CN116602268B - Application of gene knockout mutant zebra fish in preparation of animal model for reducing pigment - Google Patents
Application of gene knockout mutant zebra fish in preparation of animal model for reducing pigment Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
- A01K67/027—New breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
- A01K67/0276—Knockout animals
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- 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
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- 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
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/07—Animals genetically altered by homologous recombination
- A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
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- 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
- A01K2227/40—Fish
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- 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
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- 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
Abstract
The invention discloses an application of a gene knockout mutant zebra fish in preparing a pigment reduction animal model, which comprises the mutant zebra fish, wherein the mutant zebra fish is 11 bases of mutation from 69 th to 103 th in 1 st exon of a krt5 gene. The heterozygote and homozygote types of the constructed krt5 mutant zebra fish are mainly as follows: the number of melanocytes decreases. The gene knockout mutant zebra fish constructed by the invention shows a disease phenotype of reduced pigment, and can be used as an animal model for researching and screening pigment-reduced diseases such as vitiligo, nevus, white spot, hypopigmentation, tinea versicolor, hypopigmented mushroom-like granulation and the like. The successfully established animal model for reducing pigment has the characteristics of inheritability, short period, low cost, high flux and the like, and can provide an effective way for early screening, diagnosis research and drug screening of the pigment-reducing diseases.
Description
Technical Field
The invention relates to the technical field of biology and new medicines, in particular to application of a gene knockout mutant zebra fish in preparing a pigment reduction animal model.
Background
Pigment diseases are very common in Fitzpatrick III-V skin type people, and the disease types mainly comprise chloasma, vitiligo, leukoplakia and the like, so that the pigment diseases have obvious negative effects on beauty. Because of the limited treatment means of the pigmentary diseases, factors such as the refractory nature, high recurrence and long-term treatment requirement of the comprehensive diseases, the pigmentary diseases seriously influence the social life and psychological health of patients. Therefore, the method for researching the pathogenesis of the pigment diseases and constructing a reasonable research model has important scientific and social values for treating the pigment diseases.
Pigmentation of the skin is determined by both melanogenesis and the process of transfer of melanin from melanocytes to surrounding keratinocytes. Melanin metabolism is a complex, delicate process that consists essentially of the migration and differentiation of melanocytes, the development of melanosomes and the process of melanogenesis, the transfer of melanosomes to keratinocytes, and the redistribution and degradation of melanin in keratinocytes. Melanocytes are located only in the basal layer of the skin, and the 36 keratinocytes surrounding it constitute one epidermal melanocyte unit, but the mechanism of how melanosomes are transferred from melanocytes to keratinocytes is not clear. Keratin 5 (keratin 5, krt 5) belongs to the class II keratin family, and is expressed only in basal layer keratinocytes, and plays an important role in maintaining epidermal homeostasis and immune homeostasis. It is presumed that keratin 5 has a role in cell adhesion, uptake of melanosomes by keratinocytes, and it has been clinically found that mutation of keratin 5 gene can cause occurrence of pigment abnormality, and therefore, keratin 5 can serve as a cut-in point for studying regulatory action between keratinocytes and melanocytes.
The existing pigment deletion models comprise spontaneous systems and induction systems, and the spontaneous models comprise Smyth line chickens, sinclair pigs, grey allele horses, various mouse models and the like. The induction model includes a pigment deletion model constructed by chemically inducing melanocyte stress, immunizing mice with melanocyte antigens and an immune adjuvant to activate endogenous immune cells, or genetically modifying mice to increase the frequency of melanocyte-reactive T cells, transgenic mice expressing a single T Cell Receptor (TCR) specific for a particular melanocyte antigen in their T cells. Spontaneous models can be used as a new means of studying pigment diseases, but have the disadvantage that maintenance costs can be high and that intensive research is difficult to carry out due to the limited available reagents compatible with these species. The induction model is developed on mice, has the advantages of low propagation and maintenance cost, is suitable for researching mechanisms and therapeutic interventions for driving disease progression, but the melanocytes of the mice are limited in hair follicles, and the melanocytes of the hair follicles only synthesize eumelanin, so that the method is more suitable for being used as a subject for researching hair pigment.
Therefore, we propose the use of knockout mutant zebra fish in the preparation of animal models with reduced pigment to solve the above problems.
