KR20160042266A - Animal model for lymphoma and uses thereof - Google Patents

Abstract

The present invention relates to: an animal model which has lymphoma and in which Peli 1 gene is overexpressed; a method for producing the animal model with lymphoma; and a screening method for a drug involved in preventing or treating lymphoma, including a step of screening substances capable of lowering the level of mRNA for the Peli 1 gene or the level of protein expressed therefrom in the animal model with lymphoma.

Description

Lymphoma model animals and uses thereof < RTI ID = 0.0 >

The present invention relates to a lymphoma model animal and its use. More particularly, the present invention relates to a lymphoma model animal in which the Peli 1 gene is overexpressed, a method for producing the above lymphoma model animal, And screening for a substance capable of lowering the level of the protein expressed from the lymphocyte. The present invention also relates to a method for screening a drug for preventing or treating lymphoma.

Lymphoma is a malignant tumor that occurs in lymphoid tissue and may occur in non-lymphoid tissue. Non-lymphatic lymphoma is called non-lymphatic lymphoma, and it is known to occur mainly in the nose and throat, or in the gastrointestinal tract or brain. The United States accounts for 2.1% of total cancer incidence and 2.8% of the United Kingdom. Korea accounts for 4%, which is relatively high in the Orient. In particular, depending on the location of the lymphoma, it can be briefly divided into Hodgkin's disease and non-Hodgkin's lymphoma. In Korea, the majority of non-Hodgkin's lymphoma is occupied, more than 95% of non-Hodgkin's lymphoma is mostly malignant lymphoma, Hodgkin's lymphoma is only 5% of malignant lymphoma, and the frequency of Hodgkin's lymphoma in Western countries is about 40 %. The non-Hodgkin's lymphoma is a lymphoma of the immune system originating from T cells, B cells, or histiocytes, but most of the non-Hodgkin's lymphoma (80-90%) is B cell origin and the remainder is T or NK cell origin. Is very rarely known. Among non-Hodgkin's lymphomas, diffuse large B-cell lymphoma (Diffuse large B-cell lymphoma, 4 to 5 times larger than bovine lymphocytes, mixed with cells with and without nucleated) is the most common type. And 40%.

A common way to treat lymphoma is by administering an anticancer alone, or by combining radiation therapy with chemotherapy. In the case of recurrence after attempting such a standard treatment method, bone marrow transplantation may be performed while increasing the dose of anticancer drug. In malignant lymphoma, the number of reactive immune cells around the tumor is large, and because morphological analysis alone can not distinguish between tumor cells and reactive cells, diagnosis of malignant lymphoma is more difficult than any other tumor. Therefore, the development of a diagnostic marker that simultaneously satisfies both sensitivity and specificity is urgently required for the diagnosis of malignant lymphoma. In the late 1990s, the development of a target treatment for CD20 on the surface of B cells (Rituximab) showed a new breakthrough in the treatment of B-cell lymphoma. The development of Rituximab improved its treatment performance, Of the total. In addition, WO 1999/038525 discloses a kit comprising a composition for predicting the diagnosis or prognosis of a lymphoma comprising a biomarker Pellino 1 gene mRNA or an agent for measuring the protein level thereof.

On the other hand, studies on genes for treating or diagnosing the above lymphoma have been actively conducted. Korean Patent Registration No. 1182974 discloses a method for diagnosing or diagnosing a lymphoma comprising the biomarker Pellino 1 gene mRNA or a protein level measuring method thereof A kit comprising a composition for prediction is disclosed.

Under these circumstances, the present inventors have made extensive efforts to develop a model animal suitable for use in studying a therapeutic agent for lymphoma. As a result, Peli 1 gene was overexpressed in each organ of a transgenic mouse overexpressing the Peli 1 gene, The inventors confirmed that mice can be used as model animals for the treatment of lymphoma and completed the present invention.

One object of the present invention is to provide an animal model of lymphoma that overexpresses the Peli 1 gene.

Another object of the present invention is to provide a model animal capable of developing lymphoma by the administration of doxycycline.

It is still another object of the present invention to provide a method for producing the model animal.

It is still another object of the present invention to provide a screening method for a prophylactic or therapeutic agent for lymphoma, which comprises screening a substance capable of lowering the mRNA level of the Peli 1 gene or the level of the protein expressed therefrom using the model animal .

