KR101767456B1 - Transgenic zebrafish expressing a liver-specific hIL-6 gene and method for producing thereof - Google Patents

Abstract

The present invention relates to a method for screening candidate substances for the treatment of liver cancer using the transformed zebrafish, the zebrafish, and a method for predicting the therapeutic effect on liver cancer of a liver cancer treatment agent, and more particularly, A method for screening candidate substances for treatment of liver cancer using the zebrafish, and a method for predicting a therapeutic effect on liver cancer in a liver cancer treatment agent. The transgenic zebrafish according to the present invention expresses the hIL-6 gene in a liver-specific manner to promote chronic inflammation of the liver and to form a hepatic tumor. When the hIL-6 gene is expressed, a fluorescent protein is also expressed, It is possible to visually observe the correlation between hIL-6 gene and liver cancer by the growth stage of zebrafish. Therefore, the transgenic zebrafish can be useful for research on the development of therapeutic agents for liver cancer.

Description

Transgenic zebrafish expressing a liver-specific hIL-6 gene and method for producing thereof

The present invention relates to a transformed zebrafish, a method for screening a candidate substance for liver cancer treatment using the zebrafish, and a method for predicting the therapeutic effect of a liver cancer therapeutic agent on liver cancer, and more particularly, to a liver-specific hIL-6 gene. It relates to an expressing transgenic zebrafish, a method for producing the zebrafish, a method for screening a candidate substance for liver cancer treatment using the zebrafish, and a method for predicting the therapeutic effect of a liver cancer therapeutic agent on liver cancer.

Malignant tumors of the digestive system, including the stomach, colon, liver, colon, pancreas, and biliary tract, account for 50% of all cancers and are a major cause of death from cancer. The survival rate has been greatly improved by early detection through recent screening, and the 5-year survival rate for gastric and colon cancer reaches 65%, but the 5-year survival rate for liver cancer is around 10% and less than 5% for pancreatic biliary cancer.

In the past decades, through oncology research using white paper models, various oncogenes related to the process of cancer have been discovered, and their molecular biological mechanisms and signaling pathways have been largely uncovered. Through this, the progression of the tumor was identified, as well as providing a target for a new therapeutic drug. In fact, a number of therapeutic agents targeting oncogenes or tumor-related proteins have been developed, showing excellent anticancer effects, and concomitantly creating enormous added value. More specifically, in the case of pancreatic cancer, mutations in EGFR (epidermal growth factor receptor) are reported to be 30-60%, and as it was found that activation of EGFR plays an important role in the occurrence of pancreatic cancer, a small molecule drug (Erlotinib) that inhibits this has been found. It has been developed and is being used in clinical practice. Various oncogenes have been discovered in liver cancer, and new therapeutic targets are being developed as a result of research on related signaling systems, and sorafenib, a multi-kinase inhibitor, has an excellent therapeutic effect in patients with advanced liver cancer. Is showing.

However, the transgenic white paper model has problems that require a lot of initial cost, maintenance cost, and time. Due to these problems, studies related to cancer are being limited, and the development of related therapeutic agents is also being delayed. In particular, the manufacturing and research of the domestic transgenic white paper model remains at a rudimentary stage, and the gap with advanced countries is also significant.

On the other hand, the zebrafish ( Danio rerio ) is a three-year tropical freshwater fish, has 25 pairs of chromosomes, and has the genes preserved even though it was separated from the common ancestor of humanity 300 million years ago in evolution, it possesses almost all the genes that humans have. have. Due to these characteristics, zebrafish began to be used as a developmental biology model in the 1980s, and its value as a tumor biology model emerged from the 2000s.

Zebrafish cost less than other animal models, and as vertebrates, they are genetically very close to humans compared to fruit flies or linear animals. In addition, a large number of fertilized eggs are produced, enabling highly efficient research, and the embryo is transparent and has a short development period of 48 hours, allowing real-time observation. In particular, when organ-specific expression of a biomarker, such as a fluorescent protein, can be observed in real time in a living body, it is possible to track a specific organ at the cell level in the embryonic stage. In addition, since in vitro fertilization is carried out, it is easy to manipulate genes by injecting genetic material easily into the egg yolk of a fertilized egg.

Using these biological features of zebrafish, zebrafish are widely used as experimental models, and a number of papers related to tumor biology have been published since 2000, but no research has been conducted to use zebrafish as a liver cancer model.

The present inventors prepared a transgenic zebrafish that specifically expresses the hIL-6 gene, which plays an important role in the occurrence of liver cancer by promoting chronic inflammation of the liver, and the zebrafish induces chronic inflammation specifically in the liver, and It was confirmed that this occurred, and the present invention was completed.

An object of the present invention is to provide a zebrafish model that specifically expresses a human interleukin-6 (hIL-6) gene.

In addition, an object of the present invention is to provide a method of manufacturing the zebrafish model.

In addition, an object of the present invention is to provide a method for screening candidate substances for the prevention or treatment of liver cancer using the zebrafish model.

In addition, an object of the present invention is to provide a method for predicting the therapeutic effect of a liver cancer therapeutic agent for liver cancer using the zebrafish model.

In order to achieve the above object, the present invention provides a zebrafish model that specifically expresses the human interleukin-6 (hIL-6) gene.

In addition, the present invention,

1) Transgenic zebrafish (Tg (LFABP-Gal4/UAS-RFP)) expressing a liver-specific fluorescent protein and a vector containing human interleukin-6 (hIL-6) gene sequences were introduced. Transgenic zebrafish (Tg (LFABP-Gal4/UAS-RFP/) expressing a liver-specific human interleukin-6) gene by crossing (Tg (UAS-hIL6-CG)) Preparing UAS-hIL6-CG));

2) introducing a transformant gene of the transformed zebrafish (Tg (LFABP-Gal4/UAS-RFP/UAS-hIL6-CG)) prepared in step 1) into a zebrafish fertilized egg; containing, zebrafish Provides a model manufacturing method.

In addition, the present invention,

1) treating the zebrafish model with a test substance;

2) measuring the degree of tumor in the hepatocytes of the zebrafish model of step 1); And

3) Selecting a test substance whose tumor degree in the hepatocytes measured in step 2) has decreased compared to the control group not treated with the test substance; comprising, a method for screening a candidate substance for preventing or treating liver cancer is provided. do.

In addition, the present invention,

1) treating a liver cancer therapeutic agent to the zebrafish model of any one of claims 1 to 5; And

2) measuring the progression of liver cancer in the zebrafish model of step 1), comprising: a method for predicting a therapeutic effect of a liver cancer therapeutic agent on liver cancer.

The transgenic zebrafish according to the present invention promotes chronic inflammation of the liver by expressing the hIL-6 gene specifically for the liver and forms a liver tumor, and when the hIL-6 gene is expressed, a fluorescent protein is also expressed, thereby transforming It is possible to visually observe the correlation between the hIL-6 gene and liver cancer at each stage of zebrafish growth. Therefore, the transgenic zebrafish can be usefully used in research on the development of a substance for treating liver cancer.

1A is a diagram showing a cleavage map of a vector used in preparation of a transgenic zebrafish.
1B to M are diagrams confirming whether transgenic zebrafish express hIL-6 specifically for liver.
(Black arrow: hepatic sinusoid of liver, red arrow: intrahepatic vessel)
Figure 2 is a diagram confirming the formation of tumors in the liver of the transgenic zebrafish.
(Red arrows: dysplastic foci, black arrows: binucleated cells)
FIG. 3 is a diagram showing the degree of expression of genes related to the signaling pathway of the transformed zebrafish by RT-PCR (FIG. 3B) and western blot (FIG. 3C ), and graphed ( Fig. 3A).
4 is a diagram illustrating the degree of expression of proteins related to induction of chronic inflammation, apoptosis, and promotion of cell proliferation in the liver of a transgenic zebrafish expressing hIL-6.
5 is a diagram illustrating the degree of expression of regulators of the JAK/Stat3 signaling pathway in the liver of transgenic zebrafish expressing hIL-6.
6 is a diagram illustrating the cause of liver cancer induced by hIL-6 overexpression by treatment with a JAK/Stat3 signaling pathway inhibitor on transgenic zebrafish.

The present invention provides a zebrafish model that specifically expresses the human interleukin-6 (hIL-6) gene.

In addition, the present invention is to provide a method of manufacturing the zebrafish model.

In addition, the present invention provides a method for screening candidate substances for preventing or treating liver cancer using the zebrafish model.

In addition, the present invention provides a method for predicting the therapeutic effect of a liver cancer therapeutic agent for liver cancer using the zebrafish model.

Hereinafter, the present invention will be described in detail.

In the present invention, the term "interleukin-6" refers to a cytokine named B-cell stimulating factor-2 (BSF-2) or interferon β2 (hereinafter referred to as IL-6), and B lymphocyte passage It was discovered as an undifferentiated factor involved in cell activity. The cancer-related IL-6 signaling system has a lot to do with Stat3, an intermediate mediator. The Stat3 is involved in various types of cancer such as myeloma, breast cancer, prostate cancer, brain tumor, head and neck carcinoma, melanoma, leukemia and lymphoma, especially chronic myeloid and multiple myeloma. In addition, human interleukin-6 (hIL-6) of the present invention may be interleukin-6 isolated from humans.

In the present invention, the term “liver-specific” refers to a characteristic that is not expressed or expressed to a low degree in organs other than the liver, but is expressed to a very high degree in the liver. ), but is not limited thereto. In addition, in the present invention, the term “liver target sequence” refers to a base sequence encoding a protein exhibiting the liver-specific characteristics.

