KR20200001022A - IRX1 Knock-out Transgenic Zebrafish Model and Method for Producing Thereof - Google Patents

Abstract

The present invention relates to a zebrafish model knock-out of the IRX1 gene (zebrafish) model and its use, and more particularly to the transgenic zebrafish knocked out Irx1a and Irx1b gene, method of producing the zebrafish, the A method of screening a candidate substance for cancer treatment using zebrafish and a method of predicting a therapeutic effect on cancer of cancer treatment agents are provided. Transgenic zebrafish knocked out of Irx1a and Irx1b according to the present invention are deficient in Irx1a and Irx1b function, resulting in tumors having tumor biological properties very similar to human tumors. Therefore, the zebrafish cancer model of the present invention is an excellent animal model for studying tumor generation, metastasis, proliferation, etc. in vivo, and can be customized to patients through efficient screening of cancer therapeutic agents and prediction of cancer therapeutic effects on cancer. It can be useful for developing treatment strategies.

Description

IRX1 Knock-out Transgenic Zebrafish Model and Method for Producing Thereof}

The present invention relates to an IRX1 gene-knockout transgenic zebrafish model and its use.

Malignant tumors of the digestive system, including the stomach, colon, liver, colon, pancreas, and biliary tract, account for 50% of all cancers and are the leading cause of death from cancer. Survival has been greatly improved by early detection through recent screenings. Gastric and colorectal cancer have a five-year survival rate of 65%, but liver cancer has a five-year survival rate of around 10% and pancreatic bile duct cancer.

In the past decades, oncology studies using white paper models have revealed a variety of oncogenes involved in the cancer process, and much of their molecular biology and signaling pathways have been identified. This not only elucidated tumor progression but also provided a target for new therapeutic drugs. Indeed, many therapeutic agents that target oncogenes or proteins associated with tumors have been developed to show excellent anticancer effects and concomitantly create enormous added value. More specifically, 30-60% of EGFR (epidermal growth factor receptor) mutations have been reported in pancreatic cancer, and it has been found that the activation of EGFR plays an important role in the development of pancreatic cancer. It is developed and used in clinical practice. Various tumor genes have been found in liver cancer, and new therapeutic targets have been developed as a result of studies on related signaling systems. Sorafenib, a multi-kinase inhibitor, has excellent therapeutic effects in patients with advanced liver cancer. Is showing.

However, the transformation white paper model has a problem that the initial cost, maintenance cost and time-consuming. Because of these problems, studies related to cancer are being limited, and the development of related treatments is being delayed. In particular, the manufacturing and research of the domestic transformation white paper model is in its infancy and the gap with developed countries is considerable.

The zebrafish ( Danio rerio ), a three-year-old tropical freshwater fish, has 25 pairs of chromosomes and retains almost all human genes, even though its evolution was separated from the common ancestors of humankind 300 million years ago. have. Because of these characteristics, zebrafish began to be used as a developmental biology model in the 1980s, and its value as a tumor biology model has been highlighted since the 2000s.

Zebrafish are less expensive than other animal models and are genetically closer to humans than vertebrates or linear animals as vertebrates. In addition, the production of a large number of fertilized eggs can be researched efficiently, the embryo is transparent and has a short development period of 48 hours can be observed in real time. In particular, the expression of organ-specific biomarkers such as fluorescent proteins enables the observation of specific organs in real time in the living body, and has the advantage of being able to track at the cellular level in the embryonic stage. In addition, because of in vitro fertilization, it is easy to manipulate genes by easily injecting genetic material into the yolk of fertilized eggs.

The zebrafish is used as an experimental model by using the biological characteristics of the zebrafish, and since 2000, a number of papers on tumor biology have been published.

On the other hand, genome editing (Genome Editing) is a technique for freely correcting the genetic information of life. Advances in the life sciences and advances in genomic sequencing technology allow us to gain a broad understanding of a variety of genetic information. For example, understanding of genes for breeding plants, diseases and growth, gene mutations that cause various human genetic diseases, and production of biofuels has already been secured, but they can be used directly to improve life and improve human diseases. Further technological advances are necessary to reach the therapeutic level. Genome editing technology can dramatically expand the scope of use by changing the genetic information of animals, plants, and microorganisms, including humans. Genetic shears are molecular tools that are designed to precisely cut the desired genetic information and play a key role in genome editing technology. Like the next generation sequencing technology that has taken gene sequencing to the next level, gene shears are becoming a key technology for expanding the speed and scope of genetic information utilization and creating new industries.

Genetic scissors developed so far can be divided into three generations in that order. First-generation genetic scissors are ZFN (Zinc Finger Nuclease), second-generation genetic scissors are TALEN (Transcription Activator-Like Effector Nuclease), the most recently studied CRISPR (CrisPR, clustered regularly interspaced short palindromic repeat) -Cas9 (CRISPR-associated ) Is the third generation gene shear (Crispr / Cas9, RGEN).

However, extensive studies have not been conducted to explain the optimization and effectiveness of the zebrafish tumor model that knocked out specific genes related to cancer suppression as a tool for practical drug screening using the genetic scissors.

Therefore, in order to establish a more efficient carcinogenic model and substantially replace the conventional animal model, development of a genetic scissors-based zebrafish cancer model has emerged as an important task.

Accordingly, the present inventors have made extensive efforts to develop more efficient, economical and easy-to-use animal models as animal models for use in cancer drug screening, and replace the conventional mammalian animal models. As a result, transgenic zebrafish knocked out of Irx1a (Iroquois related homeobox 1a) and Irx1b genes, known as tumor suppressor genes, were prepared using gene scissors (TALEN and Crispr / Cas9 system), and lack of the Irx1a and Irx1b genes. Carcinoma is induced in the transgenic zebrafish, and the tumor biological properties of the carcinoma were confirmed to be not only very similar to human, but also various human-like tumors, thereby completing the present invention.

One object of the present invention to provide a zebrafish (Danio rerio) cancer model knocked out (Iknock-out) Irx1a and Irx1b gene.

Another object of the present invention is to provide a method for screening a candidate substance for preventing or treating cancer using the zebrafish cancer model.

In addition, another object of the present invention to provide a method for predicting the therapeutic effect of cancer cancer drug using the zebrafish cancer model.

Hereinafter, the present invention will be described in more detail.

In order to achieve the above object, according to an aspect of the present invention, the present invention Irx1a (Iroquois related homeobox 1a, Gene bank: NC_007127.7; mRNA: NP_997067.1) and Irx1b (Iroquois related homeobox 1b, Gene bank: NC_007130.7; mRNA: NP_571898.1) provides a zebrafish ( Danio rerio ) cancer model knocked out gene.

