockout efficiency, and a series of target spot sequences are as follows:
hoxb1bTarget(5’-3’):
GGAATGTTGCATTACACACC；
GGTGATTAAAAGAGTAACCC；
hoxb8bTarget(5’-3’):
GGGAGTTCTCGGAGGTAGAG；
GGCAGACCATCTGTGCTGTA；
GGAAACATCTTGGGCCAGGA；
the sequence of the gRNA of the determined target spot is
hoxb8b：GGGAGTTCTCGGAGGTAGAG(SEQ ID NO:1)；
hoxb1b：GGAATGTTGCATTACACACC(SEQ ID NO:2)。
3) And (3) target site specificity detection:
the target targets are subjected to specificity detection through an NCBI website, zebra fish is taken as an example, sequences of the target targets are compared with the zebra fish genome, and if sequences identical with the target targets appear at other positions of the genome, redesign is needed.
4) Primer design
By using a double-outer-side primer PCR detection method, two pairs of primers are respectively designed on both sides of a hoxb8b target spot and on both sides of a hoxb1b target spot, and PCR amplified fragments are 449bp and 344bp respectively. The total length of the hoxbb gene cluster is 25.5kb, when the primers hoxb8bF/hoxb1b R are used for amplification, a band of about 400bp can be amplified in a genome which is successfully deleted, and a target band cannot be amplified in a genome which is not successfully deleted. Simultaneously, a hoxb8b F/R single-sided primer is used as a control, wherein the sequences of the detection primers are respectively
The upstream primer hoxb8 b-F: CGCTCGTACTGTAGCCTACTT (SEQ ID NO: 8);
the downstream primer hoxb8 b-R: GACGGTCCCCGAGCAATAAA (SEQ ID NO: 9);
the upstream primer hoxb1 b-F: TATTCGGACCTGTGACCTGAT (SEQ ID NO: 10);
the downstream primer hoxb1 b-R: AGTATCATTATGCTGTGCCGGA (SEQ ID NO: 11).
5) The transcription kit is special for a T7 promoter, and the sequences of gRNA upstream primers are as follows:
T7 promoter Target site scaffold：5’-TAATACGACTCACTATANNNNNNNNNNNNNNN NNNNNGTTTTAGAGCTAGAAATAGC；
the downstream primer is gRNA reverse primer with the sequence 5'-AAAAAAAGCACCGACTCGGTGCCAC-3' (SEQ ID NO:5)
2. Synthetic gRNA
A. Shake the fungus
B. Extracting plasmid 14h
C. High fidelity enzymatic PCR
D. Reaction system:
Figure BDA0003723334640000071
2％Agarose 120V，30min。
purification of PCR product (DNA Clean & Concentrator-5) method and 3-4 tubes mixing one tube purification
In vitro transcription of MAXiScriptT7 (see in particular MAXiScript) TM Kit Instruction Manual，Ambion)
Reaction system:
Figure BDA0003723334640000072
heating in 37 deg.C water bath for 80min, adding 1 μ L TURBO DNase, and heating in 37 deg.C water bath for 15 min.
LiCl/ethanol precipitation
1) Add 2.5. mu.L of 4M LiCl (5M plus 2. mu.L, 7.5M plus 1.5. mu.L), 100. mu.L of 100% ethanol to the above system;
2) incubating the mixed solution at-80 ℃ for more than 1 h;
3) placing into a centrifuge, centrifuging at 12000rpm at 4 deg.C for 15 min;
4) discarding the supernatant, rinsing twice with 70% ethanol pre-cooled on ice;
5) standing at room temperature for about 5min, volatilizing residual ethanol, and dissolving the precipitated RNA with RNase-free water;
6) the gRNA concentration was measured using Nanodrop and RNA quality was detected using electrophoresis.
3. Synthesis of Cas9mRNA
For details, see Protocol of
Figure BDA0003723334640000083
T7 Ultra Kit, briefly as follows:
A. carrying out amplification culture on the stored Cas9 plasmid bacterial liquid, and shaking the bacteria for about 16 h;
B. extracting plasmid, measuring concentration, and electrophoretically detecting plasmid quality
C.XbaI digestion of linearized plasmid
Enzyme digestion system:
Figure BDA0003723334640000081
the reaction was carried out at 37 ℃ for 30 min.
D.1% Agarose gel electrophoresis 110v, 30min to determine whether the enzyme digestion is complete
E. Purification of
Purification procedures refer to DNAClean & Concentrator-5, ZYMO RESEARCH kit.
MmessagemMACHINE T7 Ultra kit in vitro transcription
The reaction conditions refer to the specification, and the whole experimental process is carried out in an Rnase-free environment.
Reaction system:
Figure BDA0003723334640000082
and oscillating, uniformly mixing and centrifuging, carrying out water bath at 37 ℃ for 2-3 h, then adding 1 mu L DNase, uniformly mixing, carrying out water bath at 37 ℃ for 15min, and removing the DNA template.
F. + PolyA tail
1) Reaction system:
Figure BDA0003723334640000091
2) reserving 2.5 mu L of mixed solution as a reference;
3) mixing the above mixed solution with +4 μ L E-PAP, and gently mixing;
4) heating in water bath at 37 deg.C for 45 min;