The present inventors have focused on Peli 1 (pellino homolog 1) gene while carrying out various studies to develop a model animal suitable for the treatment of lymphoma. The Peli 1 gene is known as a gene encoding a pellino homolog 1 protein, which is known as E3 ubiquitin conjugation enzyme. The mouse overexpressing the gene has a markedly decreased cumulative survival rate, and the lung, liver, intestine, pancreas, prostate, spleen, 43 ~ 66% of the tumors were naturally occurring, and lymphoid organs such as lymph nodes, fire plates, spleen, and thymus were found to develop only in the transgenic mice. In addition, it was confirmed that inhibiting the expression of the Peli 1 gene promotes the expression of the p53 or p21 gene, which is a cancer suppressor gene, and can lower the incidence of cancer and the mortality due to cancer.

Therefore, it was found that the model animal in which the Peli 1 gene was overexpressed could be used for screening a therapeutic agent for lymphoma.

As one embodiment for achieving the above-mentioned object, the present invention provides a lymphoma model animal in which the Peli 1 gene is overexpressed.

The term "Peli 1 (pellino homolog 1) gene " of the present invention means a gene encoding a pellino homolog 1 protein known as E3 ubiquitin conjugation enzyme. In the case of human, it is present on chromosome 2, It is present on the chromosome. Specific base sequences and protein information of the protein or gene are known from NCBI (NCBI Reference Sequence: NP_065702, NM_020651). In the present invention, the polynucleotide of SEQ ID NO: 1 was used as the Peli 1 gene.

The term "lymphoma" of the present invention is also referred to as lymph node cancer, and includes B-lymphocytes, T-lymphocytes, natural killer, NK cells, Reed-Sternberg cells, (Popcorn cells). It is a kind of cancer disease that can occur anywhere in the body and can occur in certain areas with non-Hodgkin's lymphoma, which is difficult to predict the progression of the tumor. It is distinguished from Gleevec lymphoma.

The term "lymphoma model animal" of the present invention means a model animal produced by manipulating a gene so that a lymphoma can be developed.

In the present invention, the lymphoma model animal can be interpreted as a model animal produced to overexpress the Peli 1 gene in a host animal. Examples of the host animal include, but are not limited to, a mouse, a rat, a rabbit, , Chimpanzee, and the like, and the overexpression of the Peli 1 gene can be performed using an expression vector containing the Peli 1 gene. Preferably, the expression vector containing the Peli 1 gene is introduced into the embryo And fertilization of the fertilized egg in the uterus of a surrogate animal, and then giving birth. In the present invention, the expression vector shown in FIG. 1A was used as an expression vector containing the Peli 1 gene.

According to one embodiment of the present invention, a vector comprising a human early cytomegalovirus enhancer (CMV) enhancer, a β-actin promoter and a poly-A (β-globin gene polyadenylation sequence) is linked to a Myc-epitope Pell 1 expression vector (FIG. 1A) was introduced by introducing the Pellino 1 gene (SEQ ID NO: 1) into the embryo of a mouse, and the microinjected embryos were implanted into the uterus of a surrogate mouse, And mice in which the Peli 1 gene was overexpressed were selected using the PCR method among the born mice (FIG. 1B). Western blot analysis was performed on each organs of the selected mice. As a result, it was confirmed that Peli 1 protein was overexpressed in each organs of the selected mice (FIG. 1C). The cumulative survival rate of the mice overexpressing the Peli 1 gene is remarkably reduced (Fig. 2a), and about 43 to 66% of naturally occurring cancers are induced in the lung, liver, intestine, pancreas, prostate, spleen and thymus 2b and 2c), lymph nodes, fire plates, spleen, thymus and the like (Table 1). On the other hand, as a result of identifying the carcinomas developed in the above-mentioned mice, the thymus, spleen and lymph nodes showed a histologically similar structure to that of lymphoma (Fig. 3A) (Fig. 3B). Especially, in spleen cells, not only the ratio of B cells increased but also the activity of B cells increased (Fig. 3C), and lymphoma developed in thymus and spleen tissues ), And that B cells infiltrated into liver tissue and lung tissue actively proliferate (FIG. 3 e), indicating that overexpression of Peli 1 gene can induce the development of B cell lymphoma.