In the present invention, the term "transformation" refers to expressing a foreign gene by directly introducing a plasmid-type DNA into a cell physicochemically or by infecting a virus into a host cell. In the present invention, the term "transformed zebrafish" refers to It refers to a zebrafish whose genome has been modified by the integration of the target nucleotide sequence.

According to an embodiment of the present invention, a transgenic zebrafish expressing human interleukin-6 (hIL-6) specifically for the liver is inflamed or tumors formed in the liver, and when the hIL-6 gene is expressed, a fluorescent protein Is also expressed. Therefore, the transgenic zebrafish can be usefully used in research on the development of liver cancer therapeutic substances by visually observing the correlation between the hIL-6 gene and liver cancer during the growth process.

The present invention provides a zebrafish model that specifically expresses the human interleukin-6 gene in the liver.

The zebrafish model of the present invention may be a zebrafish transformed by a vector including a liver target sequence and a vector including a human interleukin-6 (hIL-6) gene.

As an example, the zebrafish model of the present invention may be manufactured by a manufacturing method including the following steps.

1) Transgenic zebrafish (Tg (LFABP-Gal4/UAS-RFP)) expressing a liver-specific fluorescent protein and a vector containing human interleukin-6 (hIL-6) gene sequences were introduced. Transgenic zebrafish (Tg (LFABP-Gal4/UAS-RFP/UAS) expressing a liver-specific human interleukin 6) gene by crossing (Tg (UAS-hIL6-CG)) -hIL6-CG)); And

2) introducing the transgenic zebrafish (Tg (LFABP-Gal4/UAS-RFP/UAS-hIL6-CG)) prepared in step 1) into a zebrafish fertilized egg.

In the above manufacturing method, the vector used in step 1) refers to a DNA strand, which is usually double-stranded, which can be inserted into external DNA. The vector includes, but is not limited to, a plasmid or a virus.

In the above manufacturing method, the fluorescent protein in step 1) is a protein that emits light in a living body, and includes a green fluorescent protein, a yellow fluorescent protein, and the like, preferably a red fluorescent protein. And, more preferably, it may be a fluorescent protein expressed by the introduction of a vector represented by the following cleavage map 3, consisting of the nucleotide sequence represented by SEQ ID NO: 3, but is not limited thereto.

[Cleavage Map 3]

In the manufacturing method, the transgenic zebrafish (Tg (LFABP-Gal4/UAS-RFP) expressing a fluorescent protein specifically for the liver in step 1) is a vector represented by the cleavage map 3 and a vector represented by SEQ ID NO: 1. It may be transformed with a vector represented by the following cleavage map 1 consisting of a nucleotide sequence.

[Cleavage Map 1]

In the above production method, the transformed zebrafish (Tg (UAS-hIL6-CG)) into which the vector containing the human interleukin-6 (hIL-6) gene sequence of step 1) is introduced is a base represented by SEQ ID NO: 2 It may be transformed with a vector represented by the following cleavage map 2 consisting of a sequence.

[Cleavage Map 2]

In the above manufacturing method, the introduction of the fertilized egg in step 2) includes a microinjection method, a stem cell insertion method, an insertion method using a retrovirus, and a sperm-mediated gene transfer method, and may preferably be a microinjection method, but is limited thereto. It does not become.

The zebrafish model of the present invention can visually identify the location where the transduced gene is expressed by expressing a fluorescent protein specifically for the liver, and by expressing the hIL-6 gene specifically for the liver to form inflammation or tumor in the liver. , It can be used to clarify the mechanism of occurrence of liver cancer, and to screen for liver cancer treatment substances.

In addition, the present invention provides a method of manufacturing the zebrafish model. The method of manufacturing a zebrafish model of the present invention,

1) Transgenic zebrafish (Tg (LFABP-Gal4/UAS-RFP)) expressing a liver-specific fluorescent protein and a vector containing human interleukin-6 (hIL-6) gene sequences were introduced. Transgenic zebrafish (Tg (LFABP-Gal4/UAS-RFP/UAS) expressing a liver-specific human interleukin 6) gene by crossing (Tg (UAS-hIL6-CG)) -hIL6-CG)); And

2) introducing a transformant gene of the transformed zebrafish (Tg (LFABP-Gal4/UAS-RFP/UAS-hIL6-CG)) prepared in step 1) into a zebrafish fertilized egg. It may include.

In the above manufacturing method, the transformed zebrafish in step 1) may be a zebrafish transformed by a vector represented by a cleavage map 1 to 3.

In the above manufacturing method, the fertilized egg of step 2) is a fertilized egg produced through crossing of a male and a female zebrafish, and may preferably be a fertilized egg of 1-2 cell stage.

In the above manufacturing method, the introduction of the fertilized egg in step 2) includes a microinjection method, a stem cell insertion method, an insertion method using a retrovirus, and a sperm-mediated gene transfer method, and may preferably be a microinjection method. It is not limited.

In addition, the present invention provides a method for screening a candidate substance for treatment of liver cancer using the zebrafish model. The method for screening a candidate substance for the treatment of liver cancer of the present invention,

1) treating the zebrafish model with a test substance;

2) measuring the degree of tumor in the hepatocytes of the zebrafish model of step 1); And

3) selecting a test substance in which the degree of tumor in the hepatocytes measured in step 2) is decreased compared to the control group not treated with the test substance.

In the screening method, the zebrafish model of step 1) may be a zebrafish transformed by a vector represented by a cleavage map 1 to 3.

In the screening method, the test substance in step 1) is a substance processed to determine whether the compound is effective in treating liver cancer, and includes a compound, a peptide, a peptidomimetic, a matrix analog, an aptamer, an antibody, etc. However, it is not limited thereto.

In the above screening method, measuring the degree of tumor in the hepatocytes in step 2) means analyzing whether a tumor is formed in the hepatocytes of the zebrafish and, if so, what the characteristics are. More specifically, clear cell body, eosinophilic, pasophilic cell body, LCC (large cell change), hyaline body, eosinophilic granular cytoplasm, It may be to analyze whether the characteristics of tumor formation, including rounded nuclei, prominent nucleoli, and dysplastic foci, are present, and the level of expression of genes related to tumor formation is determined by RTPCR ( Reverse Transcription Polymerase Chain Reaction), Northern blot, cDNA microarray hybridization reaction, and in situ hybridization reaction. It may be measured through methods such as immunoprecipitation, radioimmunoassay (RIA), enzyme immunoassay (ELISA), immunohistochemistry, Western Blotting, and flow cytometry (FACS). , Is not limited thereto.

In the screening method, the selection step of step 3) quantitatively or qualitatively analyzes the measurement result of step 2) to compare the tumor degree of the hepatocytes of the zebrafish model treated with the test material and the control without treatment with the test material. It may be to select a test substance having a reduced degree of fixation compared to the control group.

In addition, the present invention provides a method for predicting the therapeutic effect of a liver cancer therapeutic agent for liver cancer using the zebrafish model. The method for predicting the therapeutic effect of the liver cancer therapeutic agent of the present invention for liver cancer

1) treating the zebrafish model with a liver cancer therapeutic agent; And

2) measuring the progression of liver cancer in the zebrafish model of step 1).

In the method for predicting treatment effect, the zebrafish model of step 1) may be a zebrafish transformed by a vector represented by a cleavage map 1 to 3.

In the method for predicting the therapeutic effect, the therapeutic agent for liver cancer in step 1) is known to be effective in treating liver cancer or is a newly developed therapeutic agent, and preferably includes a compound, a peptide, a peptidomimetic, a matrix analog, an aptamer, an antibody, etc. And, more preferably, it may be a JAK/Stat3 inhibitor. The JAK/Stat3 inhibitor may support a compound that targets, decreases or inhibits JAK/Stat3 signaling or its sub-modulators, and may be involved in the JAK/Stat3 signaling process.

In the method for predicting the therapeutic effect, the step of measuring the progression of liver cancer in step 2) is to determine the expression level of genes related to tumor formation, RTPCR (Reverse Transcription Polymerase Chain Reaction), Northern blot, cDNA microarray hybridization. It can be measured through methods such as reaction and in situ hybridization reaction, and the degree of activity of proteins related to tumor formation is determined by immunoprecipitation, radioimmunoassay (RIA), and enzyme immunoassay (ELISA). ), immunohistochemistry, Western Blotting, and flow cytometry (FACS), but are not limited thereto.

The JAK/Stat3 signal of the present invention is a signaling process that regulates immune responses essential for signaling by cytokines, interleukins, and growth factors. The JAK/Stat3 signaling process plays an important physiological role in the differentiation, proliferation, and survival of cells, but when the pathway is abnormally regulated, diseases such as inflammatory abnormalities and cancer progress. Thus, regulation of the JAK/Stat3 pathway can also be a target for treating inflammatory diseases and cancer.

The sub-modulator of the JAK/Stat3 signal of the present invention is a modulator that regulates the JAK/Stat3 signaling process, and includes, but is not limited to, Socs1, Appa, Cis, Pim1, and the like.

The inhibitor of the present invention refers to a compound that targets, decreases or inhibits the JAK/Stat3 signal or its sub-modulators, and is involved in the JAK/Stat3 signal transduction process, and is a compound, peptide, peptidomimetic, and matrix analog. , Aptamers, antibodies, and the like, but are not limited thereto.

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 merely illustrative of the present invention, and the contents of the present invention are not limited by the following examples and experimental examples.

