KR20160088997A

KR20160088997A - Composition for Preparing an Immuno-Deficient Zebrafish Model and Use Thereof

Info

Publication number: KR20160088997A
Application number: KR1020150008019A
Authority: KR
Inventors: 박승우; 정인혜
Original assignee: 연세대학교 산학협력단
Priority date: 2015-01-16
Filing date: 2015-01-16
Publication date: 2016-07-27

Abstract

The present invention relates to a composition for the production of immunodeficient zebrafish, a method for producing immunodeficient zebrafish using the same, and immunodeficient zebrafish prepared thereby
Jebriphys established using the composition of the present invention can efficiently perform xenotransplantation from human body due to lack of immune function, and the transplanted tumor tissue and the like have tumor-like biological characteristics very similar to primary tumors. Thus, the Jebriphys established using the composition of the present invention can be usefully used as an effective screening means for an excellent animal model or tumor therapeutic composition for studying tumor formation, metastasis, proliferation, etc. in In vivo .

Description

Composition for the preparation of immunodeficient zebrafish models and their uses {Composition for Preparing an Immuno-Deficient Zebrafish Model and Use Thereof}

The present invention relates to a method of producing an immunodeficiencyed zebrafish model using a nuclease targeting a Prkdc (protein kinase, DNA-activated, catalytic polypeptide) zebrafish-like gene.

In the animal disease model for identifying candidate drug candidates, mammals have advantages such as genetic and physiological similarities with humans, but are not suitable for large-scale screening due to technical support, high cost and stringent housing requirements in animal facilities. Thus, there has been a need for alternative laboratory animals with high fertility, low cost, and simple housing requirements. Since the establishment of animal models in the early 1980s, zebrafish (Danio rerio) has been developed as an important organism for drug screening. Not only does zebrafish have significant advantages over other vertebrate models because it is screenable in a 96-well or 384-well plate format, the genome is about 80% homologous to the human genome, It is further advantageous compared to popular small organism models such as Drosophila melanogaster (60%) and Caenorhabditis elegans (36%). Zebrafish costs less than 1% of mice, and embryos are recognized as a useful tool for studying the behavior of xenografted tumor cells such as dissemination and tumor-induced angiogenesis.

Currently, patient-derived xenotransplantation models are predominantly dependent on SCID / Nude mice, but the current xenotransplantation success rate is now only 40-60%, and it is not reasonable to use it as a means for mass screening because of the high cost per individual. Therefore, even though a zebrafish model that is superior in terms of success rate and cost reduction is in the spotlight, the problem of immune regulation to be solved for xenotransplantation has not been studied yet. Development of a zebrafish with an immunodeficiency phenotype has emerged as an important task in order to establish a more efficient heterologous zebrafish model and substantially replace conventional SCID / Nude mice.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

As an animal model for use in patient-derived xenografts (PDTX), the present inventors have made extensive efforts to develop a more efficient and economical animal model that replaces conventional mammalian animal models. As a result, when the gene is knocked out using a nuclease that specifically recognizes the genbank accession number XM_009303401.1 gene of zebrafish, a zebrafish object lacking immune function is produced, and the zebrafish The present invention has been completed by discovering that xenografted tumors are very similar to primary tumors and their tumor biological properties.

It is therefore an object of the present invention to provide compositions for the production of immunodeficient zebrafish.

It is another object of the present invention to provide a method for producing an immunodeficient zebrafish and an immunodeficient zebrafish prepared using the same.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention provides a nucleic acid encoding a nucleotide sequence that specifically recognizes one or more exon regions included in the nucleotide sequence of Genbank Accession No. XM_009303401.1 or nucleotides encoding the nucleases As an active ingredient, an immunosuppressive zebrafish.

As an animal model for use in patient-derived xenografts (PDTX), the present inventors have made extensive efforts to develop a more efficient and economical animal model that replaces conventional mammalian animal models. As a result, when the gene is knocked out using a nuclease that specifically recognizes the genbank accession number XM_009303401.1 gene of zebrafish, a zebrafish object lacking immune function is produced, and the zebrafish Xenotransplanted tumors have found that the primary tumor and tumor biology are very similar. Thus, the Jebriphys established using the composition of the present invention can be used as an effective screening tool for an excellent animal model or tumor therapeutic composition for studying tumor formation, metastasis, proliferation and the like in in vivo .