According to another embodiment of the present invention, a transformed cell line (Peli 1 MEF) in which the expression of Peli 1 gene is inhibited is prepared (FIG. 4A), and as the expression of Peli 1 gene is inhibited in the prepared cell line, (Fig. 4b). Inhibiting the expression of Peli 1 gene through transgenic mice resulted in a decrease in the incidence of cancer associated with Peli 1 gene and the mortality due to the cancer (Fig. 4C), suppression of the expression of Peli 1 gene promotes the expression of the p53 or p21 gene, which is a cancer suppressor gene, to decrease the incidence of cancer and the mortality due to cancer. Therefore, It can be used for treatment.

As another embodiment for achieving the above-mentioned object, the present invention provides a model animal capable of developing lymphoma by the administration of doxycycline.

In the model animals prepared above, B cell lymphoma is highly likely to develop, and this can be used as an animal model of cancer-onset model, particularly of B-cell lymphoma. However, since the model animal develops B-cell lymphoma at a substantially constant time, there is an inconvenience in that it is necessary to reflect the drug experiment plan from the birth time of the model animal in order to perform drug experiment using the model animal . As a result, various studies have been conducted to produce an improved model animal capable of regulating the overexpression time of the Peli 1 gene. As a result, when the doxycycline is administered, the Peli 1 gene is overexpressed but when the doxycycline is not administered, Lt; RTI ID = 0.0 > lymphoma < / RTI >

In the present invention, a model animal capable of inducing lymphoma by administration of the above-mentioned doxycycline can be produced by crossing an animal model of lymphoma and an animal model of rtTA (reverse tetracycline transactivator) in which the produced Peli 1 gene is overexpressed. At this time, the lymphoma model animal in which the Peli 1 gene is overexpressed can be produced by introducing the expression vector shown in FIG. 5A into the embryo and giving birth to the introduced embryo.

The rtTA model animal is a transgenic mouse produced by introducing the rtTA gene after the R26 promoter. The rtTA expressed from the rtTA model animal is activated by binding with the doxycycline, and the activated rtTA can bind to the TRE promoter and act as a transcription factor. Pseudomonas pTRE-myc-Peli 1; rtTA progeny mice can be obtained by crossing a peli 1 transgenic (pTRE-myc-Peli1) model animal with a rtTA (reverse tetracycline transactivator) model animal. When the latter mouse is administered with doxycycline, It is possible to induce the expression of myc-Peli 1 located downstream of the TRE promoter, and when the administration of the above doxycycline is stopped, the expression of myc-Peli 1 is inhibited by the inactivation of rtTA.

According to one embodiment of the present invention, a transgenic mouse capable of overexpressing the Peli 1 gene has been modified to produce a model animal capable of developing cancer at a desired time, and the overexpression of Peli 1 gene is induced by administration of doxycycline (FIG. 5A). Transgenic mice to which doxycycline inducible Peli 1 transgenic mice were transfected were prepared (FIG. 5A). From these transgenic mice, mice expressing Peli 1 gene (FIG. 5b). The selected mice were crossed with rtTA (reverse tetracycline transactivator) mice to obtain transgenic mice in which overexpression of Peli 1 gene was induced upon administration of doxycycline, from which Peli 1 and rtTA were simultaneously (Fig. 5C). As a result of the overexpression of Peli 1 protein in each organs of the selected mice, when the doxycycline was administered to the transgenic mouse (Peli 1 / rtTA) in which overexpression of the Peli 1 gene was induced, Peli 1 gene was expressed in the liver, adrenal gland, colon, kidney, prostate, pancreas and liver, whereas expression of Peli 1 gene was detected in lung, heart and skin. Expression of Peli 1 gene in brain, (Fig. 5C and Table 2). Thus, it was found that the transgenic mouse prepared above was a mouse in which Peli 1 was overexpressed by the administration of doxycycline.

As another embodiment for achieving the above object, the present invention provides a method for producing a model animal capable of causing the lymphoma.

The method for producing a model animal capable of developing lymphoma provided by the present invention comprises the steps of: (a) introducing an expression vector comprising the Peli 1 gene into a fertilized egg of a host animal; And (b) conferring the introduced embryo to the surrogate mother's uterus and giving birth to the progeny animal.