According to a preferred embodiment of the invention, the zebrafish arm model is constructed in a method comprising the following steps:

(a) preparing a transgenic zebrafish (Irx1a ^-/- ) in which Irx1a (Iroquois related homeobox 1a) gene is knocked out using an engineered nuclease;

(b) preparing a transgenic zebrafish (Irx1b ^{− / +} ) knocked out of Irquois related homeobox 1b using an engineered nuclease; And

(c) The transgenic zebrafish (Irx1a ^{− / −} ) knocked out of the Irx1a gene of step (a) and the transgenic zebrafish (Irx1b ^{− / +} ) of the knocked out Irx1b gene of step (b) are crossed to Irx1a And preparing a transgenic zebrafish (Irx1a ^-/- / Irx1b ^-/- ) in which the Irx1b gene is knocked out.

That is, the present invention is characterized by providing a method for producing a zebrafish model in which cancer occurs through deletion or alteration of Irx1a and Irx1b genes.

In the present invention, "zebrafish model" or "animal model" refers to an animal having a disease in a form very similar to that of human, that is, zebrafish. The significance of disease model animals in human disease research is due to physiological or genetic similarities between humans and animals. Biomedical Disease Models in Disease Research Animals provide research materials for various causes, pathogenesis, and diagnosis of disease, and research on disease model animals to identify genes involved in the disease and to understand their interactions. In this regard, basic data for judging the possibility of practical application can be obtained by examining the actual efficacy and toxicity of the new drug candidate.

In the present invention, a method for preparing a zebrafish cancer model using a method called gene editing, genome editing, or genome engineering technology has been developed.

In the present invention, the gene to be genetically modified is Irx1 (Iroquois related homeobox 1).

Irx1 gene is a homeobox gene found in Drosophila in the early 1990s and is known to be involved in pattern formation when it occurs. The Irxa1 gene is located on human chromosome 3.p25 and produces a protein of 213 amino acids. In zebrafish, it is divided into Irx1a and b.

The genetic correction technique is characterized by using a target specific nuclease.

As used herein, the term "target specific nuclease" may be a nuclease capable of recognizing and cleaving a specific position of DNA on a desired genome. The nuclease may include a nuclease fused with a domain that cleaves a domain that recognizes a specific target sequence on the genome.

In the present invention, the target specific nuclease is an engineered nuclease.

According to a preferred embodiment of the present invention, the genetic scissors are selected from the group consisting of ZFN (Zinc finger nuclease), TALEN (transcription activator-like effector nuclease) and RGEN (RNA-guided engineered nuclease).

That is, a transcription activator like effector nuclease (TALEN), a zinc-finger nuclease, in which a TAL activator-like effector domain derived from a plant pathogenic gene, a domain that recognizes a specific target sequence on the genome, and a cleavage domain are fused. (zinc-finger nuclease), or RGEN (RNA-guided engineered nucclease) or CRISPR / Cas9 including CRISPR / Cas9 (Clustered regularly interspaced short palindromic repeat / CRISPR-associated) derived from the microbial immune system CRISPR Can be.

For the purposes of the present invention, the method using TALEN and / or Crispr / Cas9 may perform simple and more desirable results, but is not particularly limited thereto.

As used herein, the term "transformation" refers to the introduction of plasmid-formed DNA directly into a cell physicochemically or to infect a host cell with a virus to express a foreign gene, and the term "transformation zebrafish" in the present invention. Refers to zebrafish whose genome has been modified by integration of the desired nucleotide sequence.

As used herein, the term "Transcription activator-like effector nuclease" (TALEN) refers to a nuclease capable of recognizing and cleaving a target region of DNA, and is prepared by fusion of a TAL effector DNA binding domain and a DNA cleavage domain to each other. It is an artificial restriction enzyme. In the present invention, the TALEN also includes a form in which the TAL effector DNA binding domain and the DNA cleavage domain are linked through a linker. Here, the TAL effector DNA binding domain is engineered to bind quickly to any desired DNA sequence, and when combined with the DNA cleavage domain, the TAL effector DNA binding domain is specifically bound to a desired DNA sequence, thereby bringing about an effect of cleaving a desired DNA site.

In the present invention, the terms "TAL effector nuclease" and "TALEN" are compatible. TAL effectors are known to be proteins that are secreted through their Type III secretion system when Xanthomonas bacteria are infected with various plant species. The protein may bind to a promoter sequence in the host plant to activate expression of plant genes to aid bacterial infection. The protein recognizes plant DNA sequences through a central repeat domain consisting of up to 34 different numbers of amino acid repeats. Thus, TALE is believed to be a new platform for tools of genome engineering. However, in order to construct a functional TALEN with genome-correcting activity, a few key parameters that have not been known to date should be defined. i) the minimum DNA-binding domain of TALE, ii) the length of the spacer between two half-sites constituting one target region, and iii) a linker or fusion junction that connects the FokI nuclease domain to dTALE ).

TALE domains of the invention refer to protein domains that bind to nucleotides in a sequence-specific manner via one or more TALE-repeat modules. The TALE domain includes, but is not limited to, at least one TALE-repeat module, more specifically 1 to 30 TALE-repeat modules. In the present invention, the terms "TAL effector domain" and "TALE domain" are compatible. The TALE domain may comprise half of the TALE-repeat module. The full text disclosed in WO / 2012/093833 or US Patent Publication 2013-0217131 in connection with the TALEN is incorporated herein by reference.

As used herein in connection with knockouts targeting the Irx1a and Irx1b genes, “TALEN targeting the Irx1a gene” refers to a method of binding to a specific sequence of the Irx1a gene to result in cleavage of the Irx1a gene.

In the present invention, when targeting the Irx1a gene, TALEN preferably specifically cleaves exon 1 of the Irx1a gene, and more preferably, binds to a nucleotide sequence of SEQ ID NO: 3 located in exon 1 of the Irx1a gene. 1 TALEN and the second TALEN that binds to the nucleotide sequence of SEQ ID NO: 4, more preferably to cut the base sequence site (SEQ ID NO: 2) in exon 1 to which the TAL effector DNA binding domain can bind.

In one embodiment of the present invention, the Irx1a gene is the entire nucleotide sequence of Irx1a cDNA represented by SEQ ID NO: 1, and binds to the nucleic acid sequence of the first TALEN and SEQ ID NO: 4 having a DNA binding domain that binds to the nucleic acid sequence of SEQ ID NO: 3 All of the second TALENs having the DNA binding domain were cleaved from the base sequence region (SEQ ID NO: 2) in exon 1 to which the TAL effector DNA binding domain can bind.