Example 1: Breeding and breeding of zebrafish

Zebrafish, native to India, were bred in a water tank equipped with a closed circulation filtration system that maintained a temperature of 28.5 ℃ and a humidity of 30 to 70%. The daylight cycle was adjusted by performing light treatment for 14 hours a day and dark treatment for 10 hours. Mature zebrafish were fed twice a day with a mixture of Freshwater Aquarium Flakefood (Tetra Werke, Melle, Germany) and live brine shrimp (San Francisco Bay Brand, Inc., Newark, CA, USA). At about 2 to 3 months, they became adults capable of mating. On the afternoon before mating, females and males were placed in a dedicated mating cage, and light was given to induce spawning the next morning. After that, the male and female mated, and the female spawned hundreds of fertilized eggs. The 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 a Petri dish, and generated in an incubator at 28.5°C. Using a dissecting microscope, fertilized eggs at each stage of development were selected according to morphological changes over time and fixed with 4% paraformaldehyde.

Example 2: Preparation of zebrafish expressing hIL-6 gene specifically for liver

In zebrafish, in order to construct a transformation platform in which genes are specifically expressed in liver from the stage of development, liver fatty acid binding protein (LFABP) was selected and the regulatory sequence (5' upstream 2.9kb) was cloned. In order to analyze the specificity of the LFABP regulatory sequence, the LFABP-GFP construct was constructed and LFABP-Gal4/UAS-RFP transgenic zebrafish were prepared and confirmed. The IL-6 gene, which plays an important role in liver cancer by promoting chronic inflammation of the liver, was selected, and the UAS-hIL6-CG transformant was separately established and crossed with the LFABP-Gal4/UAS-RFP transformant. A transformant line expressing hIL-6 (LFABP-Gal4 / UAS-RFP / UAS-hIL6-CG) was prepared. The expression of hIL-6 induced chronic invasion of inflammatory cells in the liver, resulting in damage and regeneration of hepatocytes, and various dysplasias from 3 months of age. At 6 months, AFP-positive hepatocellular carcinoma along with chronic inflammatory changes occurred.

The primers used to prepare the zebrafish are shown in Table 1 below.

[Table 1]

In Table 1, the sequence recognized by the restriction enzyme is underlined, and F-UAS-Seq was used to identify the transformed zebrafish.

Example 3: zebrafish fertilized egg microinjection

The transgene of the zebrafish prepared in Example 2 was injected into the egg yolk of the 1-2 cell stage fertilized eggs obtained in Example 1 by using a microinjector. As a result, the transgene was introduced into the cytoplasm through the contact hole between the cell and the egg yolk, and then inserted into the chromosome during the division period. In order to increase the efficiency of transformation, a transgene and a transferase mRNA (transposase mRNA) were injected together, and the expressed transposase recognized the Tol2 right/left arm and forced the transgene into the chromosome. . Thereafter, parental zebrafish (F0 founder fish) were reared as adults, and then fertilized eggs were produced to select a transformed zebrafish (transgenic F1 progeny) model.

Experimental Example 1: Induction of chronic inflammation by continuous hIL-6 expression

In order to analyze the phenotype of the transgenic zebrafish prepared in Example 3 according to the expression of hIL-6, riboprobe was synthesized through in vitro transcription to perform in-situ hybridization (ISH). After PCR was performed to amplify cDNA produced using primers from zebrafish RNA, a digoxigenin-labelled anti-sense mRNA probe was synthesized using a T7 IVT kit (Roche). . The sequence of the primers used at this time is 5'-ATAACGCGTACCATGAACTCCTTCTCCACAAGC-3' in the forward direction and 5'-ATAGCTAGCCTACATTTGCCGAAGAGCCC-3' in the reverse direction. After fertilization, embryos at each stage were put in 4% paraformaldehyde (PBS, pH 8.0) and fixed at 4°C for 12 hours or longer. After 24 hours, the embryos at the bud stage (10 hours after fertilization) were put in emryonic water to which 0.2mM PTU (phenylthiourea) was added to prevent pigment formation. Then, the embryos were washed three times for 5 minutes with 1X PBST (1XPBS, 0.1% Tween-20) and 100% methanol was changed twice, and then in methanol for a long period of storage to prevent damage to the embryos- Stored at 20°C. Thereafter, the embryos were washed three times with 1X PBST, treated with Proteinase K for 30 minutes, fixed again with 4% paraformaldehyde, and washed with 1X PBST. Next, ^{add 500 µl of HYB ★★} (50% formamide, 5X SSC, 0.1% Tween-20) and leave for 5 minutes at 65°C. HYB ^® (HYB ^★★ , 5 mg/ml torula RNA, 50µg/ml heparin , Citricacid, pH 6.0) was added to 200 µl, prehybridized at 65°C for 2-4 hours, and then 1µl of probe was added and hybridized overnight at 65°C. After removing the solution containing the probe, 75%, 50%, and 25% HYB*/2X SSC solutions were exchanged every 10 minutes in the order of maintaining 65°C. Thereafter, the solution was changed with 2X SSCT and left at the same temperature for 30 minutes, and then the probes that could not be hybridized were removed by grinding two or more times with 0.2X SSCT at 30 minute intervals, and then washed 5 times with 1X PBST for 15 minutes at room temperature. Then, the blocking solution (PBST + 5% goat serum) was treated at room temperature for at least 1 hour, and the anti-digoxigenin antibody (Roche) was diluted to 1/5000 in the blocking solution and then treated for at least 2 hours. After washing 5 times with 1X PBST for 15 minutes each, NBT/BCIP (Roche Diagnostics GmbH) was added to react at room temperature in a state of blocking light, and then color development was confirmed through a microscope, and the results are shown in C, D, F in FIG. And G. After checking the result, it was washed with 1X PBST and then put in 100% glycerol and stored at 4°C.

In addition, in order to analyze the tissue samples of the transgenic zebrafish prepared in Example 3, 50 transgenic zebrafish aged 3 months or longer were subjected to Tris-buffered tricaine (3-aminobenzoic acid ethylester, pH 7.0; Sigma) 20X. Was euthanized. Thereafter, for the histological change screening study, all organs were fixed in 4% paraformaldehyde for 24 hours, alcoholxylene was treated on the fixed organs, and paraffin embedded to make 4 μm slide sections. H&E staining was performed and the results of observation with an optical microscope are shown in H to M of FIG. 1.

As shown in C, D, F, and G of FIG. 1, since the transgenic zebrafish into which the hIL-6 gene was introduced was stained purple specifically for the liver, it can be seen that the hIL-6 gene was introduced. Accordingly, it can be seen that in the transgenic zebrafish prepared in Example 3, the hIL-6 gene is strongly expressed liver-specifically from 4 days after fertilization to adults.

In addition, as shown in H to M of Fig. 1, compared to the H of Fig. 1, which is a control group, the transgenic zebrafish had inflammation in the liver, especially hepatic sinusoid (black arrow) and intrahepatic Various degrees of inflammatory infiltration occurred in blood vessels (intrahepatic vessels, red arrows). In addition, when the invasion was severe, inflammation caused the breakdown of the hepatic lobules, and the discovery of some intrahepatic portal tracts was excluded.Through this, hIL-6 was continuously expressed in the liver of the transgenic zebrafish, leading to chronic hepatitis You can see that it is.

Experimental Example 2: Liver tumor formation with dysplastic foci in zebrafish expressing hIL-6

In order to confirm the formation of inflammation in the liver cells of the transgenic zebrafish prepared in Example 3, H&E staining of the liver tissue of the transgenic zebrafish was performed in the same manner as in Experimental Example 1, and the results are shown in FIGS. Shown in.

In addition, protein expression was confirmed through immunohistochemistry (IHC) in order to confirm the formation of tumors in hepatocytes of the transformed zebrafish. More specifically, the tissue cut to 4 μm was deparaffinized using xylene and then dehydrated using 70%, 80%, 90%, and 100% ethanol. It was immersed in 0.3% H ₂ O ₂ for 20 minutes, reacted for 15 minutes, and washed 3 times for 5 minutes each with 1X PBST. After treatment with blocking solution (10% normal goat serum) for an hour, wash 3 times with 1X PBST for 5 minutes each, and primary antibodies are rabbit anti-IL6 (1:200), 7 mouse anti-proliferating cell nuclear antigen (PCNA) (1:2000), goat anti-AFP (1:200) (Santa Cruz, CA), and the primary antibody was treated at room temperature for one hour. After washing three times with 1X PBST for 5 minutes each, a secondary antibody, goat anti-rabbit alexa fluor 488 (Molecular Probes, Eugene, USA) was treated and reacted at room temperature for 1 hour. After treating the ABC solution (Vector Laboratories) at room temperature for 1 hour, washing twice in 1X PBST for 10 minutes each, and using 3'-diaminobenzidine tetrachloride (DAB, peroxidase substrate kit DAB (Vector Laboratories)). After treatment, it was washed 3 times with 1st distilled water for 10 minutes each. After washing, 85%, 90%, and 100% ethanol were treated for 2 minutes each, then xylene (Sigma) was treated for 5 minutes, and then, when xylene dries, it was sealed and observed with a fluorescence microscope (Olympus BX51, Japan). The results are shown in J to L of FIG. 2.