5) after 1% gel electrophoresis at 150v for 20min, tailing (1. mu.L gel loading Buffer + 1. mu.L RNA) G.Cas9 mRNA purification was performed by aqueous LiCl precipitation.
The experimental steps are as follows:
Precipitation of RNA from Solution at a Concentration≥400ng/μL
1) adding 50 mu L LiCl into the Cas9mRNA added with the A tail, and uniformly mixing;
2) precipitation at-80 ℃ for >1 h;
3) centrifuging at 14000rpm for 15min at 4 ℃;
4) discarding the supernatant, washing the precipitate with 70% ethanol pre-cooled on ice for 2 times;
5) with an appropriate amount of RNase-free H 2 And dissolving the O.
H. Quality detection
And (4) measuring the product concentration by using the Nanodrop, and detecting whether the RNA band size is correct and the RNA quality is qualified or not by electrophoresis.
Cas9mRNA and gRNA microinjection and knockout efficiency detection
Cas9mRNA and gRNA are prepared into mixed liquid according to a certain proportion, under the general condition, the Cas9mRNA is 300-600 pg, the gRNA is 80-100 pg, and the mixed liquid is injected into a zebra fish one-cell stage animal pole. The same pair of fish eggs is injected with some uninjected eggs to be used as a control in the knockout assay.
A. Extracting genome by an alkaline lysis method. 3 groups of 48hpf (48 hours post fertilization) phenotypically normal embryos were injected, 5 eggs +50 μ L per group
50mM NaOH, 95 ℃ for 10min, and oscillating; again 95 ℃ for 10min, then 5. mu.L of 1M Tris.HCl (pH. apprxeq.8.0) was added to neutralize the NaOH, 12000rpm/min, and centrifuged for 6 min.
PCR system:
Figure BDA0003723334640000101
after 34cycles, 2% Agarose 120V was run for 30 min.
C, electrophoresis of PCR products:
mainly utilizes a double-outer-side primer PCR detection method to respectively design two pairs of primers on both sides of a hoxb8b target spot and both sides of a hoxb1b target spot, and PCR amplified fragments are 449bp and 344bp respectively. The total length of the hoxbb gene cluster is 25.5kb, when the primers of the hoxb8b F/hoxb1b R are used for amplification, a band of about 400bp can be amplified in a genome which is successfully deleted, and a target band cannot be amplified in a genome which is not successfully deleted. A single-sided primer hoxb8b F/R was also used as a control.
D. Visible light imaging and fluorescence imaging of F1 zebrafish embryos
Zebra fish embryos were placed in MS222 at a concentration of 150mg/L for 60s and then anesthetized. When placed in a low temperature environment, the heart stops beating. They were placed in side and abdomen views, respectively, of 1% 2-hydroxyethyl agarose (Sigma Aldrich, st. louis, MO, USA, a9414) to complete confocal microscopy imaging (zeiss LSM710, germany).
5 zebra fish whole embryo in situ hybridization experiment
The procedure for in situ hybridization using digoxigenin-labeled probe was performed according to standard procedures.
5.1 Probe Synthesis
(1) Probe primers are designed in a region spanning introns of a zebra fish gene coding region (CDS), and a T7 promoter is added in front of a downstream primer. And (3) carrying out in-vitro transcription after purifying a PCR product by taking cDNA (complementary deoxyribonucleic acid) of the wild zebra fish as a template for PCR.
(2) Synthesis of vmhc Probe Using in vitro transcription kit
5.2 Zebra fish embryo Collection
If more than 48hpf embryos are collected, PTU (1-phenyl-2-thiourea) is added at 24hpf to inhibit the growth of melanin in zebrafish. The embryos were collected in 1.5mL centrifuge tubes, the culture water in the centrifuge tubes was aspirated as much as possible, then 1mL of 4% paraformaldehyde was added, and the shaker was shaken overnight at 4 ℃. The following day, washing was performed 2 times with 1 XPBST for 5min each, and then dehydration was performed with 30%, 50%, 70%, 100% ethanol in this order. And storing at-20 deg.C for a long period.
5.3 in situ hybridization experiments all over the three day period
Day 1:
1) rehydrating, taking out fixed embryo from refrigerator at-20 deg.C,
70% ethanol 1 × 5min
50% ethanol 1 × 5min
30% ethanol 1 × 5min
1×PBST 2×5min
2) Proteinase K treatment
Preparing protease K into 10 mug/mL by using 1 xPBST, treating 5dpf zebra fish embryo in the patent by using the protease K for 30min, cleaning the zebra fish embryo by using 1 xPBST for 5min after the treatment is finished, fixing the zebra fish embryo by using 4% paraformaldehyde for 20min, and finally cleaning the zebra fish embryo by using 1 xPBST for 5 times, wherein each time is 5 min.