In this case, the host animal is the same as described above, and the expression vector may be the expression vector shown in FIG. 1A or FIG. 5A. In addition, the step of confirming whether the Peli 1 gene is overexpressed in the later-born animal can be further included. The overexpression of the Peli 1 gene can be confirmed by RT-PCR or Western blot analysis. In particular, when overexpression of the Peli 1 gene is confirmed by the RT-PCR method, the primer base pairs of SEQ ID NOS: 2 and 3 can be used.

In addition, in order to produce a model animal in which the Peli 1 gene is overexpressed under specific conditions, it may further comprise a step of crossing the born animal with a rtTA (reverse tetracycline transactivator) model animal to obtain a second generation progeny animal have.

Specifically, a method for producing a model animal capable of inducing lymphoma by administration of a doxycycline according to the present invention comprises the steps of: (a) introducing an expression vector comprising a Peli 1 gene into a fertilized egg of a host animal; (b) implanting the introduced embryos into the uterus of a surrogate mother and giving birth to the first-generation descendant animal; And (c) crossing the obtained first generation progeny animal with an rtTA (reverse tetracycline transactivator) model animal to obtain a second generation progeny animal.

In another aspect of the present invention, the present invention provides a method for screening a substance capable of lowering the mRNA level of Peli 1 gene or the protein expressed therefrom using the model animal, The present invention provides a screening method for a prophylactic or therapeutic agent.

Specifically, the screening method for a prophylactic or therapeutic agent for a lymphoma of the present invention comprises the steps of: (a) administering a lymphoma treatment candidate substance to the lymphoma model animal; And (b) confirming the prognosis while raising the lymphoma model animal to which the candidate substance is administered.

The term "prognosis" of the present invention means prediction of progress or recovery of the disease. For the purpose of the present invention, the prognosis is determined by administering a candidate substance capable of treating a lymphoma generated in a patient to a lymphoma model animal of the present invention, and then checking the progress of the lymphoma to determine whether the candidate substance can treat the lymphoma But it is not limited to this. For example, the prognosis can be confirmed by measuring morphological changes, changes in genetic environment, changes in marker protein expression levels, etc. The morphological environment includes invasiveness, proliferation index, microvessel density, etc. And the expression level of a gene encoding Peli 1, p53, p21, CD3, B220, Ki67, CD20, CD23 and the like can be cited as a genetic environment. Examples of the marker protein include Peli 1, p53, p21, CD3, B220, Ki67, CD20, CD23 and the like can be used.

In the lymphoma model animal of the present invention, naturally occurring lymphoma can be developed without treating a specific cause of cancer, so that it can be widely used in the development of an agent for the prevention or treatment of effective lymphoma.

