CN116602268B - Application of gene knockout mutant zebrafish in preparing animal models of pigmentation reduction - Google Patents

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

Application of gene knockout mutant zebrafish in preparing animal models of pigmentation reduction

Technical field

The present invention relates to the technical fields of biology and new medicine. More specifically, the present invention relates to the application of gene knockout mutant zebrafish in the preparation of pigment reduction animal models.

Background technique

Pigmentary diseases are very common in people with Fitzpatrick III-V skin types. The main types of diseases include chloasma, vitiligo, and vitiligo, which have a significant negative impact on beauty. Due to the limited treatment options for pigmented diseases, combined with factors such as the disease's refractory nature, high recurrence, and need for long-term treatment, it has seriously affected the social life and mental health of patients. Therefore, studying the pathogenesis of pigmented diseases and constructing reasonable research models have important scientific and social value for the treatment of pigmented diseases.

Skin pigmentation is determined by a combination of melanogenesis and the transfer of melanin from melanocytes to surrounding keratinocytes. Melanin metabolism is a complex and delicate process, which mainly includes the migration and differentiation of melanocytes, the development of melanosomes and the process of melanogenesis, the transfer of melanosomes to keratinocytes, and the transfer of melanin to keratinocytes. Four parts: redistribution and degradation. Melanocytes are only located in the basal layer of the skin and form an epidermal melanin unit with 36 surrounding keratinocytes. However, the mechanism of how melanosomes are transferred from melanocytes to keratinocytes is not clear. Keratin 5 (krt5) belongs to the type II keratin family and is only expressed in basal layer keratinocytes. It plays an important role in maintaining epidermal homeostasis and immune homeostasis. It is speculated that keratin 5 plays a role in cell adhesion and keratinocyte uptake of melanosomes. It has been clinically found that mutations in the keratin 5 gene can lead to pigment abnormalities. Therefore, keratin 5 can be used as a tool to study keratinogenesis. An entry point into the regulatory interaction between cells and melanocytes.

Existing pigment loss models include spontaneous systems and induced systems. Spontaneous models include Smyth chickens, Sinclair pigs, gray allele horses and various mouse models. Induction models include chemically inducing melanocyte stress, immunizing mice with melanocyte antigens and immune adjuvants to activate endogenous immune cells, or genetically modifying mice to increase the production of melanocyte-reactive T cells. Frequently, transgenic mice express a single T-cell receptor (TCR) specific for a specific melanocyte antigen in their T cells to create a pigment-deficient model. Spontaneous models may serve as a new means of studying pigmented diseases, but have the disadvantage that maintenance costs can be high and in-depth studies are more difficult to perform due to the limited availability of reagents compatible with these species. Induced models are mostly carried out in mice, which have the advantages of low reproduction and maintenance costs and are suitable for studying the mechanisms driving disease progression and therapeutic intervention. However, mouse melanocytes are limited to hair follicles, and their hair follicle melanocytes only synthesize true melanin. It is more suitable as an object of hair pigment research.

Therefore, we proposed the application of gene knockout mutant zebrafish in preparing animal models of reduced pigmentation to address the above issues.

Contents of the invention

In order to overcome the above-mentioned shortcomings of the prior art, embodiments of the present invention provide the application of gene knockout mutant zebrafish in preparing animal models with reduced pigmentation, so as to solve the problems raised in the above-mentioned background technology.

In order to achieve the above object, the present invention provides the following technical solution: the application of gene knockout mutant zebrafish in the preparation of pigment reduction animal models, including mutant zebrafish, and the mutant zebrafish is targeted at the first exon of the krt5 gene Zebrafish with 11 base mutations from 69 to 103.

In a preferred embodiment, the animal model of reduced pigmentation is a krt5 gene mutant zebrafish, and the symptoms of the mutant zebrafish are:

Decreased number of melanocytes;

Melanocyte area decreases;

Melanocyte synapses are inhibited and cell morphology becomes rounded.

In a preferred embodiment, the pigment reduction animal model is caused by abnormal expression of krt5 gene.

In a preferred embodiment, the symptoms of hypopigmentation are relieved by treatment with a melanosis-promoting type of drug.

In a preferred embodiment, the melanosis-type drugs include psoralen, UVB, JAK inhibitors, etc.