3) Hybridization of
Firstly, pre-hybridization is carried out, embryos are placed in hybridization solution without probes, the hybridization solution is placed in a 65 ℃ hybridization furnace for 2-3 h, then vmhc probes are added, and hybridization is carried out in the 65 ℃ hybridization furnace for about 14 h.
Day 2:
firstly, washing a sample, eluting to 1 xPBST, and then sealing the sample by using a sealing solution containing 1% of lamb serum and 2% of BSA, wherein the sealing time is generally 2-3 h. After blocking, the anti-digoxin antibody was added at a ratio of 1:3000 and shaken overnight at 4 ℃.
Day 3:
1) taking out the sample from a shaking table at 4 ℃, and washing the sample for 6 × 15min by using 1 × PBST;
2) AP solution 1 × 5 min;
3) preparing a color developing solution;
4) finally, performing light-resistant color development by using a color development liquid prepared by NBT-BCIP;
5) after the color development was completed, the reaction was terminated with 1 XPBST, and then the embryos were photographed in 3% methylcellulose. After single embryo identification, the photographed samples were measured for ventricular length using ImageJ (NIH, USA) and mapped and analyzed for significant differences using GraphPad 6.0.
6 results of the experiment
6.1 successful construction of hoxbb Gene Cluster mutant
The genotype detection results of the double lateral primers of the zebra fish F0, F1 and F2 generations show that the hoxbb gene cluster is successfully knocked out. Sequencing peak plots showed that the genomic sequence between the hoxb8b gene target and the hoxb1b gene target was deleted, demonstrating successful knock-out (figures 1, 2, 3).
6.2 photographing of phenotype observation of mutant zebrafish of hoxbb F2
(1) Heterozygous mutations of the hoxbb gene cluster were intercrossed (intercrosss), collected after oviposition and cultured for early embryo development, and significant cardiac malformations such as pericardial enlargement and abnormal cardiac cyclization were observed at 5dpf (fig. 4).
(2) In order to determine the phenotype of the hoxbb gene cluster mutant, 5dpf of the hoxbb gene cluster deletion mutant and a wild type heart are taken for observation, a common optical microscope and a fluorescence microscope are used for photographing, the heart morphology of the mutant is abnormal, trabecular defects are generated, and then genotype identification is carried out (figure 4 and figure 5).
(3)hoxbb -/- The mutant zebrafish appeared homozygous lethal, meeting mendelian inheritance rule of about 11dpf (days post fertilization) and all died (fig. 5).
(4) At 5dpf, wild type zebrafish and hoxbb -/- The zebra fish vmhc gene is hybridized in situ, and the result shows that 5dpf zebra fish hoxbb is obtained -/- The ventricles were abnormal, showing elongation and thinning of the ventricular morphology compared to the control group (fig. 6).
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Sequence listing
<110> Shanghai ocean university
Preparation method and application of <120> zebra fish hoxbb gene cluster deletion mutant
<141> 2022-06-30
<160> 11
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 1
gggagttctc ggaggtagag 20
<210> 2
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 2
ggaatgttgc attacacacc 20
<210> 3
<211> 57
<212> DNA
<213> Artificial sequence ()
<400> 3
taatacgact cactataggg agttctcgga ggtagaggtt ttagagctag aaatagc 57
<210> 4
<211> 57
<212> DNA
<213> Artificial sequence ()
<400> 4
taatacgact cactatagga atgttgcatt acacaccgtt ttagagctag aaatagc 57
<210> 5
<211> 25
<212> DNA
<213> Artificial sequence ()
<400> 5
aaaaaaagca ccgactcggt gccac 25
<210> 6
<211> 120
<212> DNA
<213> Artificial sequence ()
<400> 6
taatacgact cactataggg agttctcgga ggtagaggtt ttagagctag aaatagcgga 60
cagattcatg tcctggacgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttt 120
<210> 7
<211> 120
<212> DNA
<213> Artificial sequence ()
<400> 7
taatacgact cactatagga atgttgcatt acacaccgtt ttagagctag aaatagcgga 60
cagattcatg tcctggacgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttt 120
<210> 8
<211> 21
<212> DNA
<213> Artificial sequence ()
<400> 8
cgctcgtact gtagcctact t 21
<210> 9
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 9
gacggtcccc gagcaataaa 20
<210> 10
<211> 21
<212> DNA
<213> Artificial sequence ()
<400> 10
tattcggacc tgtgacctga t 21
<210> 11
<211> 22
<212> DNA
<213> Artificial sequence ()
<400> 11
agtatcatta tgctgtgccg ga 22