The method of introducing the TALEN of the present invention into the cell may use a known method for introducing nucleic acid into the cell, and as known in the art, suitable standard techniques such as electroporation, calcium phosphate co- Precipitation (calcium phosphate co-precipitation), retroviral infection (retroviral infection), microinjection (microinjection), DEAE textlan (DEAE-dextran) and cationic liposome method can be used, such as, but not limited to. At this time, the circular vector may be cut with an appropriate restriction enzyme and introduced into a linear vector form.

As used herein, the term "RGEN" refers to a nuclease comprising a target DNA specific guide RNA and a Cas protein as components.

The term used herein in connection with knockouts targeting the Irx1a and Irx1b genes, “RGEN (or Crispr / Cas9) targeting the Irx1b gene”, refers to a method of binding to a specific sequence of the Irx1b gene resulting in cleavage of the Irx1b gene. Means.

In addition, in the present invention, the Irx1b gene is the entire sequence of Irx1b cDNA represented by SEQ ID NO: 5, and when targeting the Irx1b gene, RGEN (Crisper / Cas9) preferably specifically cleaves exon 2 of the Irx1b gene. More preferably, it may be a guide RNA binding to 5'-CCAAGAGCGCTACCAGAGAAA-3 '(SEQ ID NO: 6) located in exon 2 of the Irx1b gene.

In the present invention, the Crispr / Cas9 (RGEN) is a target DNA specific guide RNA or DNA encoding the guide RNA; And it can be applied to the cell in the form of an isolated Cas protein or a nucleic acid encoding the Cas protein, but is not limited thereto.

In a more specific embodiment, the Crispr / Cas9 in the present invention can be applied to cells in the form of 1) target DNA specific guide RNA and isolated Cas9 protein, 2) DNA encoding the guide RNA and nucleic acid encoding Cas9 protein. have.

Delivery to the cells in the form of 1), also referred to as RNP delivery, but is not limited thereto.

When applied in the form of an isolated guide RNA, the guide RNA may be transcribed in vitro, but is not limited thereto.

In addition, the DNA encoding the guide RNA and the nucleic acid encoding the Cas9 protein may be used as the separated nucleic acid itself, and may exist in the form of a vector including the expression cassette for expressing the guide RNA, and / or Cas9 protein. However, the present invention is not limited thereto.

The vector may be a viral vector, a plasmid vector, or an Agrobacterium vector. Examples of the viral vector may include Adeno-associated virus (AAV), but are not limited thereto.

The DNA encoding the guide RNA and the nucleic acid encoding the Cas9 protein may be present in individual vectors or in one vector, but are not limited thereto.

Each application aspect described above may apply to all of the more specific aspects described in the specification.

In the present invention, the Crispr / Cas9 (RGEN) may include a guide RNA or DNA encoding the specific binding to a specific sequence of the Irx1b gene, and a Cas9 protein or a nucleic acid sequence encoding the same, It is not limited to this. As used herein, the term "Cas9 protein" is a major protein component of the CRISPR / Cas9 system, which is a protein capable of forming an activated endonuclease or nickase.

The Cas9 protein may form a complex with crRNA (CRISPR RNA) and tracrRNA (trans-activating crRNA) to show its activity.

The guide RNA may be in the form of a dual RNA (single RNA) or a single-chain guide RNA (sgRNA) including a crRNA (CRISPR RNA) and a tracrRNA (trans-activating crRNA) that specifically binds to the target sequence represented by SEQ ID NO: 6 Can be.

Cas9 protein or gene information can be obtained from known databases such as GenBank of the National Center for Biotechnology Information (NCBI). In addition, the Cas protein may be a Cas protein derived from Streptococcus, more specifically, a Streptococcus pyogens, more specifically a Cas9 protein. In addition, the Cas protein may be a genus Campylobacter, more specifically, a Cas protein derived from Campylobacter jejuni, and more specifically, a Cas9 protein. However, the present invention is not limited to the examples described above.

In addition, the Cas protein is used in the present invention as a concept including all of the variants that can act as an endonuclease or nickase activated in cooperation with the guide RNA in addition to the native protein. The variant of Cas9 protein may be a mutant form of Cas9 wherein the catalytic aspartate residue has been changed to any other amino acid. Specifically, the other amino acid may be alanine, but is not limited thereto.

In the present invention, the Cas protein may be a recombinant protein.

The term "recombinant", when used to refer to a cell, nucleic acid, protein or vector, etc., for example, introduces a heterologous nucleic acid or protein or alters a native nucleic acid or protein, or is derived from a modified cell. Cell, nucleic acid, protein, or vector modified by the cell. Thus, for example, a recombinant Cas protein can be made by reconstructing a sequence encoding the Cas protein using a human codon table.

The Cas protein or nucleic acid encoding the same may be in a form that allows the Cas protein to function in the nucleus.

The isolated Cas protein may also be in a form that is easy to introduce into the cell. For example, the Cas protein may be linked to a cell penetrating peptide or a protein transduction domain. The protein transfer domain may be, but is not limited to, poly-arginine or HIV derived TAT protein. Cell penetrating peptides or protein delivery domains are known in the art in addition to the examples described above, so those skilled in the art are not limited to these examples, and various examples can be applied to the present invention.

In addition, the nucleic acid encoding the Cas protein may additionally include a nuclear localization signal (NLS) sequence. Therefore, the expression cassette including the nucleic acid encoding the Cas protein may include an NLS sequence in addition to a regulatory sequence such as a promoter sequence for expressing the Cas protein. However, this is not limitative.

Cas proteins may be linked with tags that are advantageous for separation and / or purification. For example, a small peptide tag such as a His tag, a Flag tag, an S tag, or a GST (Glutathione S-transferase) tag, a MBP (Maltose binding protein) tag, or the like may be connected depending on the purpose, but is not limited thereto.

As used herein, the term "guide RNA" means a target DNA specific RNA, and may bind to a Cas protein to lead the Cas protein to the target DNA.

In the present invention, the guide RNA includes two RNAs, namely dual RNA (crRNA) comprising a crRNA (CRISPR RNA) and tracrRNA (trans-activating crRNA) as a component; Or a form comprising a first site comprising a sequence capable of base pairing with a sequence complementary to a sequence in the target DNA and a second site comprising a sequence interacting with the Cas protein, more specifically of crRNA and tracrRNA The main part may be single-chain RNA (sgRNA) in fused form.

The sgRNA includes a sequence portion (also referred to as a spacer region, a target DNA recognition sequence, a base pairing region, etc.) capable of forming a base and a base pair complementary to the sequence in the target DNA, and a hairpin structure for Cas protein binding. can do. More specifically, the composition may include a moiety having a sequence capable of forming a base pair with a sequence complementary to a sequence in the target DNA, a hairpin structure for Cas protein binding, and a terminator sequence. The structure described above may be present in sequence from 5 'to 3'. However, it is not limited thereto.