In a preferred embodiment, the krt5 gene knockout mutant zebrafish is prepared by the following method:

Step S1: Design and construct sgRNA for the krt5 gene target site;

Step S2: Co-inject active sgRNA and Cas9 mRNA into the animal pole of 1-cell stage zebrafish embryos, randomly select 5 to 20 embryos, and confirm that the Founder embryo cells carry the allele of the target gene mutation;

Step S3: Culture the injected embryos to obtain krt5 gene knockout zebrafish mutants.

In a preferred embodiment, in step S1, CRISPR Cas9 technology is used to design the sgRNA of the krt5 gene target site in 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 embryos are cultured to obtain krt5 gene knockout zebrafish mutant F ₀ ;

The krt5 gene knockout zebrafish mutant F ₀ was side-crossed with wild-type zebrafish to obtain the stably inherited krt5 gene knockout zebrafish mutant F ₁ generation heterozygotes;

The krt5 knockout zebrafish mutant F _1st generation heterozygotes were selfed, and the krt5 knockout zebrafish mutant F _2nd generation heterozygotes krt5 ^+/- and homozygous krt5 ^-/- were obtained.

In a preferred embodiment, the mutant zebrafish is used in a melanin research model.

Technical effects and advantages of the present invention:

1. Currently, most melanin research models focus directly on melanocytes. Keratinocytes adjacent to melanocytes also play an important role in melanosis. krt5 is a cellular molecule expressed only in keratinocytes. Construct the krt5 gene The knockout pigment research model provides a good model for observing the interaction between keratinocytes and melanocytes in melanin.

2. Zebrafish is an increasingly attractive and convenient vertebrate model organism. It has a high degree of genetic and organ system similarity with humans. At the same time, zebrafish has similar skin structure and melanin formation process to humans. It is also highly similar to humans; in addition, the body of zebrafish larvae is transparent, and melanocytes on the surface of its body can be simply observed and evaluated without using complicated experimental procedures. Therefore, zebrafish serves as an ideal model to study melanogenesis and migration.

3. The pigment-deficient zebrafish model of krt5 gene mutation constructed by the present invention can be used as a visual animal disease model to study related pigment reduction diseases and the regulatory effects of keratinocytes and melanocytes. It will provide vitiligo, vitiligo, hypochromia, etc. Provide basic scientific research support for disease treatment and drug discovery.

Description of drawings

Figure 1 shows the sequencing map of the wild-type zebrafish target region;

Figure 2 shows the sequencing map of the target region of krt5 gene targeting F ₀ generation zebrafish;

Figure 3 shows the sequencing map of the target region of krt5 gene targeting F ₁ generation zebrafish;

Figure 4 shows the phenotype imaging of krt5 gene mutant zebrafish at 24 hpf;

Figure 5 shows the phenotype imaging of krt5 gene mutant zebrafish at 48 hpf;

Figure 6 shows the phenotype imaging of krt5 gene mutant zebrafish at 72 hpf;

Figure 7 shows the phenotypic imaging of krt5 gene mutant zebrafish at 96 hpf;

Figure 8 shows the statistics of the number of melanocytes in krt5 gene mutant zebrafish at 48 and 72 hpf;

Figure 9 shows the measurement of melanin content in krt5 gene mutant zebrafish at 96 hpf;

Figure 10 shows the detection of tyrosinase activity in krt5 gene mutant zebrafish at 96 hpf;

Figure 11 shows the detection of expression levels of genes related to melanin production/migration in krt5 gene mutant zebrafish.

Detailed ways

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention.

Referring to Figure 1-11, the application of gene knockout mutant zebrafish in preparing animal models of pigmentation reduction, including mutant zebrafish, which targets positions 69 to 103 in exon 1 of the krt5 gene Zebrafish with 11 base mutations, specifically:

Before mutation: 5' CCTCGTAGACGACAGAAGCCATGTCTACTTCCTTCA 3';

After mutation: 5’ACGACAGAAGCCATAAGCCATGTCTACTTCCCTTC 3’.

The animal model of pigmentation loss is krt5 gene mutant zebrafish, and the symptoms of this mutant zebrafish are:

Decreased number of melanocytes;

Melanocyte area decreases;

Melanocyte synapses are inhibited and cell morphology becomes rounded.