Disclosure of Invention
In order to overcome the above-mentioned drawbacks of the prior art, embodiments of the present invention provide an application of a knockout mutant zebra fish in preparing a reduced pigment animal model, so as to solve the problems set forth in the above-mentioned background art.
In order to achieve the above purpose, the present invention provides the following technical solutions: application of gene knockout mutant zebra fish in preparing animal models with reduced pigment comprises mutant zebra fish, wherein the mutant zebra fish is 11 bases of mutation from 69 th to 103 th in 1 st exon of the krt5 gene.
In a preferred embodiment, the reduced pigment animal model is a mutant zebra fish of the krt5 gene, and the mutant zebra fish exhibits symptoms of:
a reduction in melanocyte number;
a decrease in melanocyte area;
melanocyte synapses are inhibited and cell morphology becomes rounded.
In a preferred embodiment, the reduced pigment animal model is caused by abnormal expression of the krt5 gene.
In a preferred embodiment, the hypopigmentation symptoms are relieved after treatment by the use of a medicament that promotes the type of melanin pigmentation.
In a preferred embodiment, the melanosis type drug comprises malaytea scurfpea fruit, UVB, JAK inhibitors and the like.
In a preferred embodiment, the krt5 knockout mutant zebra fish is prepared by:
step S1: designing and constructing sgRNA of a krt5 gene target site;
step S2: co-injecting active sgRNA and Cas9 mRNA into a 1-cell-phase zebra fish embryo animal pole, randomly selecting 5-20 embryos, and confirming that a foundation embryo cell carries alleles of target gene mutation;
step S3: culturing the embryo after injection to obtain the mutant of the krt5 gene knockout zebra fish.
In a preferred embodiment, in step S1, the CRISPR Cas9 technique is used to design the sgRNA of the target site of the krt5 gene at the first 100 bases of the first exon of the krt5 gene, and then the sgRNA is synthesized.
In a preferred embodiment, in step S3, the injected embryo is cultured to obtain a mutant of the krt5 knockout zebra fish F 0 ；
Knockout of the krt5 Gene into zebra fish mutant F 0 Laterally crossing the wild zebra fish to obtain a stably inherited krt5 gene knockout zebra fish mutant F 1 Replacing heterozygotes;
mutant F of krt5 gene knockout zebra fish 1 Selfing the heterozygote to obtain a krt5 gene knockout zebra fish mutant F 2 Heterozygote krt5 +/- And homozygote krt5 -/- 。
In a preferred embodiment, the mutant zebra fish is applied to a melanin study model.
The invention has the technical effects and advantages that:
1. at present, a research model of melanin is mainly focused on melanocytes directly, keratinocytes adjacent to the melanocytes play an important role in melanin deposition, krt5 is a cell molecule only expressed in the keratinocytes, and a research model of pigment with the krt5 gene knockdown is constructed, so that a good model is provided for observing interaction of the keratinocytes and the melanocytes in the melanin deposition.
2. Zebra fish is an increasingly attractive and convenient vertebrate-model organism that has a high degree of genetic and organ system similarity to humans; meanwhile, zebra fish has a skin structure similar to that of human beings, and the formation process of melanin is also highly similar to that of human beings; in addition, the bodies of the young zebra fish are transparent, and the melanocytes on the body surfaces of the young zebra fish can be simply observed and evaluated without using complex experimental procedures. Thus, zebra fish can be an ideal model for studying melanogenesis and migration.
3. The constructed pigment-deficient zebra fish model with the krt5 gene mutation can be used as a visual animal disease model for researching related pigment reduction diseases and keratinocyte and melanocyte regulation effects, and provides basic scientific support for treating diseases such as vitiligo, leukoplakia, pigment reduction diseases and the like and discovering medicines.