Claims (8)

1. The preparation method of the zebra fish hoxbb gene cluster deletion mutant is characterized by comprising the following steps of:
s1, determining target positions at two ends of a hoxbb gene cluster, and designing target gRNA sequences before a hoxb8b start codon and after a hoxb1b stop codon respectively;
s2, designing and synthesizing an upstream primer T7-hoxb8b-sfd, an upstream primer T7-hoxb1b-sfd and a downstream primer tracrrev of two pairs of gRNAs;
s3, performing PCR amplification by using a gRNA framework plasmid as a template and using primers T7-hoxb8b-sfd, T7-hoxb1b-sfd and a downstream primer tracrrev;
s4, respectively carrying out in vitro transcription on the PCR products obtained in the step S3, and transforming to obtain hoxb8b-gRNA and hoxb1 b-gRNA;
s5, microinjecting the purified gRNA and Cas9mRNA into a zebra fish single-cell embryo, extracting DNA after culturing for 24h, respectively designing two pairs of detection primers on two sides of a hoxb8b target spot and two sides of a hoxb1b target spot, detecting by using a double-outer-side primer PCR method, and determining that a hoxb gene cluster knocks out positive F 0 Culturing to adult fish;
s6, step S5 screening the heritable parents F 0 The stable genetic F1 is obtained by outcrossing and cultured until sexual maturity, at F 1 Obtained from intercrossed fish 1/4F 2 Homozygous mutant by pairing F 2 Sciaenops ocellatus tail detection and sequencing verification, at F 2 And generating to obtain stably inherited zebra fish hoxbb gene cluster deletion mutant.
2. The method for preparing the zebrafish hoxbb gene cluster deletion mutant according to claim 1, wherein in step S1, the sequence of the target gRNA of hoxb8b is shown as SEQ ID NO. 1, and the sequence of the target gRNA of hoxb1b is shown as SEQ ID NO. 2.
3. The method for preparing the zebra fish hoxbb gene cluster deletion mutant as claimed in claim 1, wherein in step S2, the sequences of the upstream primer T7-hoxb8b-sfd, T7-hoxb1b-sfd and the downstream primer gRNA reverse primer are shown as SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 5, respectively.
4. The method for preparing the zebrafish hoxbb gene cluster deletion mutant according to claim 1, wherein in step S4, the sequence of hoxb8b gRNA is shown as SEQ ID NO. 6, and the sequence of hoxb1b gRNA is shown as SEQ ID NO. 7.
5. The method for preparing the zebrafish hoxbb gene cluster deletion mutant as recited in claim 1, wherein in step S5, the detection primers comprise: the upstream primer hoxb8b-F shown as SEQ ID NO. 8, the downstream primer hoxb8b-R shown as SEQ ID NO. 9, the upstream primer hoxb1b-F shown as SEQ ID NO. 10 and the downstream primer hoxb1b-R shown as SEQ ID NO. 11.
6. The method for preparing deletion mutants of the zebra fish hoxbb gene cluster as claimed in claim 1, wherein in step S5, the PCR amplified fragments of the double outer primers are 449bp and 344bp respectively, the total length of the hoxbb gene cluster is 25.5kb, the primers are amplified by using the hoxb8b F/hoxb1b R pair, and the single-sided primer of the hoxb8b F/R pair is used as a control, so that the band of 300-450bp amplified in the successful genome is deleted, and the band of interest cannot be amplified in the genome which is not deleted successfully.
7. The method for preparing the deletion mutant of the zebrafish hoxbb gene cluster according to any one of claims 1 to 6, characterized in that the zebrafish hoxbb is subjected to deletion -/- The mutant causes early pericardial cavity edema and abnormal heart cyclization angle of zebra fish and has homozygous lethality, and the mutant has ventricular abnormality and elongated and thinned ventricular length 5 days after fertilization.
8. Use of a deletion mutant of the zebrafish hoxbb gene cluster obtained by the method for preparing the deletion mutant of the zebrafish hoxbb gene cluster according to any one of claims 1 to 7 as an animal model for studying the biological functions of the hoxbb gene cluster and diseases associated with the deletion of the hoxbb gene cluster.
CN202210758285.XA 2022-06-30 2022-06-30 Preparation method and application of zebra fish hoxbb gene cluster deletion mutant Pending CN115074386A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202210758285.XA CN115074386A (en) 2022-06-30 2022-06-30 Preparation method and application of zebra fish hoxbb gene cluster deletion mutant