The nucleases used in the present invention recognize deletion, deletion, and indel, frame shift, or inversion by recognizing a specific region of a gene of known nucleotide sequence and cleaving it. And any nuclease system capable of inhibiting or eliminating the inherent function or activity of the gene as a result.

According to a specific embodiment of the present invention, the nuclease used in the present invention comprises:

(a) a pair of endonuclease; And

(b) an amino acid sequence linked to one of the abovementioned (a) and specifically binding to a nucleotide sequence at a first position on any one of the exon regions included in the nucleotide sequence of Genbank Accession No. XM_009303401.1 Included transcription activator-like (TAL) effector domains:

(c) a TAL effector domain linked to one of (a) and comprising an amino acid sequence that specifically binds to a nucleotide sequence at a second position on the exon region, wherein the first and second positions are 15 -25 bp in length and are 10-20 bp apart from each other.

As used herein, the term " nucleotide " is a deoxyribonucleotide or ribonucleotide present in single-stranded or double-stranded form and includes analogs of natural nucleotides unless otherwise specifically indicated (Scheit, Nucleotide Analogs , John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews , 90: 543-584 (1990)).

The term " specifically recognized " in the present invention means selectively recognizing it by interaction with the target base sequence of the present invention, and is used with the same meaning as " specifically binding. &Quot; The TAL effector used in the present invention has a central repeat domain consisting of 34 amino acids, and the 12th and 13th amino acid residues are composed of various amino acids and are called RVD (repeat variable diresidue). Each RVD serves to identify a specific DNA base and recognizes and binds specifically to NI = A, NN = G, NG = T, and HD = C (Boch J, et al. Science 326 5959): 1509-1512 (2009)). Thus, if there is information about the target DNA sequence, a TAL effector amino acid sequence that specifically binds to the target DNA sequence can be determined based on such a code.

According to the present invention, the exon targeted in the present invention can be any one of 83 exons included in the nucleotide sequence of Genbank Accession No. XM_009303401.1, and the exon can be used for the purpose of knocking out the XM_009303401.1 gene And can be appropriately selected by those skilled in the art. According to a specific embodiment of the present invention, the exon consists of the nucleotide sequence of SEQ ID NO: 5. According to the present invention, the nucleotide sequence of Sequence Listing 5 is the nucleotide sequence of Exon 3 of XM_009303401.1 gene.

Thus, the first and second positions of the exon 3 are respectively the left TAL effector and the right TAL effector, wherein a pair of endonuclease, each of which is connected thereto, has a 10-20 bp (Spacer) portion.

According to a specific embodiment of the present invention, the first and second positions of the present invention are 10-20 bp apart, more specifically 12-16 bp apart, most specifically 12-14 bp apart.

According to a specific embodiment of the present invention, the nucleotide sequences of the first and second positions of the present invention are the nucleotide sequences of the first and second sequences of the sequence listing, respectively.

More specifically, the amino acid sequence that specifically binds to the nucleotide sequence of the first sequence of the sequence listing comprises the amino acid sequence of the third sequence of the sequence listing.

More specifically, the amino acid sequence that specifically binds to the nucleotide sequence of the second sequence of Sequence Listing includes the amino acid sequence of Sequence Listing 4.

According to a specific embodiment of the present invention, the endonuclease used in the present invention is FokI endonuclease.

According to the invention, it may be used in the form of each of the endonuclease and TAL effector protein of the present invention, by transforming with the DNA encoding them respectively with vector may be delivered to the target cell, in vitro transcription May be injected directly into the capped mRNA form.

According to another aspect of the present invention, there is provided an immunodeficient zebrafish comprising a step of injecting the above-described composition of the present invention into a zebrafish, a zebrafish cell or a zebrafish embryo, And a method of manufacturing the same.

Since the composition used in the present invention has already been described above, the description thereof is omitted in order to avoid excessive overlapping.

As used herein, the term " gene transporter " means an agent for introducing and expressing a desired target gene into a target cell. The ideal gene transducer is harmless to the human body or the cell, it is easy to mass-produce, and it is necessary to be able to efficiently transfer the gene.

As used herein, the term " gene transfer " means that a gene is carried into a cell and has the same meaning as transduction of a gene into a cell. At the tissue level, the term gene transfer has the same meaning as the spread of a gene. Accordingly, the gene carrier of the present invention can be described as a gene penetration system and a gene diffusion system.