Any form of guide RNA may be used in the present invention, provided that the guide RNA comprises a major portion of crRNA and tracrRNA and a portion capable of forming base pairs with complementary portions of the target DNA.

The crRNA may be hybridized with the target DNA.

RGEN may consist of Cas protein and dual RNA, or may consist of Cas protein and sgRNA. In addition, the RGEN is a nucleic acid encoding a Cas protein and a nucleic acid encoding a dual RNA; Alternatively, the nucleic acid encoding the Cas protein and the nucleic acid encoding the sg RNA may be used as components, but are not limited thereto.

The guide RNA, specifically crRNA or sgRNA, comprises a sequence capable of forming a base pair with a sequence complementary to the sequence in the target DNA, the 5'-terminus of an upstream site of the crRNA or sgRNA, specifically the crRNA of the sgRNA or dualRNA May include one or more additional nucleotides. The additional nucleotide may be guanine (G), but is not limited thereto.

More specifically, although not particularly limited, the genetic correction of the present invention includes a guide RNA or DNA encoding the guide RNA specifically binding to a specific sequence of Irx1b gene; And introducing a nucleic acid or Cas9 protein encoding the Cas9 protein into the cell.

The target sequence that can be used for knockout of the Irx1b gene by the RGEN method may be a 5′-CCAAGAGCGCTACCAGAGAAA-3 ′ sequence (SEQ ID NO: 6) present in Irx1b exon 2, but is not necessarily limited thereto.

In an embodiment of the present invention, in order to knock out Irx1a and Irx1b genes, which are target gene target sequences to be corrected, the Irx1a gene is removed using a method based on the TALEN system, which is one of the nucleases, and among the nucleases. By removing the Irx1b gene using the RGEN method based on one CRISPR / Cas9 system, the Irx1a and Irx1b gene-deletion models were finally constructed.

In other words, mRNA generated from the TALEN construct that recognizes the Irx1a gene target sequence and Cas9 mRNA and gRNA generated by the Crispr / Cas9 system that recognizes the Irx1b gene target sequence were used.

The method may be (1) a method of producing and delivering a gRNA (single guided RNA) recognizing a target sequence in exon 2 of the Cas9 protein and Irx1b purified after overexpression in bacteria by in vitro transcription. (2) Alternatively, a method of transferring plasmid DNAs expressing these Cas9 proteins and gRNAs into cells and expressing them in cells may be used, but is not necessarily limited thereto.

In addition, the method for intracellular delivery of protein, RNA, or plasmid DNA required for the present invention includes electroporation, liposomes, viral vectors, nanoparticles, as well as PTD (Protein translocation domain) fusion protein methods. Various methods known in the art can be used, but are not limited to the above examples.

According to a preferred embodiment of the present invention, the transformed zebrafish (Irx1a ^{− / −} ) knocked out of the Irx1a gene is transformed with a recombinant vector having a cleavage map described in FIG. La, and the transformed zebrafish knocked out of the Irx1b gene (Irx1b ^{− / +} ) is transformed with the recombinant vector having the cleavage map described in FIG. Lb.

In the present invention, the zebrafish cancer model of the present invention comprises a recombinant vector having a cleavage map described in FIG. 1A, ie, a TALEN construct for Irx1a gene knockout; Or a recombinant vector having the cleavage map described in FIG. 1B, ie, the Crispr / Cas9 construct for Irx1b gene knockout.

In the present invention, the term "vector" refers to a gene construct comprising a nucleotide sequence of a gene operably linked to a suitable regulatory sequence to be able to express the gene of interest in a suitable host, wherein the regulatory sequence initiates transcription Capable promoter, any operator sequence for regulating such transcription, and a sequence regulating termination of transcription and translation. The vector of the present invention is not particularly limited as long as it can replicate in cells, and any vector known in the art may be used, and may be, for example, a plasmid, cosemid, phage particle, or viral vector.

In the present invention, the term “recombinant vector” refers to an expression vector of a target polypeptide capable of expressing the target polypeptide with high efficiency in a suitable host cell when the coding gene of the target polypeptide to be expressed is operably linked. The recombinant vector may be expressible in a host cell. The host cell may preferably be a eukaryotic cell, and according to the type of host cell, appropriate selection of expression control sequences such as promoters, terminators, enhancers, and the like for membrane targeting or secretion, etc. And various combinations depending on the purpose.

The recombinant vector is characterized in that the base sequence of the Irx1a and Irx1b gene, respectively, by modifying, ie inserting or deleting the nucleotide sequence, preferably in the case of the Irx1a gene in the target nucleotide sequence present in exon 1 Insertion or deletion may occur, and in the case of the Irx1b gene, modification, ie insertion or deletion, may occur in the target sequence present in exon 2.

If the present invention is intended to knock out the Irx1a and Irx1b gene is included in the present invention without limitation, preferably the recombinant vector has a cleavage map described in Figures 1a and 1b, knockout of the Irx1a and Irx1b gene of the present invention If it is a structure of the vector which can be achieved, it is not limited to this.

According to a preferred embodiment of the present invention, the zebrafish cancer model of the present invention is liver cancer, stomach cancer, pancreatic cancer, gallbladder cancer, colon cancer, ovarian cancer, breast cancer, lung cancer, small cell lung cancer, blood cancer, bone cancer, skin cancer, head or neck cancer, skin Or intraocular melanoma, uterine cancer, rectal cancer, anal muscle cancer, colon cancer, breast cancer, fallopian tube carcinoma, endometrial carcinoma, cervical cancer, vaginal cancer, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, From adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocyte lymphoma, bladder cancer, kidney cancer, ureter cancer, CNS tumor, CNS lymphoma, spinal cord tumor, brainstem glioma and pituitary adenoma Selected cancers develop, and more preferably cancers selected from the group consisting of liver cancer, stomach cancer, pancreatic cancer, gallbladder cancer, colon cancer and ovarian cancer.

In one embodiment of the present invention, the method further comprises crossing the heterozygous variant zebrafish obtained from the zebrafish fertilized eggs with each other to obtain homozygous variant zebrafish.

According to the present invention, the present invention can perform cross-crossing between heterozygous variant zebrafish to obtain homozygous variant zebrafish. For example, a zebrafish fertilized egg (F0) injected with the genetic scissors system of the present invention is bred with adult heterozygous mutant progeny (F1) obtained by breeding with wild type to obtain a heterozygous mutant progeny (F2) Conjugates can be crossed to obtain homozygous variant offspring (F3).

The introduction of the fertilized egg includes a microinjection method, a stem cell insertion method, an insertion method using a retrovirus, and a sperm-mediated gene delivery method, and the like, and preferably, the microinjection method is not limited thereto.

The fertilized egg is a fertilized egg produced through the mating of male and female zebrafish, preferably a fertilized egg of 1-2 cells.