As shown in Fig. 2A to I, oncosis (A to E) accompanied by a clear cell body, eosinophilic, and basophilic cell body in the liver of a transgenic zebrafish, And tumor formation characteristics (F and G) such as a large cell chage and a hyaline body were observed. More specifically, characteristics of typical liver cancer (HCC) such as eosinophilic granular cytoplasm, rounded nuclei, and prominent nucleoli were found (D), and the 6-month-old zebrafish as a whole. Liver cancer (HCC) appeared (I).

In addition, as shown in J and K of Fig. 2, the transgenic zebrafish showed dysplatic foci in hepatocytes (J and K), and showed a high degree of αFP expression in tumor cells (L ).

Through this, it can be seen that hIL-6 was continuously expressed in transgenic zebrafish hepatocytes, resulting in chronic hepatitis and liver cancer (HCC).

Experimental Example 3: Regulatory mechanism of inflammation and tumor development by hIL-6 overexpression

In order to analyze the mechanism of inflammation and tumor development of the transgenic zebrafish model prepared in Example 3, RT-PCR and western blot were performed.

Experimental Example 3-1: Performing RT-PCR

The liver tissue of the 3-month-old transgenic zebrafish model prepared in Example 3 was removed, homogenized, and then the entire RNA was extracted using an RNeasy mini-kit (QIAGEN Inc., Valencia, CA, USA). . The purified RNA sample was synthesized as cDNA by reverse transcription (RT). The PCR reaction conditions used at this time are shown in Table 2 below.

[Table 2]

After completion of reverse transcription, real-time PCR was performed using the synthesized cDNA, and the degree of expression of the gene was quantitatively evaluated, and the results are shown in A and B of FIG. 3. ABI 7300 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) was used for real-time PCR, and the amount of PCR product was quantified by a relative quantification method (-2ΔΔCt) using a threshold cycle (Ct) value.

Experimental Example 3-2: Western blot performance

The liver tissues of the transgenic zebrafish experimental group and the control group were sampled, and lysis buffer (1% NP, 100 mM MgCl ₂ , 1 M Tris-HCl (8.0), 100 mM sodium fluoric, 100 mM DTT, 2) mM sodium orthovanadate, 1 μg/μl aprotinin, 10 μg/μl pepstatin, 50 mM Hepes) was added each 500 μl to an ependrof tube, put in an ice box, and reacted at 4° C. for 30 minutes, Centrifugation was performed under conditions of 12000 rpm, 4°C, and 20 minutes. After centrifugation, a portion of the supernatant, which is a cell substrate, was taken and protein analysis was performed using a BCA reagent (BCATM Protein Assay Kit, Pierce). First, 100 µl/well of BCA reagent was taken in a 96-well plate, and then a standard reagent (BSA, 2 mg/ml) was added for each concentration (2000, 1000, 500, 250, 125, 62.5 and 31.2 µg/ml) was added by 10 µl each, and then 10 µl of samples were added at a time, and absorbance was measured at 562 nm using a microplate reader. After preparing the 10% gel, SDS-PAGE was performed at 90V for 3 hours, and the gel plate loaded on the nitrocellulose membrane (Bio-rad) was carefully moved, and then stirred at 250mA, 4°C for 2 hours and 30 minutes. A membrane transfer was performed using. Thereafter, the band was stained with 0.2% ponceau s reagent (Biobasic Inc.) for 1 minute, washed several times with distilled water, and then the protein band loaded on the membrane was confirmed. After fixing the film in a film development kit for film development, a SuperSignal West Pico Chemiluminescent Substrate reagent (Pierce) was sufficiently applied to the film, and then reacted at room temperature for 5 minutes. Thereafter, the film was reacted to the film in a dark room for a certain period of time, and the protein band was confirmed using a developer and a fixation reagent (Kodak GBX), and the results are shown in C of FIG. 3.

3A and 3B, the transgenic zebrafish up-expressed c-Myc, which is known to regulate cell growth, and MAPK1 and cyclin D1 (CcnD1), which are known as cell survival and cell cycle regulators, and inflammation. The expression of IL1b and IL12a, which are known as indicators of the response, was also increased. Eif10, Appa, Socs, Pim1, and Cisd, which are sub-regulators of the JAK/Stat3 and P13K/Akt signaling pathways, were also up-expressed.

In addition, as shown in C of FIG. 3, pMAPK, mP13K and pStat3, sub-regulatory proteins of the JAK/Stat3 and P13K/Akt signaling pathways, were expressed to a high degree in the liver of the transgenic zebrafish.

Through this, it can be seen that overexpression of hIL-6 in transgenic zebrafish induces proliferation and inflammation of hepatocytes, and increases the expression of regulators of the JAK/Stat3 and P13K/Akt signaling pathways, resulting in liver cancer. .

Experimental Example 4: Identification of signaling pathways and mechanisms of hepatic tumor formation of sub-regulators

Expression patterns of sub-regulators using immunohistochemistry (IHC) and in-situ hybridization (ISH) to specifically confirm the mechanism of liver cancer incidence of the transgenic zebrafish prepared in Example 3 above. Was confirmed.

Immunohistochemistry (IHC) was performed in the same manner as in Experimental Example 3, and the primary antibodies used at this time were rabbit anti-IL6 (1:200), rabbit anti-Caspase 3 (1:100), and mouse anti-proliferating. cell nuclear antigen (PCNA)(1:2000), Anti-AKT1 (phospho T308)(1:200)(Abcam, Cambridge, MA), Mouse anti-phospho-PI3K (1:100) Rabbit antibodies, phospho-Tuberin/ TSC2 (1:200), phospho-mTOR (1:200), phospho-4EBP1 (1:200), phospho-p70RS6K(1:200), phospho-Stat3 (1:200), β-actin (1:300 )(Cell Signaling ,Danvers, MA), anti-AFP(C-19) (1:200)(Santa cruz, CA), Anti-Caspase-3a (p17)(NT) (1:500), Anti-MAPK -1/2 (CT), Z-FishTM (1:500), Anti-Cyclin-D1 (NT) (1:500) (ANASPEC, Fremont, CA), and the results are shown in A to H and Figs. It is shown in A to D of 5.

In vivo hybridization (ISH) was performed in the same manner as in Experimental Example 1, and in particular, the analysis of inflammatory infiltrating cells was performed by an ISH experiment using a leukocyte marker, and the results are shown in FIGS. Shown in. The nucleotide sequence of the primers for preparing riboprobes used when performing the ISH method is shown in Table 3 below.

[Table 3]

As shown in Figure 4, it can be seen that apoptosis occurs in hepatocytes expressing hIL-6 through the Caspase3 antibody reaction (B). In addition, CD4-positive and CD8-positive T cells appeared together in hepatic parenchyma (J, L), and Ighm-positive B cells and Spi1-positive bone marrow cells were also associated with hepatic sinusoids. It was found in some portal tracts of the liver (N, P). Expression of PCNA, pMARK, and CyclinD1, which are cell proliferation markers, were uniformly expressed in the areas accompanied by dysplastic foci and non-dysplastic foci (D, F, H). Through this, it can be seen that inflammation and inflammatory invasion accompanied by cell damage are occurring in the zebrafish liver where hIL-6 is overexpressed.

In addition, as shown in FIG. 5, it was found that all cells expressing hIL-6 reacted with pStat3 (D), and the expression of pStat3 was most strongly in the site accompanied by dysplastic foci. (B, C). In addition, invasive inflammatory cells were non-responsive to pStat3, and sub-regulators of the JAK/Stat3 signaling pathway such as Socs1, Appa, Cis, and Pim1 were strongly expressed in the pleiotropic proliferative site (F to I). . This suggests that the JAK/Stat3 pathway is the main cause of liver cancer.

Experimental Example 5: Identification of the cause of liver cancer induced by hIL-6 through JAK/Stat3 pathway inhibitor treatment

In the transgenic zebrafish model prepared in Example 3, in order to confirm whether the JAK/Stat3 signaling pathway is the cause of liver inflammation and liver cancer, the signaling pathway was suppressed. More specifically, PI3K inhibitors BKM120 and Stat3 inhibitors Nicolsamide and Stattic were treated with transgenic zebrafish, and molecular cytological analysis of sub-regulators was performed. In the course of treatment, these inhibitors could be fatal to zebrafish, so they were treated at no more than 25% and gradually increased their concentration to induce inhibition.

The RT-PCR results performed in the same manner as in Experimental Example 3-1 are shown in FIG. 6A, and the immunohistochemistry (IHC; immunohistochemistry) and in vivo hybridization (ISH) performed in the same manner as in Experimental Example 4 ; in situ hybridization) is shown in Fig. 6B.

As shown in Fig. 6, treatment with Stat3 inhibitor showed a gradual decrease in expression of sub-regulators of the JAK/Stat3 pathway, and BKM120 did not inhibit the PI3K pathway (A). The results of IHC and ISH assays also show that the expression of sub-regulators of the JAK/Stat3 pathway such as Appa1, Cis, and Pim1 shows a marked decrease in expression by Stagt3 inhibitors (B), and through this, the proliferation of hepatocytes is caused by JAK/Stat3 signaling. It can be seen that it is generated by the path (B in FIG. 6, red circle).

Therefore, it can be seen that the JAK/Stat3 signaling pathway in transgenic zebrafish is a direct cause of liver cancer induced by overexpression of hIL-6.