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210758285.XA CN115074386A (en) 2022-06-30 2022-06-30 Preparation method and application of zebra fish hoxbb gene cluster deletion mutant

Publications (1)

Publication Number Publication Date
CN115074386A true CN115074386A (en) 2022-09-20

Family

ID=83254878

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210758285.XA Pending CN115074386A (en) 2022-06-30 2022-06-30 Preparation method and application of zebra fish hoxbb gene cluster deletion mutant

Country Status (1)

Country Link
CN (1) CN115074386A (en)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109628454A (en) * 2019-01-30 2019-04-16 上海海洋大学 The construction method of zebra fish glycogen storage disease gys1 and gys2 gene mutation body

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109628454A (en) * 2019-01-30 2019-04-16 上海海洋大学 The construction method of zebra fish glycogen storage disease gys1 and gys2 gene mutation body

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
KAZUYA YAMADA等: "An atlas of seven zebrafish hox cluster mutants provides insights into sub/neofunctionalization of vertebrate Hox clusters", 《DEVELOPMENT》, vol. 148, no. 11 *
张绪帅: "CRISPR/Cas9系统介导的七鳃鳗和斑马鱼基因组编辑方法的建立与优化", 《中国优秀硕士学位论文全文数据库 基础科学辑》, pages 13 - 19 *
李佳: "CRISPR/Cas9技术在斑马鱼基因修饰中的应用", 《生命科学》, vol. 27, no. 1, 31 January 2015 (2015-01-31), pages 29 *

Similar Documents

Publication Publication Date Title
CN108660161B (en) Method for preparing chimeric gene-free knockout animal based on CRISPR/Cas9 technology
CN109628454B (en) Construction method of zebra fish glycogen storage disease gys1 and gys2 gene mutant
US20200375157A1 (en) Construction method for animal model for retinitis pigmentosa diseases and application
CN110904103A (en) Zebra fish mutant with GRNa gene knockout function and preparation method thereof
CN110484549B (en) Genome targeted modification method
CN110923265A (en) Construction method of mouse model for conditionally overexpressing HPV E7 gene at H11 site
CN110541002A (en) method for constructing zebra fish asap1b gene knockout mutant by using CRISPR/Cas9 technology
CN110643636A (en) Megalobrama amblycephala MSTNa &amp; b gene knockout method and application
CN113088521A (en) Construction method of Ahnak2 gene knockout animal model based on CRISPR/Cas9 technology
CN113584079A (en) Establishment of zebra fish heart specific marker strain applied to calcium ion imaging
CN113234756A (en) Construction method of LAMA3 gene knockout animal model based on CRISPR/Cas9 technology
CN114480497B (en) Construction and application method of ep400 gene knockout zebra fish heart failure model
CN111500589A (en) Pig SOX10 mutant gene causing inner ear hypoplasia and application thereof
CN115074386A (en) Preparation method and application of zebra fish hoxbb gene cluster deletion mutant
CN115261360A (en) Method for constructing gata6 gene knockout zebra fish model
CN110592122B (en) Zebra fish naalad2 gene promoter and application thereof
CN113106102A (en) Method for constructing POU4F3 gene deletion type mutant zebra fish animal model
CN109694885B (en) Method for preparing PI3K gamma whole-body knockout mode mouse based on CRISPR/Cas9 technology, application thereof and kit
CN113373150A (en) sgRNA of targeting dat gene and application thereof
CN113774128B (en) Gja8 application of gene mutation site in preparation of product for diagnosing cataract disease
CN113388639B (en) Method for breeding zebra fish vmhcEGFP-KI strain by gene knock-in
CN114934069A (en) Construction method and application of double-mutant zebra fish line with kctd10 and tbx5a genes
CN114868707B (en) Zebra fish model for metabolic encephalopathy and arrhythmia diseases and application thereof
CN114908098A (en) Preparation method and application of zebra fish hoxb1a gene deletion mutant
CN114934075A (en) Screening method and application of major gene in zebra fish heart development related gene cluster deletion mutant

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