In other words, the present inventors made genetically modified animals in a simpler manner by using a genetic scissors system, and effectively produced a cancer-generating transgenic zebrafish model caused by a deficiency of the Irx1 gene.

In addition, according to another aspect of the present invention, the present invention provides a method for screening a candidate substance for preventing or treating cancer, comprising the following steps:

(a) treating the test substance with the zebrafish cancer model described above;

(b) measuring the extent of the tumor in the cells of the zebrafish cancer model of step (a); And

(c) selecting the test substance whose degree of tumor in the cells measured in step (b) is reduced compared to the control group not treated with the test substance.

In the screening method, the test material of step (a) is a material to be treated to determine whether it is effective in treating cancer, and may include a compound, a peptide, a peptide mimetic, a substrate analog, an aptamer, an antibody, and the like. But it is not limited thereto.

In the screening method, measuring the extent of the tumor in the cell of step (b) means analyzing whether the tumor has been formed in the cells of zebrafish and, if so, what its characteristics are.

More specifically, the clear cell body (clear cell body), eosinophils (eosinophilic), basophilic cell body (pasophilic cell body), LCC (large cell change), hyaline body (eosinophilic granular cytoplasm), It may be to analyze the characteristics of tumor formation, including rounded nuclei, prominent nucleoli, dysplastic foci, etc. It may be measured by a method such as reverse transcription polymerase chain reaction (PCR), northern blot, cDNA microarray hybridization reaction, and in situ hybridization reaction. Immunoprecipitation, radioimmunoassay (RIA), enzyme immunoassay (ELISA), immunohistochemistry, western blotting and flow cytometry ( It may be measured through a method such as FACS), but is not limited thereto.

In the screening method, the screening step of step (c) may be performed by quantitatively or qualitatively analyzing the measurement result of step (b) to treat the zebrafish cancer model treated with the test substance and the cells of the control group not treated with the test substance. By comparing the degree of tumor may be to select a test material whose tumor degree is reduced compared to the control.

According to one embodiment of the present invention, transgenic zebrafish knocked out of Irx1a and Irx1b form tumors in liver, small intestine, pancreas and ovarian tissue.

Therefore, by observing tumor incidence and extent, it can be usefully used for research on cancer treatment substance itself and its development.

Since the method of the present invention uses the above-described transgenic zebrafish cancer model knocked out of Irx1a and Irx1b, the description thereof is omitted in order to avoid excessive complexity of the present specification.

In addition, according to another aspect of the present invention, the present invention provides a method for predicting the therapeutic effect against cancer of a cancer therapeutic agent comprising the following steps:

(a) treating a cancer therapeutic agent with the zebrafish cancer model described above;

(b) measuring the progress of cancer in the zebrafish cancer model of step (a); And

(c) In step (b), when the cancer of the zebrafish model is advanced, it is predicted that the cancer treatment effect of the cancer treatment agent does not exist, and when the cancer of the zebrafish model is not advanced, the cancer treatment effect of the cancer treatment agent Predicting that is present.

In the method of predicting the therapeutic effect, the cancer treatment agent of step (a) is known or newly developed therapeutic agent, preferably compounds, peptides, peptide mimetics, matrix analogs, aptamers, antibodies, etc. It includes.

In the method of predicting treatment effect, the step of measuring the progression of liver cancer in step (b) may include RT-PCR (Reverse Transcription Polymerase Chain Reaction), Northern blot, cDNA microarray hybridization, and in situ hybridization, and the like, and can measure the activity of tumor-associated proteins in immunoprecipitation, radioimmunoassay (RIA), enzymes. An immunoassay (ELISA), immunohistochemistry (immunohistochemistry), Western blot (Western Blotting) and flow cytometry (FACS) may be measured by such methods, but is not limited thereto.

Since the method of the present invention uses the above-described transgenic zebrafish cancer model knocked out of Irx1a and Irx1b, the description thereof is omitted in order to avoid excessive complexity of the present specification.

Transgenic zebrafish knocked out of Irx1a and Irx1b according to the present invention are deficient in Irx1a and Irx1b function, resulting in tumors having tumor biological properties very similar to human tumors. Therefore, the zebrafish cancer model of the present invention is an excellent animal model for studying tumor generation, metastasis, proliferation, etc. in vivo, and can be customized to patients through efficient screening of cancer therapeutic agents and prediction of cancer therapeutic effects on cancer. It can be useful for developing treatment strategies.

Figure 1 shows a method for producing Irx1a / b knockout zebrafish model using the TALEN of the present invention.
Figure 2 shows the phenotypic and pathological conditions in the liver, small intestine, pancreas and ovarian tissue of the 12-month-old transgenic zebrafish isomorphism of the present invention.
Figure 3 shows the development of hyperplasia of intestinal epithelium in the epidermis of 19 months of age of transformed zebrafish isomorphism intestinal tissue of the present invention.
Figure 4 shows the development of intrahepatic cholangiocarcinoma in the liver tissue epidermis 12-18 months of age of transformed zebrafish isomorphism of the present invention.

Hereinafter, the content of the present invention will be described in more detail through Examples and Experimental Examples. The following Examples and Experimental Examples are only illustrative of the present invention, and the content of the present invention is not limited by the following Examples and Experimental Examples.

Example

Example 1 Preparation of Transgenic Zebrafish Knock-out of Irx1a / b Gene

The present inventors selected Irx1 (Iroquois related homeobox 1) gene as a gene that plays an important role in cancer development, and knockout-transformed zebrafish model by targeting zebrafish Irx1a / b gene using genetic scissors techniques as follows. And homogeneous mutant strains were established through subcultures.

At this time, the zebrafish model is transformed with the recombinant vector having the cleavage map described in Figure la and Figure 1b below.

1-1. Breeding and Breeding Zebrafish

Zebrafish, native to India, were bred in tanks with closed circulation filtration systems that maintained a temperature of 28.5 ° C and a humidity of 30-70%. 14 hours of light treatment per day, 10 hours of dark treatment to match the daylight cycle. Mature zebrafish were fed twice a day by mixing Freshwater Aquarium Flakefood (Tetra Werke, Melle, Germany) with live brine shrimp (San Francisco Bay Brand, Inc., Newark, CA, USA). At about two to three months of age, they became adult to breed, and in the afternoon before mating, the females and males were placed in dedicated mating cages and lit to induce spawning the next morning. Thereafter, males and females mated, and the females lay hundreds of fertilized eggs. Collected fertilized eggs were washed with Ringer's solution (116 mM NaCl, 2.9 mM KCl, 1.8 mM CaCl ₂ , 5 mM HEPES, pH 7.2), transferred to Petri dishes and generated in an incubator at 28.5 ° C. Using an anatomical microscope, fertilized eggs of each developmental stage were selected according to time and morphological changes and fixed with 4% paraformaldehyde.

