KR-20260062256-A - CPT1B Knock-out Transgenic Zebrafish Model and Method for Producing Thereof

Abstract

The present invention relates to a CPT1B gene-knockout transgenic zebrafish model and its use. By injecting a CPT1B gene splicing blocking morpholino into zebrafish, it was analyzed that CPT1B gene expression was knocked down and pigment formation was inhibited. By injecting a ribonucleoprotein (RNP) composed of sgRNA targeting the CPT1B gene and Cas9 protein, it was analyzed that the CPT1B gene was knocked out and pigment formation was inhibited. By confirming that the CPT1B gene is involved in the development of melanocytes and black spots, a melanocyte-deficient zebrafish model with the CPT1B gene knocked out was constructed.

Inventors

• 이윤성
• 김만석
• 권순효
• 남의정

Assignees

• 경희대학교 산학협력단

Dates

Publication Date

20260507

Application Date

20241028

Claims (11)

1. A melanocyte-deficient zebrafish ( Danio rerio ) model with the CPT1B (Carnitine Palmitoyltransferase 1B) gene knocked out.
2. In paragraph 1, The above CPT1B gene is a model comprising the nucleotide sequence represented by SEQ ID NO. 1.
3. In paragraph 1, The above zebrafish model is a model in which the occurrence of lentigo is suppressed.
4. 1) A step of manufacturing a CPT1B gene knock-out construct; 2) a step of injecting the construct of step 1) into a zebrafish fertilized egg; and 3) A step of selecting embryos in which the CPT1B gene is knocked out; a method for preparing a melanocyte-deficient zebrafish ( Danio rerio ) model comprising.
5. In paragraph 4, A method of manufacturing in which the knockout construct of step 1) above comprises a ribonucleoprotein (RNP) composed of guide RNA and Cas9 protein.
6. In paragraph 5, A method for manufacturing in which the guide RNA of step 1) above is one or more selected from the group consisting of RNAs comprising nucleotide sequences represented by SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, and SEQ ID NO. 5.
7. In paragraph 4, A method for manufacturing in which the knockout construct of step 1) above comprises guide RNA and Cas9 protein.
8. A zebrafish ( Danio rerio ) model of skin depigmentation disease with the CPT1B (Carnitine Palmitoyltransferase 1B) gene knocked out.
9. In paragraph 8, The above-mentioned skin discoloration disorder is a model in which vitiligo or leukotrichia.
10. 1) A step of manufacturing a CPT1B gene knock-out construct; 2) a step of injecting the construct of step 1) into a zebrafish fertilized egg; and 3) A step of selecting embryos in which the CPT1B gene is knocked out; a method for preparing a zebrafish ( Danioli rerio ) model with skin depigmentation disease.
11. 1) A step of treating the test substance to the zebrafish model of skin depigmentation disease of claim 8; 2) a step of measuring the degree of pigment production in the zebrafish model of skin depigmentation disease of step 1) above; and 3) A step of selecting a test substance in which the degree of pigment production measured in step 2) above has increased compared to a control group that has not been treated with the test substance; comprising a screening method for a treatment for skin depigmentation disease.

Description

CPT1B Knock-out Transgenic Zebrafish Model and Method for Producing Thereof The present invention relates to a CPT1B gene-knockout transgenic zebrafish model and its uses. Meanwhile, the zebrafish (Danio rerio) is a three-year tropical freshwater fish with 25 pairs of chromosomes. Although it diverged from a common human ancestor 300 million years ago, its genes have been preserved, possessing almost all of the genes found in humans, making it the most widely used fish animal model for genetics and disease model research. Its genomic information is about 80% similar to that of humans, allowing for access to various genetic mutation experiments. Furthermore, it develops very quickly during the early developmental stages, and its transparent embryos make real-time observation easy. Morpholinos are high-molecular-weight oligomers that inhibit gene expression by specifically binding to RNA transcription sites or splicing sites, thereby suppressing normal translation. Meanwhile, genome editing is a technology that allows for the free modification of an organism's genetic information. Through advancements in the life sciences and the development of genome sequencing technology, we have gained a broad understanding of various types of genetic information. For instance, while we already possess a sufficient understanding of genes for the reproduction of plants and animals, disease and growth, genetic mutations causing various human genetic diseases, and biofuel production, further technological progress is essential to directly utilize this knowledge to improve living organisms and treat human diseases. Genome editing technology can dramatically expand the scope of its application by altering the genetic information of humans, animals, plants, and microorganisms. Gene scissors, which are molecular tools designed to precisely cut desired genetic information, play a pivotal role in genome editing technology. Much like next-generation sequencing technology, which advanced the field of gene sequencing to a new level, gene scissors are becoming a core technology that expands the speed and scope of genetic information utilization and creates new industrial sectors. Meanwhile, CRISPANT is a model that uses a gene editing system to directly induce mutations in the DNA of target genes to suppress their function. For efficient targeting, the Cas9 protein is typically injected into zebrafish along with four guide RNAs to induce mutations in the target genes. The inventors analyzed that CPT1B gene expression was knocked down and pigment formation was inhibited by injecting a CPT1B gene splicing blocking morpholino (Morpholino Oligomer) into zebrafish, and that CPT1B gene expression was knocked out and pigment formation was inhibited by injecting a ribonucleoprotein (RNP) composed of sgRNA targeting the CPT1B gene and Cas9 protein, thereby confirming that the CPT1B gene is involved in the development of melanocytes and black spots, and completed the present invention by constructing a melanocyte-deficient zebrafish model in which the CPT1B gene was knocked out. Figure 1 shows the results of knocking down or overexpressing the CPT1B gene in zebrafish. A: Target sites of junction-blocking MOs (Morpholino Oligomers) targeting CPT1B exon 3 and intron 3, and results of PCR performed on CPT1B upon MO injection B: Phenotype of zebrafish when the CPT1B gene is overexpressed Figure 2 shows the phenotype of zebrafish (cpt1b morphants, cpt1b crispants) with the CPT1B gene knocked down or knocked out, and the results of the phenotype recovery analysis for cpt1b morphants. A: Phenotype of early 3-day-old zebrafish embryos (cpt1b morphants) injected with MO B: Degree of lateral larval striation formation in cpt1b morphants C: Phenotype of cpt1b morphants upon co-injection of CPT1B MO and mRNA D: Target site of CPT1B gene target sgRNA E: Phenotype of zebrafish with knockout of the CPT1B gene (cpt1b crispants) Figures 3 to 5 show the results of genotype analysis for 11 zebrafish (cpt1b crispants) injected with RNA/Cas9 RNP. The terminology used in this specification is used to appropriately describe preferred embodiments of the present invention, and may vary depending on the intent of the user or operator, or the conventions of the field to which the present invention belongs. Therefore, the definitions of these terms should be based on the content throughout this specification. Throughout the specification, when a part is described as "comprising" a certain component, unless specifically stated otherwise, this means that it does not exclude other components but may include additional components. Throughout this specification, '%' used to indicate the concentration of a particular substance is (w/w) % for solid/solid, (w/v) % for solid/liquid, and (v/v) % for liquid/liquid, unless otherwise noted. The present invention provides a melanocyte-deficient zebrafish ( Danioli rerio ) model in which the CPT1B (Carnitine Palmitoyltransferase 1B) gene