To prepare the gene delivery vehicle of the present invention, the nucleotide sequence of the present invention is preferably present in a suitable expression construct. In such expression constructs, the nucleotide sequence of the invention is preferably operatively linked to a promoter. As used herein, the term " operably linked " means a functional linkage between a nucleic acid expression control sequence (e.g., an array of promoter, signal sequence, or transcription factor binding site) and another nucleic acid sequence, The regulatory sequence will regulate transcription and / or translation of the other nucleic acid sequences. In the present invention, the promoter bound to the nucleotide sequence of the present invention is preferably capable of regulating transcription of the rilacsin gene by operating in an animal cell, more preferably a mammalian cell, and includes a promoter derived from a mammalian virus And a promoter derived from the genome of a mammalian cell, such as CMV (mammalian cytomegalovirus) promoter, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, U6 promoter, HSV tk Promoter of the human IL-4 gene, promoter of the human lymphotoxin gene, promoter of the human IL-2 gene, promoter of the human IL-2 gene, promoter of the human IL- A promoter of a GM-CSF gene, but is not limited thereto.

The gene carrier of the present invention can be produced in various forms including (i) naked recombinant DNA molecules, (ii) plasmids, (iii) viral vectors, and (iv) the naked recombinant DNA molecule or Or in the form of a liposome or a niozyme containing a plasmid.

The nucleotide sequence of the present invention can be applied to all gene delivery systems used for conventional animal transformation and is preferably a plasmid, an adenovirus (Lockett LJ, et al., Clin . Cancer Res. 3: 2075-2080 ), Adeno-associated viruses (AAV, Lashford LS., Et al., Gene Therapy Technologies , Applications and Regulations Ed. A. Meager, 1999), retrovirus (Gunzburg WH, et al., Retroviral vectors. Gene Therapy Technologies , Applications and Regulations Ed. A. Meager, 1999), lentivirus (Wang G. et al., J. Clin . Invest . 104 (11): R55-62 (1999)), herpes simplex virus (Chamber R., et al., Proc ... Natl Act Sci USA 92 :. 1411-1415 (1995)), Bash Catania virus (Puhlmann M. et al, Human Gene Therapy 10: 649-657 (1999)), liposomes (Methosin in Molecular Biology, Vol. 199, SC Basu and M. Basu (Eds.), Human Press 2002) or niosomes.

In the present invention, when the gene carrier is prepared based on a viral vector, the contacting step is carried out according to a virus infection method known in the art. Infection of host cells with viral vectors is described in the above cited documents.

In the present invention, when the gene carrier is an naked recombinant DNA molecule or plasmid, the microinjection method (Capecchi, MR, Cell , 22: 479 (1980); and Harland and Weintraub, J. Cell Biol . (1987), calcium phosphate precipitation method (Graham, FL et al., Virology , 52: 456 (1973) and Chen and Okayama, Mol . Cell . Biol . 7: 2745-2752 ), Electroporation (Neumann, E. et al., EMBO J. , 1: 841 (1982) and Tur-Kaspa et al., Mol . Cell Biol . , 6: 716-718 (1986)), liposome-mediated transfection (Wong, TK et al., Gene , 10:87 (1980), Nicolau and Sene, Biochim . Biophys . Acta , 721: 185-190 1982) and Nicolau et al., Methods Enzymol . , 149: 157-176 (1987)), DEAE-dextran treatment (Gopal, Mol . Cell Biol . , 5: 1188-1190 (1985)) and the gene bend buddhism (Yang et al., Proc . Natl . Acad. Sci . , 87: 9568-9572 , And most specifically, a microinjection method can be used.

According to a specific embodiment of the present invention, the method of the present invention is carried out by introducing the composition of the present invention into a zebrafish embryo.

According to a specific embodiment of the present invention, the method of the present invention further comprises the steps of crossing the heterozygous mutant zebrafish obtained from the zebrafish embryo with each other to obtain homozygous mutant zebrafish.

According to the present invention, the present invention can carry out heterozygous mutant zebrafish interbreeding to obtain homozygous mutant zebrafish. More specifically, the zebrafish embryo (F0) injected with the composition of the present invention is cultivated in the adult fish, and the obtained heterozygous mutant F1 is crossed with the wild type to obtain the heterozygous mutant offspring (F2) The heterozygotes can be mated with each other to obtain homozygous transcript offspring (F3).