1-2. Fabrication of Irx1a Knockout Zebrafish Model Using TALEN

The present inventors produced the zebrafish knocked out by targeting the zebrafish Irx1a gene using the vector of FIG. 1A and established homozygous strains through passage breeding (FIG. 1C).

First, the present inventors prepared the TALEN construct, and then prepared the mRNA through in vitro transcription, and then injected the mRNA through microinjection into the zebrash fertilized egg. After breeding the adult language, the offspring were produced, and individuals with germline mutations in the Irx1a gene were identified through sequencing.

To prepare zebrafish knocked out of the Irx1a gene, exon 1 of Irx1a (Iroquois related homeobox 1a, Genbank numb. NC_007127.7; mRNA: NP_997067.1) was selected as the gene to be knocked out. The entire nucleotide sequence of exon 1 of the Irx1a gene of zebrafish was analyzed to determine the nucleotide site within exon 1 to which the TAL effector DNA binding domain can bind (SEQ ID NO: 2; 5'- TCCCCCAGCTGGGCTACCCG CAGTATTTAAGTGCCTCCCACGCGGTGTA CGGAGGCGACCGACCGGGA -3 '). Based on this, a first TALEN (left TALEN underline, SEQ ID NO: 3) and a second TALEN (right TALEN underline, SEQ ID NO: 4) were prepared.

In other words, the design of TALEN through the search for candidate binding sites was carried out using the software program Bogdanove laboratory (https://boglab.plp.iastate.edu/node/add/talen), and the TALEN construct targeting exon 1 of the Irx1a gene Was produced. The first TALEN, which is the upper nucleotide sequence (left) recognized by TALEN, is TCCCCCAGCTGGGCTACCCG (SEQ ID NO: 3) present in exon 1, and the second TALEN, which is the lower nucleotide sequence, is CGGAGGCGACCGACCGGGA (SEQ ID NO: 4). A 29 bp spacer was placed between the two base sequences. Constructs were prepared using the Feng Zhang method, and confirmed by analyzing the nucleotide sequence of the produced construct plasmid.

Subsequently, microtransjection of the zebrafish transformed gene prepared in Example 1-2 was microinjected into the yolk of the 1-2 cell fertilized egg obtained in Example 1-1. Subsequently, parental generations of zebrafish (F0 founder fish) are bred to adult sex and fertilized eggs are produced to confirm the transgenic F1 founder fish model with mutations in the Irx1a gene through sequencing and then passaging. Isomorphic variants were established.

On the other hand, as a result of confirming the genotype of the knockout F3 zebrafish, Irx1a ^-/- knockouts in which the Irx1a gene is knocked out, the base sequence of exon 1 to which the TAL effector DNA binding domain, which is a target sequence site, can bind It was confirmed that a frameshift mutation (underlined in bold type) having C inserted after the 40th base from the ATG start codon in the region was performed as follows (Irx1a cDNA whole nucleotide sequence is represented by SEQ ID NO: 1):

5'-ATGTCTT TCCCCCAGCTGGGCTACCCG CAGTATTTAAGTGCCTCCCACGCGGTGTA CGGAGGCGACCGACCGGGA -3 '→

5'-ATGTCTTT TCCCCCAGCTGGGCTACCCG CAGTATTTAAGTG C CCTCCCACGCGGTGTA CGGAGGCGACCGACCGGGA -3 '.

(The first TALEN (SEQ ID NO: 3) and the second TALEN (SEQ ID NO: 4) recognized by TALEN are underlined on the left and right sides, respectively.)

1-3. Fabrication of Irx1b Knockout Zebrafish Model Using RGEN (Crispr / Cas9)

The present inventors produced the zebrafish knocked out by targeting the zebrafish Irx1b gene using the vector of FIG. 1B and established homozygous strains through passage breeding (FIG. 1C).

First, the inventors prepared the guide-RNA using Crispr / Cas9, and then injected the mRNA through microinjection into the zebrafish fertilized egg together with the Cas9 mRNA. Breeding the adult language to produce the next offspring (F1) and then analyzed the nucleotide sequence of the Irx1b gene to identify individuals with mutations. Since then, homogeneous mutant stocks have been established through subcultures.

In order to prepare zebrafish knocked out of the Irx1b gene, exon 2 of Irx1b (Iroquois related homeobox 1b, Gene bank.NC_007130.7; mRNA: NP_571898.1) was selected as the gene to be knocked out. The entire base sequence of exon 2 of the Irx1b gene of zebrafish was analyzed, and based on this, SEQ ID NO: 6 (5′-CCAAGAGCGCTACCAGAGAGAAA-3 ′) was produced as a target sequence.

Subsequently, microtransjection of the zebrafish transformed gene prepared in Example 1-3 was microinjected into the yolk of the 1-2-cell fertilized egg obtained in Example 1-1. Subsequently, parental generations of zebrafish (F0 founder fish) were bred to adult sex, and fertilized eggs were produced to confirm the transgenic F1 founder fish model with mutations in the Irx1b gene through sequencing and then passage. Isomorphic variants were established.

Meanwhile, as a result of confirming the genotype of the knockout F3 zebrafish, the Irx1b- ^{/ +} knockout strain in which the Irx1b gene was knocked out, the CG was deleted after the 368th base from the ATG start codon, and the TTT was inserted into the frame shift (frameshift) mutation (underlined bold) occurred as follows (Irx1b full cDNA sequence is shown by SEQ ID NO: 5):

5'-CCAAGAGCGCTACCAGAGAAA-3 '→

5'-CCAAGAG TTT CTACCAGAGAAA-3 '

1-4. Irx1a ^-/- / Irx1b ^-/-  Knockout Zebrafish Model Making

The present inventors produce a large number of offspring (F2) by breeding a F1 heterozygous zebrafish (Frameshift) heterozygous with a frameshift mutation to a natural type, and then identify the individuals with the mutations to secure each male and female through crossbring. F3 progeny were produced, and homozygous Irx1a and Irx1b Irx1a ^-/- , Irx1b ^-/- Knockout Zebrafish was established.

In other words, Irx1a ^-/- / Irx1b ^-/- knockout strains produced Irx1a and Irx1b homozygous strains to produce progeny, and finally established Irx1a ^-/- / Irx1b ^-/- strains through subcultures. Through inbreeding of the established Irx1a ^-/- / Irx1b ^-/- knockout strains, a stable population was secured by breeding Irx1a ^-/- / Irx1b ^-/- strains in which both Irx1a and Irx1b genes were completely knocked out.