Drawings
FIG. 1 is a sequencing map of a wild type zebra fish target region;
FIG. 2 is a chart of krt5 gene targeting F 0 Sequencing a target area of the substituted zebra fish;
FIG. 3 is a chart of krt5 gene targeting F 1 Sequencing a target area of the substituted zebra fish;
FIG. 4 is a chart showing the phenotype imaging of the mutant zebra fish 24 hpf of the krt5 gene;
FIG. 5 is a chart showing the phenotype imaging of the mutant zebra fish 48 hpf of the krt5 gene;
FIG. 6 is a graph of a krt5 gene mutant zebra fish 72 hpf phenotype;
FIG. 7 is a 96 hpf phenotype image of a krt5 gene mutant zebra fish;
FIG. 8 is a chart showing the statistics of the number of melanocytes of mutant zebra fish 48 and 72 hpf of the krt5 gene;
FIG. 9 shows the measurement of the melanin content of the mutant zebra fish 96 hpf of the krt5 gene;
FIG. 10 shows the detection of the activity of the mutant zebra fish 96 hpf tyrosinase of the krt5 gene;
FIG. 11 shows the detection of the expression level of the gene related to the melanin production/migration of the mutant zebra fish of the krt5 gene.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
1-11, the use of a knockout mutant zebra fish in the preparation of an animal model for reduced pigment, comprising a mutant zebra fish that is 11 bases mutant from position 69 to position 103 in exon 1 of the krt5 gene, specifically:
before mutation: 5 'CCTCGTAGACGACAGAAGCCATGTCTACTTCCTTCA';
after mutation: 5'ACGACAGAAGCCATAAGCCATGTCTACTTCCCTTC 3'.
The pigment reduction animal model is a mutant zebra fish of a krt5 gene, and the mutant zebra fish has the following symptoms:
a reduction in melanocyte number;
a decrease in melanocyte area;
melanocyte synapses are inhibited and cell morphology becomes rounded.
The hypopigmentation animal model is caused by abnormal expression of the krt5 gene, and the hypopigmentation symptoms are relieved after treatment by using a melanosis-promoting drug, including malaytea scurfpea fruit, UVB, JAK inhibitors, etc.
The krt5 gene knockout mutant zebra fish is prepared by the following method:
step S1: designing and constructing sgRNA of a krt5 gene target site; in step S1, the CRISPR Cas9 technology is applied to design the sgRNA of the target site of the krt5 gene at the first 100 bases of the first exon of the krt5 gene, and then the sgRNA is synthesized.
Step S2: co-injecting active sgRNA and Cas9 mRNA into a 1-cell-phase zebra fish embryo animal pole, randomly selecting 5-20 embryos, and confirming that a foundation embryo cell carries alleles of target gene mutation;
step S3: culturing the embryo after injection to obtain the mutant of the krt5 gene knockout zebra fish. In the step S3, the embryo after injection is cultured to obtain the mutant F of the krt5 gene knockout zebra fish 0 ；
Knockout of the krt5 Gene into zebra fish mutant F 0 Laterally crossing the wild zebra fish to obtain a stably inherited krt5 gene knockout zebra fish mutant F 1 Replacing heterozygotes;
and (5) selfing the wild zebra fish to obtain wild zebra fish embryo WT. Mutant F of krt5 gene knockout zebra fish 1 Selfing the heterozygote to obtain a krt5 gene knockout zebra fish mutant F 2 Heterozygote krt5 +/- And homozygote krt5 -/- 。
Mutant zebra fish were used in the melanin study model.
Example 1
Cultivation of zebra fish
The breeding of wild zebra fish AB strain zebra fish into fish is provided by Nanjing Yishu pear peanut technology Co., ltd.m., a zebra fish circulation breeding system is used for culturing at 28 ℃ as shown in figure 1, the system automatically controls the conductivity of circulating water to be 500-550 mu S/cm, the pH value is 7.0-7.4, the photoperiod is 14 h light/10 h darkness, the breeding density is less than or equal to 5 tail/L, and the zebra eggs which are freshly hatched for 2 times per day are fed.
Propagation of zebra fish
Propagation of wild type zebra fish AB strain zebra fish embryos occurs in a natural pair mating manner. The zebra fish parent fish is selected according to the proportion of female fish to male fish (2:2) at night before embryo collection is required, the female fish and the male fish are placed in a spawning jar separately by a baffle plate, and a jar cover is covered.
Drawing out the partition board after the photoperiod begins in the next morning, and naturally mating male and female fish for spawning; 30 After min, embryo is collected, placed into embryo culture solution containing methylene blue, dead eggs are removed, and proper embryo is selected according to the development stage of embryo, placed into intelligent illumination incubator for culture, the temperature of the incubator is controlled at 28 ℃, and the photoperiod is set to be 14 h illumination/10 h darkness every day.
Embryos were observed 6 hours after fertilization, 12 hpf and 24 hpf, and unfertilized or incompletely fertilized dead embryos were picked out in time, with embryo culture medium changed once daily in the morning.