<110> Industry-Academic Cooperation Foundation, Yonsei University <120> Transgenic zebrafish for expressing a liver-specific hIL-6 gene and method for producing thereof <130> YONSEI_1_17P <160> 3 <170> KopatentIn 2.0 <210> 1 <211> 10035 <212> DNA <213> Artificial Sequence <220> <223> LFABP2.8kb-Gal4VP16 <400> 1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc 60 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga 120 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc 180 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc 240 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag 300 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa 360 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac 420 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg 480 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg 540 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg 600 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tgggtaccaa 660 atagtaggaa ttacccacct gtacaagtgc tgaaaacttg gatgaataag cccgtttgcc 720 attttctact gctattttaa atcttttctg tatttgtctg catttgtctt tacccttgaa 780 taaatgtttt agttgttttt tttcaattat gactgtgttt aacagacatt attacaataa 840 tatgtaatag tagtacacac tattattatg taatagtact tgttgactgt atttgagaac 900 tggaccgagt gagtgttacg tcacccattc aaaatgactt acttctggct ccaacaaaat 960 gaagttaatt cagttgccat ttttcactgt atggacatcg ccgtgttgga gctagacgtc 1020 gccaataagc aagaaagagc cgagatgcgt cgagtctgag tcaccgttcc tatggcaacc 1080 cctctaacca atcagaagta agcttgttgg aagtccacag cctaccactt gaaagcgggc 1140 tgcacaaaat ctgtcaaacg ttttgaacgt tggatgtgag agcacatact tttattaagg 1200 catctggttg gtcagtttat aatatcaaca acttgggcta cagaaaagaa aagttattac 1260 agaaattatg attaacaagt acatgttaaa taaagatttt aatatgaatg ccaccactgg 1320 agcattcatg ccatttggag cttcttcctg tttggatcac tagaaggagg aggtcactca 1380 ttacagttct catatacagt cgttggttgg ttggttggtt ggttggttgg tagattgatt 1440 gattgattga ttgattgatt gattgattga ttgattgatt gattgattga ttgattgatt 1500 gattgattga ttgattgatt gattgattga ttgattggta gtcaaaataa gaaataattt 1560 ccacagattc attacagaaa tgattaaatg catacataaa aaactggggg gggggggata 1620 caacaacaca cttaagtcac atttgcctac gtaaagaaaa gtaaagaaaa tcaatagcta 1680 tattttacat ctcctttttt tgctgtcttt taattagcct tgttttgctg ttatcttgat 1740 tgaaactgta acttcttcac ctgcttcttt tctttgtagg ttttgccagc ccagaaacag 1800 gcatggctgt gcaaggccag agcaccatgc acaatgcgct gcatgtcttc atgaacggct 1860 caatgtcctc agtccagggc tcagccaacg accccatctt cctccttcac catgctttca 1920 ttgacaggta acaaacacgt catgacatta gactgcacag tttttgacaa agttcataca 1980 atctgttgtt tatagctgct acaattagtg aagtttgtga atgtacttgg atgagcagcg 2040 aaagatcaat tgagatcaat tgttagagtt tggttgccct gcagagcaaa gaacaaaaaa 2100 taatctggtg gctttactgc gtgaggttat tattggtgga atagaaacac aaaacataat 2160 tgcatttatt tgtttaattt tttatcttat cttaactttc atcttgcata tttgtttctt 2220 acatcatttc tagcatcttt gagcgctggc taagaactca tcagcctccc cggtccatct 2280 acccacgtac caatgcacca attggccaca atgacggcta ctacatggtg ccattccttc 2340 ctctttatag gaatggagac tacctcctgt ccaacaaggc tcttggatac gagtacgcct 2400 acctgttgga cccaggtcat tgcacaacac cagaaatgcc ctctgatctg caaaagacgt 2460 gaatatctgt tcagacaccc atatccactc tgttccacac aggtcagagg tttgtccagg 2520 agttcttgac atatttgttt cttacatcat ttctagcatc tttgagcgct ggctaagaac 2580 tcatcagcct ccccggtcca tctactcacg taccaatgca ccaattggcc acaatgacgg 2640 ctactacatg gtgccattcc ttcctcttta taggaatgga gactacctcc tgtccaacaa 2700 tgctcttgga tacgagtacg cctacctgtt ggacccaggt cattgcacaa caccagaaat 2760 gccctctgat ctgcaaaaga cgtgaatatc tgttcagaca cccatatcca ctctgttcca 2820 cacaggtcag aggtttgtcc aggagttctt gacagaggtg taaaaagtac tcaaaaattt 2880 tactcaagtg aaagtacaag tacttaggga aaattttact caattaaaag taaaagtatc 2940 tggctagaat cttacttgag taaaagtaaa aaagtactcc attaaaattg tacttgagta 3000 ttaaggaagt aaaagtaaaa gcaagaaaga aaactagaga ttcttgttta agcttttaat 3060 ctcaaaaaac attaaatgaa atgcatacaa ggttttatcc tgctttagaa ctgtttgtat 3120 ttaattatca aactataaga cagacaatct aatgccagta cacgctactc aaagttgtaa 3180 aacctcagat ttaacttcag tagaagctga ttctcaaaat tgttagtgtc aagcctagct 3240 cttttggggc tgaaaagcaa tcctgcagtg ctgaaaagcc tctcacaggc agccgatgcg 3300 ggaagaggtg tattagtctt gatagagagg ctgcaaatag caggaaacgt gagcagagac 3360 tccctggtgt ctgaaacaca ggccagatgg gccctcgatc tgctgcagtt cgaactgagc 3420 atcagaatgg ggaaggaaga ggatttcagt gactttgaac gaggcatggt tgttgctgcc 3480 agatgggctg ctctgagtat ttcagaaact gctgatcttc agggattttc acgcacaacc 3540 atctctaggg tttacagaga atgatctgaa aagaggaaat atccagtgag cggcagttct 3600 gtgggtgcaa atgccttgtt gatgccagag atcagaggag aatggccaga ctggttccag 3660 ctgatagaaa ggcaacagta actcaaataa gcactcgtta caaccgagct ctgcagaaga 3720 gcatctctga acacacaaca cgtccaacct tgaggcagat gggctacagc agcagaagac 3780 cacaccgggt gccgctcctg tcagctaaga acaggaaact gaggctacaa ttcacacaga 3840 ctcaccaaaa ctggacaata tattattagc cccccttaga atttttcatt tgataatatt 3900 tttcttctgg cgaaagcctc atttgtttta tatattatag aataaaatta gtttttaata 3960 gtttttatgc cattttaagg tcaatattat tagccccttt aagctatttt ttttcgatag 4020 tctacagaac aaaccatcgg tatacaatga cttgcctaat taccctaacc tgcctagtta 4080 ccctaattaa cctagttaag cctttaaatg tcactttaag ctgtatagaa gtgtcttgaa 4140 gaatatctag tctaatatta ttgactgtca tcatggcaaa gataaaataa atcagttatt 4200 aaaactatta tgattagaaa tgtgctgaaa caatctgctc tccgataaac agaaattgaa 4260 caaaataaac aggggggcta ataaatttaa ggggttaaat aattctgatt gcaaaaaaat 4320 gatgtctgca aaactgttgc aaataattta tttgtgttga ttttaagcaa acaaattaaa 4380 tttaataaaa tacaacttaa tctgtttgtt taaattcagc cctaataaat tgtttacagc 4440 cacttaacgt aaaaaaattg agtaaatcca aggaatcatc tctgaataat ttttttcagt 4500 gtatatatat acatatatat tcttacaaaa caactcattt actttagttt aattttcagg 4560 ggcaaaaaac taaagtaatc gacgttgctt gaataaaaag tgtaattaag ggaatgaggt 4620 aacatttaac catgtgtcaa tgcagtttaa atatgccagt tagtggtata tgtttaaatg 4680 gtaagctatt caaaacttta aactaactta accagccttt tgttgtcaga ctgaacagac 4740 tctccatctg cattattaga gactaatctt tggctggatg aatgattcat ctgctgatat 4800 ttcagaatag acagattgag gctgtttcta atatgattat gcaacctgag ggtgattatt 4860 tgaagcaaac tccacagacc agcaggtcat tgaccgtcgt gtgttcaaac agagcagaaa 4920 catttgcaaa actggtctga caggagaatc cagtccagca caacacatat gctgagcaaa 4980 ctgaatcaat cctgcaggtc aactctcgtg ctttaagttt attaagatta ttttatttat 5040 ttattatttt atttatctat ttatttattt agttgtttat ttagcaatta tttgtttatt 5100 tatttgttta tttattcctg cagatcatgc cttgtgcctt tttacattta atttaatttt 5160 taatttaatt tccttttatt tttttttatt tttttatttt attttatttt acagtctgac 5220 gaatactgaa ctaaaaaccc tcagatcatg tcttatgcat ttcattttat tttatttcat 5280 tttatattat taattttaat atttttattt tacagtctga caaatactga attaaaacca 5340 tcagatcatg tctcatgcat ctaacttaac tttatttaat tcaattaaat tgtttgtttg 5400 tttgtttcct tgcatttgtt tgtttgtttt ttacaatctg acatactgga ccgaaaaaac 5460 tcagatcatg tcttatgcat ttattttatt tattttttat tttatgtcta ttttatttta 5520 tttcatctta ttttatttta cagtctgaca tatggaccaa taaatctctc agatcatgtc 5580 tcatgcattt aatttaattt ttttatttat tcatttttat ttatttattt tattatttat 5640 tttattttaa tatttatttt attttatttt attttatttt acagtctgac atactggacc 5700 aataaatctc tcagatcatg tctcatgcat tttactttta ttttattaga attagaaaga 5760 tcaaaggaac aacttttaaa atattaattc tgtatcaaaa tctcttttga tacatttaat 5820 tgatttaaaa aagcagttca cccaagaaac atttcctcac agtcgaatgg ttgtaaactt 5880 ttatgaatta ctttcacaga aaaagatttt tggaagaata ttggaaaaaa agcagccgtt 5940 gacttccata gtaacaacaa aaaatactat ggaagtcaat ggctgttttt tcaccattcg 6000 gtatcttcat tctggagcag aattttttgg gtgatctgtc cctttaagtc gtcaaatcct 6060 ggtgcaatat tccacatgca ctgacgagtc tttatttttg gtctgctact gctgtgtgtg 6120 tgagggcatt tacctcatct tctgctggag ttgatgaacg gtgggttgtt caaacagcag 6180 caggtcattg actgaactcc tctcgatata aaagctgcag atctgaagct gaccttcact 6240 ttgtgttgag cttctccaga aagccatgga gatgaagcta ctgtcttcta tcgaacaagc 6300 atgcgatatt tgccgactta aaaagctcaa gtgctccaaa gaaaaaccga agtgcgccaa 6360 gtgtctgaag aacaactggg agtgtcgcta ctctcccaaa accaaaaggt ctccgctgac 6420 tagggcacat ctgacagaag tggaatcaag gctagaaaga ctggaacagc tatttctact 6480 gatttttcct cgagaagacc ttgacatgat tttgaaaatg gattctttac aggatataaa 6540 agcattgtta acaggattat ttgtacaaga taatgtgaat aaagatgccg tcacagatag 6600 attggcttca gtggagactg atatgcctct aacattgaga cagcatagaa taagtgcgac 6660 atcatcatcg gaagagagta gtaacaaagg tcaaagacag ttgactgtat cttcgaggtc 6720 gaccccggga attcagatct ctcgagccgc ccccccgacc gatgtcagcc tgggggacga 6780 gctccactta gacggcgagg acgtggcgat ggcgcatgcc gacgcgctag acgatttcga 6840 tctggacatg ttgggggacg gggattcccc gggtccggga tttacccccc acgactccgc 6900 cccctacggc gctctggata tgtagatcga tgatgatcca gacatgataa gatacattga 6960 tgagtttgga caaaccacaa ttagaatgca gtgaaaaaaa tgctttattt gtgaaatttg 7020 tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa 7080 ttgcattcat tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt aaagcaagta 7140 aaacctctac aaatgtggta tggctgatta tgatcctcta gatcaagatc tgcgaagata 7200 cggccacggg tgctcttgat cctgtggctg attttggact gtgctgctcg cagctgctga 7260 tgaatcacat acttcctcca ttttcttcca ctgattgact gttataattt ccctaatttc 7320 caggtcaagg tgctgtgcat tgtggtaata gatgtgacat gacgtcactt ccaaaggacc 7380 aatgaacatg tctgaccaat ttcatataat gtgaaaacga ttttcatagg cagaataaat 7440 aacatttaaa ttaaactggg catcagcgca attcaattgg tttggtaata gcaagggaaa 7500 atagaatgaa gtgatctcca aaaaataagt actttttgac tgtaaataaa attgtaagga 7560 gtaaaaagta cttttttttc taaaaaaatg taattaagta aaagtaaaag tattgatttt 7620 taattgtact caagtaaagt aaaaatcccc aaaaataata cttaagtaca gtaatcaagt 7680 aaaattactc aagtacttta cacctctggt tcttgacccc ctaccttcag caagcccagc 7740 agatccacta gttctagagc gttcttgacc ccctaccttc agcaagccca gcagatccac 7800 tagttctaga gcggccgcca ccgcggtgga gctccagctt ttgttccctt tagtgagggt 7860 taattgcgcg cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc 7920 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat 7980 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc 8040 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg 8100 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag 8160 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag 8220 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc 8280 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc 8340 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc 8400 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt 8460 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg 8520 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat 8580 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag 8640 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt 8700 ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc 8760 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta 8820 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag 8880 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga 8940 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa 9000 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa 9060 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc 9120 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga 9180 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa 9240 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt 9300 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg 9360 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc 9420 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg 9480 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag 9540 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt 9600 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt 9660 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac 9720 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac 9780 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag 9840 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa 9900 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga 9960 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc 10020 cccgaaaagt gccac 10035 <210> 2 <211> 9587 <212> DNA <213> Artificial Sequence <220> <223> UAS-hIL6-CG <400> 2 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc 60 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga 120 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc 180 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc 240 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag 300 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa 360 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac 420 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg 480 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg 540 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg 600 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tgggtaccaa 660 atagtaggaa ttacccacct gtacaagtgc tgaaaacttg gatgaataag cccgtttgcc 720 attttctact gctattttaa atcttttctg tatttgtctg catttgtctt tacccttgaa 780 taaatgtttt agttgttttt tttcaattat gactgtgttt aacagacatt attacaataa 840 tatgtaatag tagtacacac tattattatg taatagtact tgttgactgt atttgagaac 900 tggaccgagt gagtgttacg tcacccattc aaaatgactt acttctggct ccaacaaaat 960 gaagttaatt cagttgccat ttttcactgt atggacatcg ccgtgttgga gctagacgtc 1020 gccaataagc aagaaagagc cgagatgcgt cgagtctgag tcaccgttcc tatggcaacc 1080 cctctaacca atcagaagta agcttgttgg aagtccacag cctaccactt gaaagcgggc 1140 tgcacaaaat ctgtcaaacg ttttgaacgt tggatgtgag agcacatact tttattaagg 1200 catctggttg gtcagtttat aatatcaaca acttgggcta cagaaaagaa aagttattac 1260 agaaattatg attaacaagt acatgttaaa taaagatttt aatatgaatg ccaccactgg 1320 agcattcatg ccatttggag cttcttcctg tttggatcac tagaaggagg aggtcactca 1380 ttacagttct catatacagt cgttggttgg ttggttggtt ggttggttgg tagattgatt 1440 gattgattga ttgattgatt gattgattga ttgattgatt gattgattga ttgattgatt 1500 gattgattga ttgattgatt gattgattga ttgattggta gtcaaaataa gaaataattt 1560 ccacagattc attacagaaa tgattaaatg catacataaa aaactggggg gggggggata 1620 caacaacaca cttaagtcac atttgcctac gtaaagaaaa gtaaagaaaa tcaatagcta 1680 tattttacat ctcctttttt tgctgtcttt taattagcct tgttttgctg ttatcttgat 1740 tgaaactgta acttcttcac ctgcttcttt tctttgtagg ttttgccagc ccagaaacag 1800 gcatggctgt gcaaggccag agcaccatgc acaatgcgct gcatgtcttc atgaacggct 1860 caatgtcctc agtccagggc tcagccaacg accccatctt cctccttcac catgctttca 1920 ttgacaggta acaaacacgt catgacatta gactgcacag tttttgacaa agttcataca 1980 atctgttgtt tatagctgct acaattagtg aagtttgtga atgtacttgg atgagcagcg 2040 aaagatcaat tgagatcaat tgttagagtt tggttgccct gcagagcaaa gaacaaaaaa 2100 taatctggtg gctttactgc gtgaggttat tattggtgga atagaaacac aaaacataat 2160 tgcatttatt tgtttaattt tttatcttat cttaactttc atcttgcata tttgtttctt 2220 acatcatttc tagcatcttt gagcgctggc taagaactca tcagcctccc cggtccatct 2280 acccacgtac caatgcacca attggccaca atgacggcta ctacatggtg ccattccttc 2340 ctctttatag gaatggagac tacctcctgt ccaacaaggc tcttggatac gagtacgcct 2400 acctgttgga cccaggtcat tgcacaacac cagaaatgcc ctctgatctg caaaagacgt 2460 gaatatctgt tcagacaccc atatccactc tgttccacac aggtcagagg tttgtccagg 2520 agttcttgac atatttgttt cttacatcat ttctagcatc tttgagcgct ggctaagaac 2580 tcatcagcct ccccggtcca tctactcacg taccaatgca ccaattggcc acaatgacgg 2640 ctactacatg gtgccattcc ttcctcttta taggaatgga gactacctcc tgtccaacaa 2700 tgctcttgga tacgagtacg cctacctgtt ggacccaggt cattgcacaa caccagaaat 2760 gccctctgat ctgcaaaaga cgtgaatatc tgttcagaca cccatatcca ctctgttcca 2820 cacaggtcag aggtttgtcc aggagttctt gacagaggtg taaaaagtac tcaaaaattt 2880 tactcaagtg aaagtacaag tacttaggga aaattttact caattaaaag taaaagtatc 2940 tggctagaat cttacttgag taaaagtaaa aaagtactcc attaaaattg tacttgagta 3000 ttaaggaagt aaaagtaaaa gcaagaaaga aaactagaga ttcttgttta agcttttaat 3060 ctcaaaaaac attaaatgaa atgcatacaa ggttttatcc tgctttagaa ctgtttgtat 3120 ttaattatca aactataaga cagacaatct aatgccagta cacgctactc aaagttgtaa 3180 aacctcagat ttaacttcag tagaagctga ttctcaaaat tgttagtgtc aagcctagct 3240 cttttggggc tgaaaagcaa tcctgcagtg ctgaaaagcc tctcacaggc agccgatgcg 3300 ggaagaggtg tattagtctt gatagagagg ctgcaaatag caggaaacgt gagcagagac 3360 tccctggtgt ctgaaacaca ggccagatgg gcccctctgc taaccatgtt catgccttct 3420 tctctttcct acagctcctg ggcaacgtgc tggttgttgt gctgtctcat cattttggca 3480 aagaattcct cgacggatct catcaagctt aggcctccaa ggcggagtac tgtcctccgg 3540 gctggcggag tactgtcctc cggcaaggtc ggagtactgt cctccgacac tagaggtcgg 3600 agtactgtcc tccgacgcaa ggcggagtac tgtcctccgg gctgcggagt actgtcctcc 3660 ggcaaggtcg gagtactgtc ctccgacact agaggtcgga gtactgtcct ccgacgcaag 3720 gtcggagtac tgtcctccga cactagaggt cggagtactg tcctccgacg caaggtcgga 3780 gtactgtcct ccgacactag aggtcggagt actgtcctcc gacgcaaggc ggagtactgt 3840 cctccgggct ggcggagtac tgtcctccgg caagggtcga ctctagaggg tatataatgg 3900 atcccatcgc gtctcagcct cactttgagc tcctccacac gaattccctc gacctcgaag 3960 acgcgtatga actccttctc cacaagcgcc ttcggtccag ttgccttctc cctggggctg 4020 ctcctggtgt tgcctgctgc cttccctgcc ccagtacccc caggagaaga ttccaaagat 4080 gtagccgccc cacacagaca gccactcacc tcttcagaac gaattgacaa acaaattcgg 4140 tacatcctcg acggcatctc agccctgaga aaggagacat gtaacaagag taacatgtgt 4200 gaaagcagca aagaggcact ggcagaaaac aacctgaacc ttccaaagat ggctgaaaaa 4260 gatggatgct tccaatctgg attcaatgag gagacttgcc tggtgaaaat catcactggt 4320 cttttggagt ttgaggtata cctagagtac ctccagaaca gatttgagag tagtgaggaa 4380 caagccagag ctgtgcagat gagtacaaaa gtcctgatcc agttcctgca gaaaaaggca 4440 aagaatctag atgcaataac cacccctgac ccaaccacaa atgccagcct gctgacgaag 4500 ctgcaggcac agaaccagtg gctgcaggac atgacaactc atctcattct gcgcagcttt 4560 aaggagttcc tgcagtccag cctgagggct cttcggcaaa tgtaggctag cggccgcgac 4620 tctagatcat aatcagccat accacatttg tagaggtttt acttgcttta aaaaacctcc 4680 cacacctccc cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt aacttgttta 4740 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat 4800 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tacatatgaa 4860 agcttaaatc agttgtgtta aataagagac attcaaaata aatgtaaatg agctctccaa 4920 atcagcagac ttaacattct ttaaaatgat tgattcaata gtgataaaaa tcaggcatag 4980 ccagttgtaa ctttagataa attacagaaa atgtcaaata cagagaaccg attctttttt 5040 atgatacatc caagcacaca tttaacacaa tccaggcaaa ccccgaattt cacagtcaca 5100 agcactgttt gtacaagagc tttgcctaag gacacacagt ctctataagt ccaggtcgtt 5160 ggtttcactc ttattttaaa catgtgacat ttttcctgcc atcctgtctt aggctgctgt 5220 ttgcttcatt ccatgtcaca ttaaattcct cagtagcacc ttttacacac acagccaatc 5280 ttttccagaa aattcaattg ctttgaagag ataatgtgtg aacaaatcca tttagaaaag 5340 gaaaattaag aatttgtaaa atcatctgta aattgttggc attcttctgt atatgaacat 5400 cacatcattt acaggtaaag gtctggtcat taattatatg acaatttact ggtattattt 5460 tgtgaaaggg gctattttca atgcgttcat ccatcctttt catccctcaa atctctcatt 5520 cacgtccccc tccccatctg cacactttat ctcattttcc accctgctgg aatctgagca 5580 cttgtgcagt tatcagggct cctgtattta ggaggctctg ggtgtccatg taggggacga 5640 acagaaacac tgcagacctt tatagaagaa caattgataa gagtcctcat acataaagac 5700 tccattagta agccagtgac ccaggagccc agaccaacag caaagcagac agtgaccatg 5760 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc 5820 gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc 5880 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc 5940 gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag 6000 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc 6060 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg 6120 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag 6180 ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc 6240 atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac 6300 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac 6360 ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg 6420 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtaaatc 6480 gatgatgatc cagacatgat aagatacatt gatgagtttg gacaaaccac aattagaatg 6540 cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt 6600 ataagctgca ataaacaagt taacaacaac aattgcattc attttatgtt tcaggttcag 6660 ggggaggtgt gggaggtttt ttaaagcaag taaaacctct acaaatgtgg tatggctgat 6720 tatgatcctc tagatcaaga tctgcgaaga tacggccacg ggtgctcttg atcctgtggc 6780 tgattttgga ctgtgctgct cgcagctgct gatgaatcac atacttcctc cattttcttc 6840 cactgattga ctgttataat ttccctaatt tccaggtcaa ggtgctgtgc attgtggtaa 6900 tagatgtgac atgacgtcac ttccaaagga ccaatgaaca tgtctgacca atttcatata 6960 atgtgaaaac gattttcata ggcagaataa ataacattta aattaaactg ggcatcagcg 7020 caattcaatt ggtttggtaa tagcaaggga aaatagaatg aagtgatctc caaaaaataa 7080 gtactttttg actgtaaata aaattgtaag gagtaaaaag tacttttttt tctaaaaaaa 7140 tgtaattaag taaaagtaaa agtattgatt tttaattgta ctcaagtaaa gtaaaaatcc 7200 ccaaaaataa tacttaagta cagtaatcaa gtaaaattac tcaagtactt tacacctctg 7260 gttcttgacc ccctaccttc agcaagccca gcagatccac tagttctaga gcgttcttga 7320 ccccctacct tcagcaagcc cagcagatcc actagttcta gagcggccgc caccgcggtg 7380 gagctccagc ttttgttccc tttagtgagg gttaattgcg cgcttggcgt aatcatggtc 7440 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg 7500 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt 7560 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg 7620 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga 7680 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat 7740 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca 7800 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc 7860 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata 7920 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc 7980 