According to another aspect of the present invention, there is provided an immunodeficient zebrafish prepared by the above-described method of the present invention.

Since the composition used in the present invention and the method using the composition have already been described above, the description thereof will be omitted in order to avoid excessive duplication.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a composition for the production of immunodeficient zebrafish, a method for producing immunodeficient zebrafish using the same, and immunodeficient zebrafish prepared thereby.

(b) Jebrific established using the composition of the present invention can efficiently perform xenogeneic transplantation from human body due to lack of immune function, and the transplanted tumor tissue and the like have tumor-like biological characteristics very similar to primary tumors.

(c) The Zebriphich established using the composition of the present invention is an excellent animal model for studying the formation, metastasis, proliferation, and the like of tumor in In vivo , and it is an effective animal screening method for the tumor screening composition and the in- And can be usefully used to establish a patient-tailored treatment strategy.

Fig. 1 is a schematic diagram showing a process of producing a TALEN construct. By linking monomers that can target each nucleotide through a complex process, we have constructed a construct that targets 18-20 specific DNA sequences.
FIG. 2 is a diagram showing the result of confirming mutation base sequences. FIG. The mutant strains were correctly cloned by TA cloning of PCR products identified as heterozygous mutants by base sequence analysis. The # 3 mutant was identified as a frame shift mutation of AC double base defects, and the # 6 mutant as a frame shift mutation due to TCTACA deletion and CATAT insertion.
Fig. 3 ISH (in situ gene for Rag1 Hybridization) results. In the Prkdc homozygous mutant, the size of the thymus is remarkably small, indicating that there is a problem in the development and differentiation of T lymphocytes.
Fig. 4 is a graph showing the results of observing the elongation of wild type and mutant zebrafish. In the Prkdc mutant, the size of the kidney is relatively small compared to the wild type, and the number of leukocytes matured in the renal tubule is reduced compared to the wild type.
Figure 5 is a picture showing the phenotype of the wild type and the mutant zebrafish. FIG. 5A shows the overall appearance of the wild-type (left) and the homozygous (right) zebrafish. In the case of the homozygous mutant, the infection caused the myofascial necrosis. FIG. 5b shows a picture showing the dropsy phenotype of the isomorphous mutant, which shows skin swelling and scales due to infection. FIG. 5c is a view showing a state where necrosis occurs due to inflammation of muscles caused by infection of the homologous zebrafish of FIG. 5a. Fig. 5d is a picture showing the brain of the infected fish, and infiltration of inflammatory cells was generally observed. FIG. 5E is a photograph showing ovarian cancer naturally occurring in the Prkdc homologous mutant.
FIG. 6 is a view showing the injection process of the cancer cells into the intraperitoneal zebrafish. A Hamilton syringe with a small volume of injection was used to puncture the abdominal cavity of an adult anesthetized for 30 seconds, then injected 20 μl, and then moved back to the tank to recover from anesthesia within 1 minute.
FIG. 7 is a diagram showing the results of xenogeneic xenograft transplantation of cancer cells. HPAC ^CMV ^- ^{RFP was} injected intraperitoneally into the abdominal cavity for 1 month and ⁵ months after injection, tissue analysis showed that the tumor was implanted and grown.
FIG. 8 is a diagram showing a schematic diagram of a xenotransplantation procedure and a method of using the Prkdc homologous SCID zebrafish strain in a patient-derived cancer cell. Cancer cells extracted from cancer patients can be xenotransplanted into SCID zebrafish, and when the mass is enlarged, the mass can be extracted and used for establishing a customized treatment strategy by acquiring various biology information.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Experimental Method

Breeding zebra fish and crossing

Zebrafish is a small tropical fish ornamental fish with a length of 3-4 cm. The zebrafish used in the experiment was raised in a water bath with a closed circulating filtration system maintaining a temperature of 28.5 ° C and a humidity of 30-70%. 14 hours a day for men and 10 hours for cancer treatment. Mature zebrafish was fed twice daily with a mixture of Freshwater Aquarium Flakefood (Tetra Werke, Melle, Germany) and a living broth shrimp (San Francisco Bay Brand, Inc., Newark, CA, USA). The zebrafish becomes an adult capable of breeding at about 2-3 months. If you give the mating cage for the afternoon of the previous day and give light the next morning, if you induce the spawning, Eggs scattered. The collected embryos were raised in a petri dish containing E3 medium for 5 days and then transferred to a fish tank for breeding.