1A is a schematic diagram showing the structure of a Peli 1 expression vector.
Fig. 1b shows the results of the stomach, liver, kidney, S. intestine, pancreas, and pancreas of non-Tg normal mice and Peli 1-Tg transformed mice. (BM) and spleen (Spleen) tissues. The results are shown in Fig.
Figure 1c is a graph showing the results of stomach, liver, kidney, S. intestine, pancreas, and pancreas of normal non-Tg and Peli 1-Tg mice. This is a western blot analysis showing the comparison of expression of Peli 1 gene in bone marrow (BM) and spleen (Spleen) tissues.
FIG. 2A shows Kaplan-Meier's results comparing the cumulative survival rates of normal non-Tg mice and Peli 1-Tg mice according to the passage of time, Curve.
FIG. 2B is a photograph showing the results of comparison between the normal non-Tg mice and the transgenic mice (Peli 1-Tg).
FIG. 2c is a graph showing the results of comparing cancer incidence rates in the normal late mice (Non-Tg, # 1, 5 and 6) and in the transgenic mice (Peli 1-Tg, # 2, 3 and 9).
FIG. 3A is a photomicrograph showing the tissues of the thymus, spleen, lymph nodes, fire plates, liver, and lungs of the normal posterior mouse (Non-Tg, # 1) and transgenic mice (Peli 1-Tg, # 9).
Figure 3b shows the ratio of B cells (B220 ⁺ ) to T cells (CD3 ⁺ ) in the spleen, lymph nodes (LNs) and thymus of normal non-Tg mice and transgenic mice (Peli 1-Tg) And FIG.
FIG. 3c is a flow cytometry graph showing the results of stimulating splenocyte-derived B cells of normal non-Tg and transgenic mice (Peli 1-Tg) to be.
Figure 3D shows the thymic and spleen tissues of normal non-Tg and transgenic mice (Peli 1-Tg) stained with hematoxylin / eosin (H & E) staining and immunocyte markers (CD3, B220, CD20 or CD23 ). ≪ / RTI >
Figure 3e shows the liver and lung tissues of normal non-Tg and transgenic mice (Peli 1-Tg) stained with hematoxylin / eosin (H & E) staining and various markers (CD3, B220, Ki67, ≪ / RTI > CD23) using an antibody.
FIG. 4A shows the structure of a vector obtained by substituting the puromycin gene for the 4th accession of the gDNA of the gDNA containing the Peli 1 gene and the structure of the transformed cell line (Peli 1 ES cell is manufactured.
FIG. 4B is a graph showing changes in the level of the protein expressed from the p53 gene and the p53 gene, which are cancer suppressor genes expressed in the wild-type embryonic fibroblast (MEF) and the transformed embryonic fibroblast (Peli 1 koMEF) (+/-) represents wild-type embryonic fibroblast (MEF), and (+/-) indicates that one of two Peli 1 genes contained in the allele is substituted with a puromycin gene (- / -) represents transfected fibroblasts (MEFs) in which all two Peli 1 genes contained in the allele are replaced with puromycin genes.
4C is a graph showing the incidence and mortality rates of buckling lymphoma of a cancer model mouse (Eμ-Myc Tg) and a cancer model mouse (Peli 1 KO; Eμ-Myc Tg) in which Peli 1 protein is not expressed.
5A is a schematic diagram showing the construction of a transformation vector for producing doxycycline inducible Peli 1 transgenic mice in which overexpression of Peli 1 gene is induced by administration of doxycycline.
5B is an electrophoresis image showing a result of selecting transgenic mice in which overexpression of the Peli 1 gene is induced. Negative indicates a negative control mouse to which no expression vector is introduced, and Positive indicates an expression vector containing no Peli 1 gene , And # 1 to # 3 represent the first-generation late-stage mice.
FIG. 5C is an electrophoresis image showing the result of selecting rtTA model animals. In FIG. 5C, (- / -) represents a control group in which no DNA is inserted, Normal represents a negative control mouse to which no expression vector is introduced, ) Represents a transgenic mouse in which one R26 gene of the allele gene is replaced with the rtTA gene, and (+ / +) represents a transgenic mouse in which both R26 genes of the allele gene are replaced with the rtTA gene, Represents a later mouse.
FIG. 5D shows the expression levels of Peli 1 protein according to the administration of doxycycline in each organ tissue of a control mouse (- / rtTA) and a transgenic mouse (Peli 1 / rtTA) in which overexpression of Peli 1 gene is induced upon administration of doxycycline Western blot analysis photograph showing the result.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples. In addition, all animal experiments used in the following examples were carried out in accordance with the International Experimental Animals (IACUC) approved by the Institutional Animal Care and Use Committee (IACUC) at Sungkyunkwan University School of Medicine (SUSM) The study was conducted in accordance with the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International) and the Institute of Laboratory Animal Resources (ILAR).

Example  One: Peli  1 transgenic mouse overexpressing a transgenic mouse

Example  1-1: Construction of Transgenic Mice

A human Peli 1 (Pellino 1) gene (SEQ ID NO: 1) in which a Myc-epitope-linked vector is inserted into a vector containing a human early cytomegalovirus enhancer (CMV), a β-actin promoter and a poly- ) Was introduced to prepare a Peli 1 expression vector (FIG. 1A).

1A is a schematic diagram showing the structure of a Peli 1 expression vector.

The prepared Peli 1 expression vector was microinjected into mouse embryos, and the microinjected embryos were implanted into the uterus of a surrogate mouse (C57BL / 6J), and then 14 posterior mice were born. Tissue samples of Stomach, Liver, Kidney, S. Intestine, Pancreas, bone marrow (BM) and Spleen of the 14 late-born mice were obtained PCR was carried out using the synthesized cDNA as a template and the primers described below to obtain the total RNAs from the respective tissues of Peli 1 gene was overexpressed (Fig. 1B). In this case, L32 was used as the internal control, and the PCR conditions were 4 times at 95 ° C for 30 seconds, 55 ° C for 30 seconds, and 72 ° C for 1 minute in the case of Peli 1, 1 min and 72 ° C for 1 min. In the case of L32, PCR was performed 34 times at 95 ° C for 30 sec, 60 ° C for 30 sec and 72 ° C for 1 min.