The young zebra fish need not be fed within 7 days after fertilization because the embryo can take nutrients from its own yolk sac.
After the experiment was completed, zebra fish at each development stage were subjected to overexposure treatment with anesthetic, thereby anesthetizing the zebra fish.
Construction of Gene knockout zebra fish
Using CRISPR/Cas9 technology, the sgrnas of the krt5 gene target site were designed and synthesized at the first 100 bases of the first exon of the krt5 gene: 5'ACGACAGAAGCCATAAGCCA 3'.
Zebra fish embryos with good fertilization state are collected, sgRNA and Cas9 mRNA are co-injected into a 1-cell-phase zebra fish embryo animal pole, and the animal pole is placed in an intelligent illumination incubator for cultivation at 28.5 ℃ and controlled to be 14 h illumination/10 h darkness every day.
After the embryo develops to a proper period, randomly selecting 5-20 embryos, extracting genome by using a zebra fish direct PCR kit, sequencing to confirm that the Foundator embryo cells carry alleles of target gene mutation as shown in the accompanying drawings 1 and 2, and culturing the injected embryos to obtain a krt5 gene knockout zebra fish mutant F 0 。
Knockout of the krt5 Gene into zebra fish mutant F 0 Crossing with wild zebra fish to obtain F 1 Culturing to sexual maturity, cutting tail to extract DNA, and sequencing to identify genotype, see figure 3.
Gene knockout zebra fish phenotype verification
The F is carried out 1 Selfing the substituted zebra fish to obtain F 2 The generation of homozygous krt5 zebra fish. Selfing with wild AB strain zebra fish, AB and F respectively 2 Crossing of the progeny krt5 homozygote, F 2 Selfing the homozygous mutant of the generation krt5 to obtain wild WT and heterozygote krt5 +/- And homozygote krt5 -/- Gene knockout zebra fish.
Microscopic observation of zebra fish embryo is carried out on 24 hpf shown in figure 4, 48 hpf shown in figure 5, 72 hpf shown in figure 6, 96 hpf shown in figure 7, and pigment change during embryo development is recorded by bright field imaging.
The results showed that the heterozygote krt5 was compared to wild zebra fish +/- And homozygote krt5 -/- The pigment cell number of zebra fish is remarkably reduced, as shown in figure 8, wherein the pigment reduction phenomenon of homozygote zebra fish is more obvious, and the main phenotypes are pigment cell number reduction, pigment cell synapse disappearance, morphological rounding and the like.
The obvious reduction of the melanocyte of the mutant zebra fish indicates that the successfully established animal model for reducing pigment has the characteristics of inheritability, short period, low cost, high flux and the like, and can provide an effective path for early screening, diagnosis research and drug screening of the pigment-reducing diseases.
Example 2
Functional verification of gene knockout zebra fish
The skin color of a person is mainly determined by melanin, and color spots, color sinks and the like are closely related to the content of the melanin. Tyrosinase is a key enzyme for melanin formation, and the content of melanin is related to the expression level of tyrosinase genes in the body.
Collecting young zebra fish developed to 96 hpf, homogenizing on ice, cracking, centrifuging at 4 ℃ and 12000 rpm, collecting precipitate, adding 1% KOH, heating at 100 ℃ to dissolve 1 h, and measuring melanin content; the supernatant was used for tyrosinase activity detection. The results show that the melanin content in the zebra fish body with the gene knocked out is obviously reduced as shown in figure 9 and tyrosinase activity is obviously reduced as shown in figure 10.
Taking young zebra fish developed to 96 hpf, placing 30 tails of each group of zebra fish into QSP centrifuge tubes for removing ribozymes of 1.5 and mL, washing with PBS for 2 times, sucking up the liquid, adding 1 mL precooled TRIzol total RNA extraction reagent, and homogenizing on ice for 2-3 min;
standing at room temperature for 10 min, adding 200 μl chloroform into the lysate, mixing, standing at room temperature for 15 min. Centrifuge at 12000 rpm for 15 min at 4℃and aspirate about 400. Mu.L of colorless upper aqueous phase and transfer to fresh EP tube.
400 mu L of isopropanol is added into the supernatant, and after being fully and evenly mixed up and down, the mixture is left to stand for 10 min at room temperature. Centrifuge at 12000 rpm for 10 min at 4℃and discard supernatant, slowly add 1 mL of 75% ethanol and wash the pellet.