Zebra fish Prkdc Search for genes

Genomic data base search (http://www.ncbi.nlm.nih.gov) searches for homologous genes of Prkdc (protein kinase, DNA-activated, catalytic polypeptide) and human SCID mouse To obtain 12623 bp coding sequence and exon structure (chromosome # 7, 86 exons) (accession number: XM_009303401.1) and genomic DNA (accession number: NC_007118) sequence).

Production of TALEN constructs for gene-specific targeting

The design of TALEN through the candidate binding site design was performed using the software program Bogdanove laboratory (https://boglab.plp.iastate.edu/node/add/talen) and the TALEN construct targeting the exon 3 of zPrkdc gene (Fig. 1). The top sequence recognized by TALEN (left) is 5'-TATGAATTTCTTAGGGGCAT-3 'and the subsequence (right) is 5'-TCCTCGGACAGTGGCTGACA-3'. A spacer of 12 bp was placed between the two base sequences. The left and right TALEN recognize the top and bottom strands of the target site, respectively, and each recognition site starts with T and recognizes the 18bp specific sequence. The construct was constructed using the Fang & Zhang method, and the nucleotide sequence of the constructed construct plasmid was analyzed and confirmed.

Zebrafish embryo microinjection

After cutting with linear DNA using restriction enzyme, capped mRNA was produced by in vitro transcription. TALEN was designed to specifically bind to the target gene in the zebrafish embryo at the first stage of embryo transfer, and DNA cleavage was induced by microinjection of the capped mRNA synthesized through in vitro transcription using mMESSAGE mMACHINE T7 ULTRA kit (Ambion) .

Identification of nucleotide sequences of Prkdc mutants by sequencing

The fertilized eggs (F0) injected with TALEN mRNA were grown in sexually progeny, and the offspring (F1) was produced. The F1 offspring were cultured in the sextetes and the caudal fin was excised. The heterozygous mutant zebrafish obtained from multiple mutation searches were crossed with wild type to produce a large number of F2 offspring, and then genotyped again to obtain a large number of heterozygous mutant strains. Heterozygotes were crossed to produce a number of F3 progeny, and then genotyped by breeding in a male sex to establish a homozygous mutant strain. Genotyping was performed by caudal fin clipping to extract genomic DNA, followed by PCR using Platinum PCR SuperMix High Fidelity (Invitrogen, 12532016) master mix. After the obtained PCR product was purified, the nucleotide sequences were compared and analyzed by sequencing. The PCR amplification program consisted of PCR initiation at 95 ° C for 10 minutes and PCR 40 cycles consisting of 95 ° C for 30 seconds, 51 ° C for 30 seconds and 72 ° C for 15 seconds. The primer sequences used in the experiments are F9-Prkdc-E3 5'-TTCGCAGGTCTCTGTCTACTGAAA-3 'and R9-Prkdc-E3 5'-CTAGTGCAACAAAGATGACATG -3'.

Total in situ hybridization (ISH)

For phenotypic analysis, riboprobe for T cell differentiation was synthesized by in vitro transcription. PCR was performed using cDNA prepared from zebrafish RNA using primers F-zRag1 5'-TTCTGAAGATGCTCCCAGAGC-3 'and R-T7zRag1 5'-CTAATA CGACTCACTATAGGGTGGCACCGTGTGATATTCTT-3' primers and amplified using T7 IVT kit (Roche) A di-glycogenin-labeled antisense mRNA probe was synthesized.

Embryos at appropriate times were fixed in 4% paraformaldehyde (PBS, pH 8.0) at 4 ° C for more than 12 hours. Embryos after 24 hours were inhibited from pigmentation by adding 0.2 mM PTU (phenylthiourea) to embryonic nic water from budstage (10 h after fertilization). The embryos were washed three times for 5 minutes with 1X PBST (1XPBS, 0.1% Tween-20), and then rewarmed twice with 100% methanol. The embryos were stored at -20 ° C in methanol. . The embryos in 100% methanol were washed 3 times with 1XPBST, treated with Proteinase K for 30 minutes, fixed again with 4% paraformaldehyde and washed with 1X PBST. 500 μl of HYB (50% formamide, 5 × SSC, 0.1% Tween-20) was placed and incubated at 65 ° C. for 5 minutes. Then, HYB (HYB, 5 mg / ml torula RNA, 50 μg / ml heparin, After prehybridization at 65 ° C for 2-4 hours, 1 μl of probe was added and hybridization was carried out overnight at 65 ° C.