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc 8040 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga 8100 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc 8160 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag 8220 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag 8280 gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag 8340 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca 8400 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga 8460 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat 8520 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga 8580 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg 8640 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga 8700 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc 8760 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac 8820 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc 8880 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc 8940 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc 9000 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt 9060 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc 9120 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg 9180 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag 9240 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat 9300 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc 9360 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa 9420 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta 9480 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa 9540 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccac 9587 <210> 3 <211> 7804 <212> DNA <213> Artificial Sequence <220> <223> UAS-RFP <400> 3 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc 60 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga 120 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc 180 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc 240 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag 300 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa 360 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac 420 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg 480 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg 540 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg 600 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tgggtaccaa 660 atagtaggaa ttacccacct gtacaagtgc tgaaaacttg gatgaataag cccgtttgcc 720 attttctact gctattttaa atcttttctg tatttgtctg catttgtctt tacccttgaa 780 taaatgtttt agttgttttt tttcaattat gactgtgttt aacagacatt attacaataa 840 tatgtaatag tagtacacac tattattatg taatagtact tgttgactgt atttgagaac 900 tggaccgagt gagtgttacg tcacccattc aaaatgactt acttctggct ccaacaaaat 960 gaagttaatt cagttgccat ttttcactgt atggacatcg ccgtgttgga gctagacgtc 1020 gccaataagc aagaaagagc cgagatgcgt cgagtctgag tcaccgttcc tatggcaacc 1080 cctctaacca atcagaagta agcttgttgg aagtccacag cctaccactt gaaagcgggc 1140 tgcacaaaat ctgtcaaacg ttttgaacgt tggatgtgag agcacatact tttattaagg 1200 catctggttg gtcagtttat aatatcaaca acttgggcta cagaaaagaa aagttattac 1260 agaaattatg attaacaagt acatgttaaa taaagatttt aatatgaatg ccaccactgg 1320 agcattcatg ccatttggag cttcttcctg tttggatcac tagaaggagg aggtcactca 1380 ttacagttct catatacagt cgttggttgg ttggttggtt ggttggttgg tagattgatt 1440 gattgattga ttgattgatt gattgattga ttgattgatt gattgattga ttgattgatt 1500 gattgattga ttgattgatt gattgattga ttgattggta gtcaaaataa gaaataattt 1560 ccacagattc attacagaaa tgattaaatg catacataaa aaactggggg gggggggata 1620 caacaacaca cttaagtcac atttgcctac gtaaagaaaa gtaaagaaaa tcaatagcta 1680 tattttacat ctcctttttt tgctgtcttt taattagcct tgttttgctg ttatcttgat 1740 tgaaactgta acttcttcac ctgcttcttt tctttgtagg ttttgccagc ccagaaacag 1800 gcatggctgt gcaaggccag agcaccatgc acaatgcgct gcatgtcttc atgaacggct 1860 caatgtcctc agtccagggc tcagccaacg accccatctt cctccttcac catgctttca 1920 ttgacaggta acaaacacgt catgacatta gactgcacag tttttgacaa agttcataca 1980 atctgttgtt tatagctgct acaattagtg aagtttgtga atgtacttgg atgagcagcg 2040 aaagatcaat tgagatcaat tgttagagtt tggttgccct gcagagcaaa gaacaaaaaa 2100 taatctggtg gctttactgc gtgaggttat tattggtgga atagaaacac aaaacataat 2160 tgcatttatt tgtttaattt tttatcttat cttaactttc atcttgcata tttgtttctt 2220 acatcatttc tagcatcttt gagcgctggc taagaactca tcagcctccc cggtccatct 2280 acccacgtac caatgcacca attggccaca atgacggcta ctacatggtg ccattccttc 2340 ctctttatag gaatggagac tacctcctgt ccaacaaggc tcttggatac gagtacgcct 2400 acctgttgga cccaggtcat tgcacaacac cagaaatgcc ctctgatctg caaaagacgt 2460 gaatatctgt tcagacaccc atatccactc tgttccacac aggtcagagg tttgtccagg 2520 agttcttgac atatttgttt cttacatcat ttctagcatc tttgagcgct ggctaagaac 2580 tcatcagcct ccccggtcca tctactcacg taccaatgca ccaattggcc acaatgacgg 2640 ctactacatg gtgccattcc ttcctcttta taggaatgga gactacctcc tgtccaacaa 2700 tgctcttgga tacgagtacg cctacctgtt ggacccaggt cattgcacaa caccagaaat 2760 gccctctgat ctgcaaaaga cgtgaatatc tgttcagaca cccatatcca ctctgttcca 2820 cacaggtcag aggtttgtcc aggagttctt gacagaggtg taaaaagtac tcaaaaattt 2880 tactcaagtg aaagtacaag tacttaggga aaattttact caattaaaag taaaagtatc 2940 tggctagaat cttacttgag taaaagtaaa aaagtactcc attaaaattg tacttgagta 3000 ttaaggaagt aaaagtaaaa gcaagaaaga aaactagaga ttcttgttta agcttttaat 3060 ctcaaaaaac attaaatgaa atgcatacaa ggttttatcc tgctttagaa ctgtttgtat 3120 ttaattatca aactataaga cagacaatct aatgccagta cacgctactc aaagttgtaa 3180 aacctcagat ttaacttcag tagaagctga ttctcaaaat tgttagtgtc aagcctagct 3240 cttttggggc tgaaaagcaa tcctgcagtg ctgaaaagcc tctcacaggc agccgatgcg 3300 ggaagaggtg tattagtctt gatagagagg ctgcaaatag caggaaacgt gagcagagac 3360 tccctggtgt ctgaaacaca ggccagatgg gcccctctgc taaccatgtt catgccttct 3420 tctctttcct acagctcctg ggcaacgtgc tggttgttgt gctgtctcat cattttggca 3480 aagaattcct cgacggatct catcaagctt aggcctccaa ggcggagtac tgtcctccgg 3540 gctggcggag tactgtcctc cggcaaggtc ggagtactgt cctccgacac tagaggtcgg 3600 agtactgtcc tccgacgcaa ggcggagtac tgtcctccgg gctgcggagt actgtcctcc 3660 ggcaaggtcg gagtactgtc ctccgacact agaggtcgga gtactgtcct ccgacgcaag 3720 gtcggagtac tgtcctccga cactagaggt cggagtactg tcctccgacg caaggtcgga 3780 gtactgtcct ccgacactag aggtcggagt actgtcctcc gacgcaaggc ggagtactgt 3840 cctccgggct ggcggagtac tgtcctccgg caagggtcga ctctagaggg tatataatgg 3900 atcccatcgc gtctcagcct cactttgagc tcctccacac gaattccctc gacctcgaag 3960 acgcgtctgt ttgggatcca ccggtcgcca ccatggcctc tttgctgaag aagaccatgc 4020 ccttcaggac caccatcgag ggcaccgtga acggccacta cttcaagtgc accggcaagg 4080 gcgagggcaa ccccctggag ggcacccagg agatgaagat cgaggtgatc gagggcggcc 4140 ccctgccctt cgccttccac atcctgtcca cctcctgcat gtacggctcc aaggccttca 4200 tcaagtacgt gtccggcatc cccgactact tcaagcagtc cctccccgag ggcttcacct 4260 gggagcgcac caccacctac gaggacggcg gcttcctgac cgcccaccag gacacctccc 4320 tggacggcga ctgcctggtg tacaaggtga agatcctggg caacaacttc cccgccgacg 4380 gccccgtgat gcagaacaag gccggccgct gggagccctc caccgagatc gtgtacgagg 4440 tggacggcgt gctgcgcggc cagtccctga tggccctgga gtgccccggc ggtcgccacc 4500 tgacctgcca cctgcacacc acctaccgct ccaagaagcc cgcctccgcc ctgaagatgc 4560 ccggcttcca cttcgaggac caccgcatcg agatcctgga ggaggtggag aagggcaagt 4620 gctacaagca gtacgaggcc gccgtgggcc gctactgcga cgccgccccc tccaagctgg 4680 gccacaactg aatcgatgat gatgatccag acatgataag atacattgat gagtttggac 4740 aaaccacaat tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg 4800 ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt 4860 ttatgtttca ggttcagggg gaggtgtggg aggtttttta aagcaagtaa aacctctaca 4920 aatgtggtat ggctgattat gatcctctag atcaagatct gcgaagatac ggccacgggt 4980 gctcttgatc ctgtggctga ttttggactg tgctgctcgc agctgctgat gaatcacata 5040 cttcctccat tttcttccac tgattgactg ttataatttc cctaatttcc aggtcaaggt 5100 gctgtgcatt gtggtaatag atgtgacatg acgtcacttc caaaggacca atgaacatgt 5160 ctgaccaatt tcatataatg tgaaaacgat tttcataggc agaataaata acatttaaat 5220 taaactgggc atcagcgcaa ttcaattggt ttggtaatag caagggaaaa tagaatgaag 5280 tgatctccaa aaaataagta ctttttgact gtaaataaaa ttgtaaggag taaaaagtac 5340 ttttttttct aaaaaaatgt aattaagtaa aagtaaaagt attgattttt aattgtactc 5400 aagtaaagta aaaatcccca aaaataatac ttaagtacag taatcaagta aaattactca 5460 agtactttac acctctggtt cttgaccccc taccttcagc aagcccagca gatccactag 5520 ttctagagcg ttcttgaccc cctaccttca gcaagcccag cagatccact agttctagag 5580 cggccgccac cgcggtggag ctccagcttt tgttcccttt agtgagggtt aattgcgcgc 5640 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca 5700 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa 5760 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag 5820 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc 5880 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct 5940 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg 6000 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc 6060 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga 6120 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct 6180 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg 6240 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag 6300 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat 6360 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac 6420 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac 6480 tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc 6540 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt 6600 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc 6660 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg 6720 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca 6780 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca 6840 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag 6900 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac 6960 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc 7020 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct 7080 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc 7140 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg 7200 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc 7260 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat 7320 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag 7380 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat 7440 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg 7500 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca 7560 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga 7620 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc 7680 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata 7740 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg 7800 ccac 7804