Centrifuging at 12000 rpm at 4deg.C for 5 min, discarding supernatant, precipitating to obtain RNA, and air drying.
And adding 20 mu L DEPC water to dissolve the sample. RNA concentration was determined and 1 μg RNA was aspirated for reverse transcription into cDNA with reference to kit instructions.
And (3) determining the expression of the tyr gene and the expression of the reference gene beta-actin by fluorescent quantitative PCR.
Real-time quantitative fluorescence detection (QPCR) with a reaction system of 10. Mu.L, specifically as follows:
the plate was centrifuged at 1000 rpm at 4℃for 1 min, and the plate was put into a real-time fluorescent quantitative PCR apparatus to perform a reaction under the following conditions.
The quantitative detection of the expression of the genes is carried out by carrying out a real-time fluorescence quantitative PCR technology (RT-qPCR) on young 96 hpf zebra fish, and the result shows that the expression level of tyr genes in mutant zebra fish is obviously reduced, as shown in figure 11.
Finally: the foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and principles of the invention are intended to be included within the scope of the invention.
Claims (5)
1. The application of the gene knockout mutant zebra fish in preparing the animal model with reduced pigment is characterized in that: the mutant zebra fish is the zebra fish with 11 bases of mutation from 69 th to 103 th positions in 1 st exon of the krt5 gene, and specifically comprises the following steps:
before mutation: 5 'CCTCGTAGACGACAGAAGCCATGTCTACTTCCTTCA';
after mutation: 5 'ACGACAGAAGCCATAAGCCATGTCTACTTCCCTTC';
the reduced pigment animal model is caused by abnormal expression of the krt5 gene;
symptoms of hypopigmentation are relieved after treatment by the use of a drug that promotes the type of pigmentation;
the melanin-promoting drugs include fructus Psoraleae, UVB, and JAK inhibitor;
the pigment reduction animal model is a krt5 gene knockout mutant zebra fish, and the mutant zebra fish has the following symptoms:
a reduction in melanocyte number;
a decrease in melanocyte area;
melanocyte synapses are inhibited and cell morphology becomes rounded.
2. The use of a knockout mutant zebra fish according to claim 1 for the preparation of an animal model with reduced pigment, characterized in that: the krt5 gene knockout mutant zebra fish is prepared by the following method:
step S1: designing and constructing sgRNA of a krt5 gene target site;
step S2: co-injecting active sgRNA and Cas9 mRNA into a 1-cell-phase zebra fish embryo animal pole, randomly selecting 5-20 embryos, and confirming that a foundation embryo cell carries alleles of target gene mutation;
step S3: culturing the embryo after injection to obtain the mutant of the krt5 gene knockout zebra fish.
3. The use of a knockout mutant zebra fish according to claim 2 for the preparation of an animal model with reduced pigment, characterized in that: in step S1, the CRISPR Cas9 technology is applied to design the sgRNA of the target site of the krt5 gene at the first 100 bases of the first exon of the krt5 gene, and then the sgRNA is synthesized.
4. The use of a knockout mutant zebra fish according to claim 2 for the preparation of an animal model with reduced pigment, characterized in that: in the step S3, the injected embryo is cultured to obtain a krt5 gene knockout zebra fish mutant F0;
laterally crossing the krt5 gene knockout zebra fish mutant F0 with wild zebra fish to obtain a stably inherited krt5 gene knockout zebra fish mutant F1 generation heterozygote;
selfing the mutant F1 generation heterozygote of the mutant of the krt5 knockout zebra fish to obtain the mutant F2 generation heterozygote and homozygote of the mutant of the krt5 knockout zebra fish.
5. The use of a knockout mutant zebra fish according to claim 1 for the preparation of an animal model with reduced pigment, characterized in that: the mutant zebra fish is applied to a melanin study model.
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| CN110904103A (en) * | 2019-10-18 | 2020-03-24 | 陕西师范大学 | Zebra fish mutant with GRNa gene knockout function and preparation method thereof |
| CN115261360A (en) * | 2022-08-02 | 2022-11-01 | 温州医科大学附属第二医院（温州医科大学附属育英儿童医院） | Method for constructing gata6 gene knockout zebra fish model |
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