After removing the probe solution, the solution was exchanged at intervals of 10 minutes in the order of 75%, 50%, and 25% HYB * / 2XSSC solution maintained at 65 ° C, and then the solution was changed to 2XSSCT. Subsequently, the probe was changed to 0.2XSSCT at least once every 30 minutes to remove the non-hybridized probe. Then, it was washed 5 times with 1X PBST at room temperature for 15 minutes. Next, the blocking solution (PBST + 5% goto serum) was treated at room temperature for at least 1 hour. Anti-di-gracilinase antibody (Roche) was diluted in the blocking solution at 1/5000 and then treated for 2 hours or more. After washing with 1X PBST 5 times for 15 minutes, NBT / BCIP (Roche Diagnostics GmbH) was added and reacted at room temperature with light blocked. After confirming the color through a microscope, it was washed with 1X PBST, and 100% glycerol And stored at 4 ° C.

Analysis of tissue samples

For analysis of zebrafish more than 2 months old, eosinophils were euthanized by Tris-buffered tricaine (3-aminobenzoic acid ethyl ester, pH 7.0; Sigma) 0.0 > aldehyde < / RTI > for 24 hours. After processing in alcohol / gel, paraffin embedding was carried out to make 4 μm slices and H & E staining was performed and observed with an optical microscope.

Cancer cell line Xenotransplantation

To identify the possibility of xenotransplantation, primary cancer cell lines were transplanted and HPAC, Panc1, CFPAC, and MiaPaca cells were used as pancreatic cancer cell lines. The CMV-RFP construct was transformed with lentivirus so that the growth of the tumor after transplantation could be observed in real time, so that red fluorescence was expressed in the cytoplasm. RFP expression was confirmed in cultured cell lines and then injected intraperitoneal injection of Prkdc homozygous zebrafish using a Hamilton syringe. The control group was heterozygous zebrafish. Fluorescence was observed at the interval of one week after the injection. After 4 weeks of transplantation, the cells were sacrificed, fixed and paraffin-embedded to form a 4 μm slice, and stained with H & E and observed under an optical microscope.

Experiment result

Prkdc rust- Out note establish

Prkdc gene target F0 zebrafish microinjected with TALEN mRNA was cultivated in male sperm and then mated to produce several F1 offspring. In the F1 zebrafish grown as an adult fish, a part of the caudal fin was excised, DNA was extracted, PCR was performed to include the target exon, and genotype analysis was performed by sequencing. (Frameshift mutation 3) and TCTACA deletion and CATAT inserted frame mutation 6 (mutant conjugate) were confirmed at the target site of exon 3 (Fig. 2).

# 3, and # 6 were mutated with wild type zebrafish to produce F2 offspring. After breeding on a male sex strain, genotype analysis was conducted to select mutant strains of each male and female, F3 progeny were produced. After F3 offspring grew into male sex, genotype analysis was performed again to establish the final homozygous mutant strain, and multiple homozygous mutant strains were obtained by crossing the homologous strain of male and female.

Phenotypic analysis

The genotypes of homozygous mutants observed by real - time microscopy showed no difference from wild type. The growth rate of the adult fishes was slightly delayed compared with that of the wild type, and the size of the individual at 2 months of age was about 10% smaller than that of the wild type. However, the size of a fully mature adult fish was like a wild type. Because the Prkdc gene affects the generation and differentiation of T and B lymphocytes, ISH was performed on the Rag1 gene to detect the thymus responsible for the ripening of T lymphocytes. Thymus was significantly smaller than that of the wild type (Fig. 3 ).

In addition, in the zebrafish, the kidney plays a hematopoietic function. In the Prkdc mutant, the size of the kidney is relatively smaller than that of the wild type, and the number of leukocytes matured among the kidney tubules is decreased as compared with the wild type (Fig.