Claims (10)

As a zebrafish model that liver-specifically expresses human interleukin-6 (hIL-6) gene,
Wherein the zebra fish model is fabricated by a method comprising the steps of:
1) a transgenic zebrafish expressing a liver fatty acid binding protein (LFABP) and a fluorescent protein and (b) a vector comprising a human IL-6 (hIL-6) (LFABP-Gal4 / UAS-RFP / UAS-hIL6-CG), which is a transgenic zebrafish that expresses the human interleukin 6 gene in a liver-specific manner, is produced by crossing the transformed zebrafish , &Lt; / RTI &
The transgenic zebrafish expressing the liver fatty acid binding protein (LFABP) and the fluorescent protein (a) comprises a vector represented by the following cleavage map 3 and a vector represented by the following SEQ ID NO: Tg (LFABP-Gal4 / UAS-RFP) transformed by the vector represented by map 1,
The transformed zebrafish into which the vector containing the human interleukin-6 (hIL-6) gene sequence of (b) has been transfected is transformed by a vector represented by the following cleavage map 2 composed of the nucleotide sequence shown in SEQ ID NO: Gt; (UAS-hIL6-CG) &lt; / RTI &gt;
[Cleft Map 1]

[Cleaved map 2]

[Cleft Map 3]

; And
2) introducing a transgenic gene of Tg (LFABP-Gal4 / UAS-RFP / UAS-hIL6-CG), which is the transformed zebrafish produced in step 1), into a zebrafish embryo.
delete delete The zebrafish model according to claim 1, wherein the zebrafish model is hepatitis or liver cancer.
delete delete 1) treating the test substance with the zebrafish model of claim 1;
2) measuring the degree of tumor in hepatocytes of the zebrafish model of step 1); And
3) selecting a test substance whose degree of tumor in the hepatocyte measured in the step 2) is lower than that of a control group not treated with the test substance; and screening the candidate substance for prevention or treatment of liver cancer.
1) treating the liver cancer treatment agent with the zebrafish model of claim 1; And
2) measuring the progress of liver cancer in the zebrafish model of step 1).
9. The method of claim 8,
And 3) a step of predicting that the treatment effect of liver cancer treatment agent does not exist when the liver cancer of the zebrafish model proceeds and the therapeutic effect of the liver cancer therapeutic agent is present if the liver cancer of the zebrafish model does not proceed To a method for predicting the therapeutic effect on liver cancer.
9. The method according to claim 8, wherein the agent for treating liver cancer is an inhibitor for inhibiting a JAK / Stat3 signal or a sub-regulator thereof.

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