Example 2. Irx1a ^-/- , Irx1b ^-/-  Knockout Zebrafish Tumor Formation Verification

The present inventors bred the Irx1a ^{− / −} and Irx1b ^{− / −} transgenic zebrafish homozygous strains prepared in Example 1 to determine whether tumors were formed in the transgenic zebrafish, 12-18 years old. Liver, small intestine, pancreatic and ovarian tissues were obtained from month ^- old Irx1a ^-/- / Irx1b ^-/- transgenic zebrafish isomorphisms to determine tumor development patterns and levels through H & E staining and immunohistochemistry (IHC). Confirmed.

The results of confirming the tumor formation of the Irx1a ^{− / −} , Irx1b ^{− / −} knockout zebrafish are shown in Table 1 below.

Table 1 Tumor Phenotype in Irx1 Gene Variants

More specifically, 4 μm thin tissue was dehydrated using 70%, 80%, 90%, 100% ethanol after deparaffinization using xylene. After soaking in 0.3% H ₂ O ₂ for 20 minutes, the reaction was carried out for 15 minutes and washed three times with 1X PBST for 5 minutes. Treatment with blocking solution (10% normal goat serum) for one hour followed by three washes of 5 min with 1 × PBST, the primary antibody was anti-PCNA, and the primary antibody was treated at room temperature for one hour. After washing 3 times with 1X PBST for 5 minutes, the secondary antibody was treated with anti-mouse Ab and reacted at room temperature for 1 hour. After treatment with ABC solution (Vector Laboratories) for 1 hour at room temperature, washed twice in 1X PBST for 10 minutes and 3'-diaminobenzidine tetrachloride (DAB, peroxidase substrate kit DAB (Vector Laboratories)) The mixture was washed three times for 10 minutes with primary distilled water. After washing for 2 minutes, each treated with 85%, 90%, and 100% ethanol, xylene (xylene, Sigma) was treated for 5 minutes, after which xylene was dried and observed with a fluorescence microscope (Olympus BX51, Japan). .

As a result, as shown in Figure 2, when the phenotype and pathological conditions in the liver, small intestine, pancreas and ovarian tissue of the 12-month-old transgenic zebrafish isomorphism were observed, not only hyperplasia of the gastrointestinal tract (hyperplasia) but also , Intrahepatic cholangiocarcinoma and mucinous cystadenocarcinoma were found, and the carcinoma showed almost identical findings at the level of histopathology with cholangiocarcinoma and ovarian cancer in humans. .

In addition, as shown in Figure 3, it was confirmed that the gastrointestinal epithelial cells (Hyperplasia of intestinal epithelium) occurred in the intestinal tissue epithelium of 19 months old transgenic zebrafish isomorphism. Compared with the control group, the gastrointestinal epithelial cell layer was thicker and the number of PCNA and mitotic markers, pHH3 immunostain positive cells, were significantly increased.

In addition, as shown in Figure 4, it was confirmed that intrahepatic cholangiocarcinoma occurred in the epidermis of the liver tissue of 12-18 months old transgenic zebrafish isomorphism. Cholangiocarcinoma was very similar at the level of carcinoma and histopathology in humans. In addition, the carcinoma showed invasive proliferation to the liver as well as the pancreas located in the surrounding area, and the positive rate of PCNA immunostaining cancer cells was significantly increased.

This suggests that the Irx1 gene acts as a tumor suppressor gene in the development of various carcinomas and occurs when the Irx1a / b is deleted.

Overall, through the above experimental results, by using a specific gene shear method for knockout of the Irx1a and Irx1b genes according to the present invention, knocking out DNA strands encoding the Irx1a and Irx1b genes in a transgenic animal effectively inducing cancer development. It was confirmed that it can be done. Therefore, the Irx1a and Irx1b gene knockout zebrafish cancer models according to the present invention can be used in the development of cancer therapeutics because they can be used in various ways such as the identification of the mechanism of cancer, the search for therapeutic substances, and the development of diagnostic methods. .