Increased disease susceptibility

Since the immune function of T and B lymphocytes is deficient, serious disease susceptibility such as infectious diseases can be expected to increase. In fact, the Prkdc homozygous mutant was found to develop various diseases such as infection, muscle necrosis during growth (Fig. 5). The most common phenotypes caused by the infection are 'Dropsy' (swelling), which causes swelling of the skin and scales. It was confirmed that infections occurred frequently due to frequent occurrence of 'Dropsy' phenotype. In some cases, spontaneous tumors such as ovarian cancer were also observed.

Cancer cell line Implantability Analysis and optimization

At 10 weeks of maturation, various cell lines were transplanted and xenotransplantation was analyzed. Pancreatic cancer cell line was used and transformed to express red fluorescence in cancer cell line so that growing tumor can be observed in real time. Zebrafish is cultivated in the environment of 28-30 ℃ due to the characteristics of tropical fish, but human cancer cell line shows optimal growth in 37 ℃ environment. Therefore, in order to provide optimal conditions for growth of the transplanting cell line, Adaptation was induced. After 1 and 2 months of cancer cell transplantation, fluorescence expression was confirmed and sacrificed and histopathologic analysis was performed. HPAC ^CMV ^- ^RFP , Panc1 ^CMV ^- ^RFP 5 x 10 ⁶ pancreatic cancer cell lines were dissolved in 200 μl of PBS and 5 x 10 ⁵ (20 μl) per individual were injected intraperitoneally (Fig. 6). Fluorescence was observed after anesthesia with tricaine solution once a week, and histological findings were observed at sacrifice 4 or 8 weeks after injection. In the heterozygous mutant strain used as a control, the cancer cell fluorescence disappeared within one week after injection, but the fluorescence image remained in the homozygous mutant, and localized gradually became clear. The histological findings analyzed after sacrifice confirmed the formation of a clear mass by the growth of cancer cells injected from the homozygous mutant (Fig. 7). 0% in heterozygous mutants and 80% in homozygous mutants. This shows that xenotransplantation of cancer cells is possible in Prkdc homozygous mutants lacking T and B lymphocyte function.

Review

Human-derived xenotransplantation (PDX) is effective for carcinomas that are difficult to obtain sufficient cancer specimens. In particular, carcinomas with a low ablation rate are good candidates for PDX for obtaining specimens to produce high quality biodata because biopsy samples can be obtained only (Fig. 8). PDX can be performed using this SCID zebrafish for pancreatic cancer and biliary cancer which have a low ablation rate in digestive system cancer.