The animal model of hypopigmentation is caused by abnormal expression of the krt5 gene. The symptoms of hypopigmentation are relieved after treatment with melanin-promoting drugs, including psoralen, UVB, JAK inhibitors, etc.

The krt5 gene knockout mutant zebrafish was prepared by the following method:

Step S1: Design and construct the sgRNA for the krt5 gene target site; in step S1, use CRISPR Cas9 technology to design the sgRNA for the krt5 gene target site in the first 100 bases of the first exon of the krt5 gene, and then synthesize the sgRNA.

Step S2: Co-inject active sgRNA and Cas9 mRNA into the animal pole of 1-cell stage zebrafish embryos, randomly select 5 to 20 embryos, and confirm that the Founder embryo cells carry the allele of the target gene mutation;

Step S3: Culture the injected embryos to obtain krt5 gene knockout zebrafish mutants. In step S3, the injected embryos are cultured to obtain krt5 gene knockout zebrafish mutant F ₀ ;

The krt5 gene knockout zebrafish mutant F ₀ was side-crossed with wild-type zebrafish to obtain the stably inherited krt5 gene knockout zebrafish mutant F ₁ generation heterozygotes;

Wild-type zebrafish were selfed to obtain wild-type zebrafish embryos WT. The krt5 knockout zebrafish mutant F _1st generation heterozygotes were selfed, and the krt5 knockout zebrafish mutant F _2nd generation heterozygotes krt5 ^+/- and homozygous krt5 ^-/- were obtained.

Mutant zebrafish as a model for melanin research.

Example 1

Zebrafish culture

Wild-type zebrafish AB strain zebrafish breeding adults were provided by Nanjing Yishu Lihua Biotechnology Co., Ltd., and were cultured at 28°C using a zebrafish circulating breeding system as shown in Figure 1. The system automatically controlled the conductivity of the circulating water to 500-550 μS/cm, pH 7.0-7.4, the photoperiod is 14 h light/10 h dark, the breeding density is ≤5 tail/L, and freshly hatched artemia eggs are fed twice a day.

Zebrafish reproduction

The reproduction of wild-type zebrafish AB strain zebrafish embryos is carried out by natural pair mating. The night before embryos need to be collected, select zebrafish broodstock according to the ratio of female:male (2:2), use partitions to separate the males and females in the spawning tank, and cover the tank.

The next morning, after starting the photoperiod, the partitions are opened, and the male and female fish naturally mate and lay eggs; 30 minutes later, the embryos are collected, placed in embryo culture medium containing methylene blue, dead eggs are removed, and appropriate ones are selected according to the development stage of the embryos. The embryos were cultured in a smart light incubator, the temperature of the incubator was controlled to 28°C, and the photoperiod was set to 14 hours of light/10 hours of darkness every day.

Observe the embryos at 6 hours, 12 hpf and 24 hpf after fertilization, pick out unfertilized or incompletely fertilized dead embryos in time, and replace the embryo culture medium every morning.

Because the embryo can obtain nutrients from its own yolk sac, larval zebrafish do not need to be fed for 7 days after fertilization.

After the experiment was completed, zebrafish at various developmental stages were overexposed to anesthetics, and the zebrafish were anesthetized and killed.

Construction of gene knockout zebrafish

Using CRISPR/Cas9 technology, the sgRNA of the krt5 gene target site was designed and synthesized in the first 100 bases of the first exon of the krt5 gene: 5’ACGACAGAAGCCATAAGCCA 3’.

Collect zebrafish embryos with good fertilization status, co-inject sgRNA and Cas9 mRNA into the animal pole of 1-cell stage zebrafish embryos, place them in a smart light incubator, and culture them at 28.5°C, with 14 hours of light/10 hours of darkness every day.

When the embryos develop to the appropriate stage, 5 to 20 embryos are randomly selected, the genome is extracted using the zebrafish direct PCR kit, and the alleles of the Founder embryo cells carrying the target gene mutation are confirmed by sequencing (see Figure 1 and Figure 2), and will be injected The embryos were cultured to obtain krt5 gene knockout zebrafish mutant F ₀ .