<110> Industry-Academic Cooperation Foundation, Yonsei University <120> IRX1 Knock-out Transgenic Zebrafish Model and Method for          Producing Thereof <130> YSU1-25p <160> 6 <170> KoPatentIn 3.0 <210> 1 <211> 1281 <212> DNA <213> Danio rerio <400> 1 atgtctttcc cccagctggg ctacccgcag tatttaagtg cctcccaggc ggtgtacgga 60 ggcgaccgac cgggagttct gactccgtcg tcccgcgcag ggagcgctga gatcgggggt 120 agcgcgtctg cggccgccgc tgcggtgtcc tcggtgttgg gcatgtaccc gtacgcacac 180 aactacagcg cctttctgcc ttacaccagc gccgatctgg ctcttttctc ccaaatgggc 240 tctcagtatg atctgaaaga cagccctgga gtccaccctg ccagcttcgc tgcccacacg 300 agtcctgcct tctaccccta cggccagttc cagtacggag accccgccag gcccaaaaac 360 gccacccggg agagcaccag caccctgaag gcctggctga acgagcacag gaaaaacccc 420 tatcccacca aaggagagaa gatcatgctg gccatcatca ccaagatgac cctcacgcaa 480 gtgtccacct ggttcgccaa cgctagaagg agactcaaga aggagaacaa ggtgacctgg 540 ggcgccagga gcaaggacga cgagaacatt tttggaagcg ataacgaagg agacgccgaa 600 aagaacgagg acgaagagga gatagatctg gagagcatag atatcgataa aatcgacgac 660 aacgatggcg atcagagcaa cgaggaggac gaggacaagc gcgcggagct ggagagcctc 720 caggagaagc gacacgcgtc caaagagccg tcggatggca acaacaacac cactagagta 780 ctgtcgcccg gtcgacaggg cggttttcaa gttccggtta gcaataagcc caaaatatgg 840 tcgttggcag aaacagcgac cagtcccgat agcgcgcaga aacctacgtc accttccgtt 900 cccgtcagcc acccggcctt cctgcccagc cacggactgt acacgtgcca gatcggcaag 960 ttccacaact ggacgaacgg cgcgtttctg ggacaaaact ctctgctaaa tgtgcgctct 1020 ttcctgggcg tcaaccagca caaccacccg gcgcagcagc agcagcagca gacggggccc 1080 gcgtccctgc aggtgctgtc ggccgggaac acggacaaga tgccggagga cctgagtcca 1140 aaacacatag atcgggaaaa tgtcctcaga aatgaatcac caacgcaacc tttaaagtcg 1200 tcgtttcgtc ctcttcatga cagtcccaga aaccagcagg aatcaacaca gagggtcctc 1260 acagcgctgt cctccgcttg a 1281 <210> 2 <211> 68 <212> DNA <213> Artificial Sequence <220> <223> Irx1a target sequence <400> 2 tcccccagct gggctacccg cagtatttaa gtgcctccca cgcggtgtac ggaggcgacc 60 gaccggga 68 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Left binding site of TALEN targeting exon 1 of Irx1a <400> 3 tcccccagct gggctacccg 20 <210> 4 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Right binding site of TALEN targeting exon 1 of Irx1a <400> 4 cggaggcgac cgaccggga 19 <210> 5 <211> 1338 <212> DNA <213> Danio rerio <400> 5 atgtcgttcc cccaactggg ttacccacag ttcctaaacg cctcccaaga ggtgtacgga 60 gcggagcgca cgggtgtaat gccgtcctct ccacgcgagg gaagctcgga caacagcgca 120 aatcccgcga ctgcagcggc tgctatcacg ccgatgctcg gcatgtatgc gaatccgtgg 180 accgtgcaaa attatagcgc cttcttacct tacaacagtg ccgagctggc cctgctctct 240 cagatggggt cgcagtatga gctgaaggat aaccccgggg ctcatcctcc aggatttgca 300 gtgcacgctg cgcccggttt ttatccatac ggccagtatc agtacggtga tccggcccgg 360 gccaagagcg ctaccagaga aaccaccagc acactgaaag catggttgca ggaacacaaa 420 aagaacccgt atcccaccaa aggtgagaag atcatgctgg ccatcatcac gaagatgacc 480 cttacgcagg tgtccacctg gttcgccaac gcgcgccgga gactcaaaaa ggagaataaa 540 gtgacctgtt gtcgcagtgc cgaggacaga gacgggcgaa ttttcgacag cgacaacgag 600 gatgatgcgg acaaaaatga cgacgaggaa gaaatcgatc tggagaccat cgatatggac 660 aaagcggagg agcccagagt ggaccagaac aaggaagcag cgaataaccc gcacgcgatt 720 gaaaacattg aaacaccgcg gatattatct tccccagcac ttaaaaacac ggacagtcct 780 atgtcgagtg gtaatagaga acagaatgtg gtcaaaagcg ttggtgaggt gtctcctggt 840 ggagcagtgt gtcaaaggcc tggaaacagc aaacccaaaa tctggtcttt ggcggagacg 900 gcaacaagtc ctgataatac gcagaaactc agtatcccgt cgacgcctgc aggcctcagg 960 gcctcacctt caacccatcc tgcttttctc tccacaaacg gaatttatac ttgccagatt 1020 gggaaactac acaattgggc gagcgccgct ttccttagtg ctaattctct gatgggtgtg 1080 cgctcacttt tgagtcaaaa cagtaacttt ccaaaccaca gtcccgttac taaccccgaa 1140 aagaaacctt cgtctgcctc tggatctcca tgtcttgaca gcgacaactc atccgacagt 1200 ttcagtccca aacataatga tcaagagaac gttcagagaa ttgaatctcc aacacttgca 1260 tcaagatcac cgtttccagt catccatgac aggtctcatc atgaaacatc acaacgcgct 1320 ttgaagacaa tttcttga 1338 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Irx1b target sequence <400> 6 ccaagagcgc taccagagaa a 21

Claims (11)

Knock-out genes Irx1a (Iroquois related homeobox 1a, Genbank numb.NC_007127.7; mRNA: NP_997067.1) and Irx1b (Iroquois related homeobox 1b, Gene bank.NC_007130.7; mRNA: NP_571898.1) Zebrafish ( Danio rerio ) cancer model.
The method of claim 1,
The zebrafish arm model, characterized in that the zebrafish arm model is produced by a method comprising the following steps:
(a) preparing a transgenic zebrafish (Irx1a ^-/- ) in which Irx1a (Iroquois related homeobox 1a) gene is knocked out using an engineered nuclease;
(b) preparing a transgenic zebrafish (Irx1b ^{− / +} ) knocked out of Irquois related homeobox 1b using an engineered nuclease; And
(c) The transgenic zebrafish (Irx1a ^{− / −} ) knocked out of the Irx1a gene of step (a) and the transgenic zebrafish (Irx1b ^{− / +} ) of the knocked out Irx1b gene of step (b) are crossed to Irx1a And preparing a transgenic zebrafish (Irx1a ^-/- / Irx1b ^-/- ) in which the Irx1b gene is knocked out.
The method of claim 2,
The genetic scissors are zebrafish cancer model, characterized in that selected from the group consisting of transcription activator-like effector nuclease (TALEN) and RNA-guided engineered nuclease (RGEN).
The method of claim 3,
The TALEN zebrafish cancer model, characterized in that it comprises a TAL effector DNA binding domain (Transcription activator-like effector DNA binding domain) and DNA cleavage domain (DNA cleavage domain) that binds to the Irx1a gene.
The method of claim 4, wherein
The TAL effector DNA binding domain is characterized in that binding to the nucleotide sequence represented by SEQ ID NO: 3 and SEQ ID NO: 4, zebrafish cancer model.
The method of claim 3,
The RGEN zebrafish cancer model, characterized in that it comprises a guide RNA and Cas9 protein that specifically binds to the sequence of the Irx1b gene.
The method of claim 6,
The guide RNA is in the form of a dual RNA (dualRNA) or single-chain guide RNA (sgRNA) comprising a crRNA (CRISPR RNA) and a tracrRNA (trans-activating crRNA) that specifically binds to the target sequence represented by SEQ ID NO: 6 The zebrafish arm model characterized by the above-mentioned.
The method of claim 2,
The transgenic zebrafish (Irx1a ^{− / −} ) knocked out of the Irx1a gene of step (a) is transformed with a recombinant vector having a cleavage map described in FIG.
1A

The method of claim 2,
Transgenic zebrafish (Irx1b ^{− / +} ) knocked out of the Irx1b gene of step (b) are transformed with a recombinant vector having a cleavage map as described in FIG.
1B.

A method for screening a candidate substance for preventing or treating cancer, comprising the following steps:
(a) treating the test substance with the zebrafish cancer model of claim 1;
(b) measuring the extent of the tumor in the cells of the zebrafish cancer model of step (a); And
(c) selecting the test substance whose degree of tumor in the cells measured in step (b) is reduced compared to the control group not treated with the test substance.
A method of predicting the therapeutic effect of a cancer drug on cancer, comprising the following steps:
(a) treating the zebrafish cancer model of claim 1 with a cancer therapeutic agent;
(b) measuring the progress of cancer in the zebrafish cancer model of step (a); And
(c) In step (b), if the cancer of the zebrafish model is advanced, it is predicted that the cancer treatment effect of the cancer treatment agent does not exist, and if the cancer of the zebrafish model is not advanced, the cancer treatment effect of the cancer treatment agent Predicting that is present.

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