The xenografts derived from endoscopic ultrasound-guided fine-needle aspiration biopsy, tissues obtained through biopsy from hepatocellular lesions or tissues obtained through surgical resection can be transplanted into the model of the present invention step by step, and human xenotransplantation can be performed. Tumor studies in vivo can be done more efficiently.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> Industry-Academic Cooperation Foundation Yonsei University <120> Composition for Preparing an Immuno-Deficient Zebrafish Model and Use Thereof <130> PN140631 <160> 9 <170> Kopatentin 2.0 <210> 1 <211> 20 <212> DNA <213> Danio rerio <400> 1 tatgaatttc ttaggggcat 20 <210> 2 <211> 20 <212> DNA <213> Danio rerio <400> 2 tcctcggaca gtggctgaca 20 <210> 3 <211> 661 <212> PRT <213> Artificial Sequence <220> <223> left TAL effector <400> 3 Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys 1 5 10 15 Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala 20 25 30 His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ile Gly 35 40 45 Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys 50 55 60 Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn 65 70 75 80 Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val 85 90 95 Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala 100 105 110 Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu 115 120 125 Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala 130 135 140 Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg 145 150 155 160 Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val 165 170 175 Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val 180 185 190 Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu 195 200 205 Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu 210 215 220 Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr 225 230 235 240 Pro Glu Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala 245 250 255 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly 260 265 270 Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys 275 280 285 Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala 290 295 300 His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly 305 310 315 320 Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys 325 330 335 Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn 340 345 350 Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val 355 360 365 Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala 370 375 380 Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu 385 390 395 400 Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala 405 410 415 Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg 420 425 430 Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val 435 440 445 Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val 450 455 460 Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu 465 470 475 480 Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu 485 490 495 Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr 500 505 510 Pro Glu Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala 515 520 525 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly 530 535 540 Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys 545 550 555 560 Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala 565 570 575 His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly 580 585 590 Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys 595 600 605 Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn 610 615 620 Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val 625 630 635 640 Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala 645 650 655 Ser Asn Gly Gly Gly 660 <210> 4 <211> 661 <212> PRT <213> Artificial Sequence <220> <223> right TAL effector <400> 4 Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys 1 5 10 15 Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala 20 25 30 His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly 35 40 45 Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys 50 55 60 Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His 65 70 75 80 Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val 85 90 95 Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala 100 105 110 Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu 115 120 125 Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala 130 135 140 Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg 145 150 155 160 Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val 165 170 175 Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val 180 185 190 Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu 195 200 205 Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu 210 215 220 Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr 225 230 235 240 Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala 245 250 255 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly 260 265 270 Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys 275 280 285 Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala 290 295 300 His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ile Gly 305 310 315 320 Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys 325 330 335 Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn 340 345 350 Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val 355 360 365 Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala 370 375 380 Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu 385 390 395 400 Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala 405 410 415 Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg 420 425 430 Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val 435 440 445 Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val 450 455 460 Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu 465 470 475 480 Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu 485 490 495 Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr 500 505 510 Pro Glu Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala 515 520 525 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly 530 535 540 Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys 545 550 555 560 Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala 565 570 575 His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ile Gly 580 585 590 Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys 595 600 605 Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His 610 615 620 Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val 625 630 635 640 Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala 645 650 655 Ser Asn Ile Gly Gly 660 <210> 5 <211> 129 <212> DNA <213> Danio rerio <400> 5 ctggggacta caggagtgga gattctgcgg gaaacgagag tggagattat gaatttctta 60 ggggcatttc tacagagaat gtcagccact gtccgaggat gggaaaagaa ctatgctgtc 120 gagcttaag 129 <210> 6 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> F9-Prkdc-E3 <400> 6 ttcgcaggtc tctgtctact gaaaa 25 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> R9-Prkdc-E3 <400> 7 ctagtgcaac aaagatgaca tg 22 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> F-zRag1 <400> 8 ttctgaagat gctcccagag c 21 <210> 9 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> R-T7zRag1 <400> 9 ctaatacgac tcactatagg gtggcaccgt gtgatattct tt 42

Claims

A nuclease that specifically recognizes one or more exon sites contained in the nucleotide sequence of Genbank Accession No. XM_009303401.1 or a nucleotide encoding the nuclease as an active ingredient is used as an active ingredient of an immunodeficient zebrafish &Lt; / RTI >

2. The composition of claim 1, wherein the nuclease comprises:
(a) a pair of endonuclease; And
(b) an amino acid sequence linked to one of the abovementioned (a) and specifically binding to a nucleotide sequence at a first position on any one of the exon regions included in the nucleotide sequence of Genbank Accession No. XM_009303401.1 Included transcription activator-like (TAL) effector domains:
(c) a TAL effector domain linked to one of (a) and comprising an amino acid sequence that specifically binds to a nucleotide sequence at a second position on the exon region, wherein the first and second positions are 15 -25 bp in length and are 10-20 bp apart from each other.

3. The composition of claim 2, wherein the exon comprises the nucleotide sequence of SEQ ID NO: 5.

3. The method of claim 2, wherein the nucleotide sequence at the first and second positions is a nucleotide sequence of a first sequence and a second sequence, respectively.

5. The method of claim 4, wherein the amino acid sequence that specifically binds to the nucleotide sequence of the first sequence listing comprises the amino acid sequence of SEQ ID NO: 3.

5. The method of claim 4, wherein the amino acid sequence that specifically binds to the nucleotide sequence of the second sequence of Sequence Listing comprises the amino acid sequence of Sequence Listing 4.

3. The composition of claim 2, wherein said endonuclease is a FokI endonuclease.

A method for producing an immunodeficiencyed zebrafish comprising the steps of injecting the composition of any one of claims 1 to 7 into a zebrafish, a zebrafish cell or a zebrafish embryo, or transforming the gene into a gene carrier into which the composition is inserted.

9. The method of claim 8, wherein the method comprises injecting a composition of any one of claims 1 to 7 into a zebrafish embryo or transforming the composition with an inserted gene carrier.

10. The method of claim 9, further comprising the step of crossing the heterozygous mutant zebrafish obtained from said zebrafish embryos with each other to obtain homozygous mutant zebrafish.

10. An immunodeficiencyed zebrafish produced by the method of any one of claims 8 to 10.