The krt5 gene knockout zebrafish mutant F ₀ was side-crossed with wild-type zebrafish to obtain F ₁ , cultured to sexual maturity, tail clipped to extract DNA, and sequenced to identify the genotype, see Figure 3.

Phenotypic verification of gene knockout zebrafish

The above F ₁ generation zebrafish were selfed to obtain F ₂ generation homozygous krt5 zebrafish. Wild-type AB strain zebrafish was used to self-cross, AB was crossed with F _2- generation krt5 homozygotes, and F _2- generation krt5 homozygotes were self-crossed to obtain wild-type WT, heterozygous krt5 ^+/- and homozygous krt5 ^-/- gene knockouts. Except for zebrafish.

At 24 hpf of embryonic development, see Figure 4, at 48 hpf, see Figure 5, at 72 hpf, see Figure 6, and at 96 hpf, see Figure 7. Microscopic observations were made on zebrafish embryos, and bright-field imaging recorded pigments during embryonic development. Variety.

The results showed that compared with wild-type zebrafish, the number of pigment cells in heterozygous krt5 ^+/- and homozygous krt5 ^-/- zebrafish was significantly reduced. See Figure 8, in which the pigment cells in homozygous zebrafish were reduced. More obviously, the main phenotypes are reduction in the number of pigment cells, disappearance of pigment cell synapses, and rounded shape.

The significant reduction of melanocytes in the mutant zebrafish indicates that the animal model of pigmentation reduction successfully established by the present invention has the characteristics of heritability, short cycle, low cost and high throughput, and can be used for early screening, diagnostic research and research on pigmentation reduction diseases. Drug screening provides an effective approach.

Example 2

Functional verification of gene knockout zebrafish

Human skin color is mainly determined by melanin, and spots and pigmentation are closely related to the content of melanin. Tyrosinase is the key enzyme for the formation of melanin. In addition, the content of melanin is also related to the expression of tyrosinase gene in the body.

We collected zebrafish juveniles that developed to 96 hpf, homogenized and lysed on ice, centrifuged at 4°C and 12,000 rpm, added 1% KOH to the precipitate, heated at 100°C for 1 hour to dissolve, and used it for the determination of melanin content; take the Clear for tyrosinase activity detection. The results showed that the melanin content in the gene knockout zebrafish was significantly reduced, as shown in Figure 9, and the tyrosinase activity was significantly reduced, as shown in Figure 10.

Take zebrafish juveniles that have developed to 96 hpf. Collect 30 zebrafish from each group into QSP centrifuge tubes containing 1.5 mL of ribonuclease. Wash twice with PBS, absorb the liquid, and add 1 mL of pre-cooled TRIzol total RNA extraction reagent. , homogenize on ice for 2-3 min;

Let stand at room temperature for 10 minutes, add 200 μL chloroform to the lysis solution, mix by inverting several times, and let stand at room temperature for 15 minutes. Centrifuge at 12,000 rpm for 15 minutes at 4°C, and transfer approximately 400 μL of the colorless upper aqueous phase into a new EP tube.

Add 400 μL isopropyl alcohol to the supernatant, mix thoroughly by inverting it upside down, and let it stand at room temperature for 10 minutes. Centrifuge at 12000 rpm for 10 min at 4°C, discard the supernatant, slowly add 1 mL of 75% ethanol, and wash the precipitate.

Centrifuge at 12,000 rpm for 5 minutes at 4°C, discard the supernatant, and precipitate RNA, and allow to dry slightly.

Add 20 µL DEPC water to dissolve the sample. Determine the RNA concentration and refer to the kit instructions, pipette 1 µg of RNA and reverse-transcribe it into cDNA.

Fluorescence quantitative PCR was used to measure the expression of tyr gene and the gene expression of internal reference gene β-actin.

Real-time quantitative fluorescence detection (QPCR), the reaction system is 10 μL, the details are as follows:

Centrifuge at 1000 rpm for 1 min at 4°C, place the plate into a real-time fluorescence quantitative PCR instrument, and perform the reaction according to the following conditions.

The expression of the above genes was quantitatively detected by using real-time fluorescence quantitative PCR (RT-qPCR) on 96 hpf zebrafish juveniles. The results showed that the expression level of the tyr gene in the mutant zebrafish was significantly reduced, see Figure 11.

Finally: The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included in the present invention. within the scope of protection.

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.