Primers for screening transgenic mice

Forward primer: 5'-GCAATAAGCAACAAAG -3 '(SEQ ID NO: 2)

Reverse primer: 5'-ATGAGCCAAATCCTGCAG -3 '(SEQ ID NO: 3)

Fig. 1b shows the results of the stomach, liver, kidney, S. intestine, pancreas, and pancreas of non-Tg normal mice and Peli 1-Tg transformed mice. (BM) and spleen (Spleen) tissues. The results are shown in Fig. As shown in FIG. 1B, Peli 1 gene was not expressed in each organ of non-Tg, but Peli 1 gene was expressed in each organ of transgenic mouse (Peli 1-Tg) , And it was confirmed that Peli 1 gene was overexpressed in three consecutive mice out of a total of 14 later mice.

Example  1-2: Peli  1 gene expression level

Western blot analysis was performed to verify overexpression of the Peli 1 gene. Approximately, tissue samples of stomach, liver, kidney, S. intestine, pancreas, bone marrow (BM) and spleen (Spleen) of the later 14 mice were obtained The lysis buffer (150 mM NaCl, 20 mM HEPES, 5 mM EDTA, 0.5% Nonidet P-40, 1 mM phenylmethanesulfonyl fluoride, 10 mM NaF, 1 mM Na ₃ Vo ₄ , 1 mM dithiothreitol and protease inhibitor cocktail) And applied to a homogenizer to disrupt the cells of each tissue to obtain a lysate. The lysate was centrifuged (14,000 rpm, 30 min, 4 ° C) to obtain a supernatant. Western blot analysis was performed using the obtained supernatant and a primary antibody (anti-Myc antibody (Roche; 1: 2,000 dilution) or anti-Peli 1 antibody (Santa Cruz; 1: 2,000 dilution) . At this time, actin was used as an internal control group.

Figure 1c is a graph showing the results of stomach, liver, kidney, S. intestine, pancreas, and pancreas of normal non-Tg and Peli 1-Tg mice. This is a western blot analysis showing the comparison of expression of Peli 1 gene in bone marrow (BM) and spleen (Spleen) tissues. As shown in FIG. 1C, the Myc-epitope-bound Peli 1 protein was not detected in each organs of the normal non-Tg mice. However, in each organ of the transgenic mice (Peli 1-Tg) Epitope-bound Peli 1 protein was detected, and it was confirmed that Peli 1 protein bound to the Myc-epitope was detected in three postnatal mice out of a total of 14 later mice.

Example  2: Peli  1 over-expressing transgenic mice

Example  2-1: Cumulative survival analysis

Twenty normal non-Tg mice and 23 transgenic mice (Peli 1-Tg) prepared by the method of Example 1-1 were cultivated for 1000 days, and the cumulative survival rate and overall survival was measured and compared (Figure 2a).

FIG. 2A shows Kaplan-Meier's results comparing the cumulative survival rates of normal non-Tg mice and Peli 1-Tg mice according to the passage of time, Curve. As shown in FIG. 2A, the cumulative survival rate of the transgenic mice (Peli 1-Tg) was significantly lower than that of the non-Tg mice, and the median survival rate was only about 80 weeks Respectively.

Example  2-2: Cause analysis of cumulative survival rate change

Normal non-Tg and transgenic mice (Peli 1-Tg) were dissected and the status of each organ was checked and compared (FIG. 2B). FIG. 2B is a photograph showing the results of comparison between the normal non-Tg mice and the transgenic mice (Peli 1-Tg). As shown in FIG. 2B, in the transgenic mice (Peli 1-Tg), naturally occurring tumors in various organs such as liver, spleen and thymus, as compared with non-Tg mice, Respectively.

Example  2-3: Cancer incidence rate  analysis

Cancer incidence was compared between normal posterior mice (Non-Tg, # 1, 5 and 6) and transgenic mice (Peli 1-Tg, # 2, 3 and 9) (FIG.

FIG. 2c is a graph showing the results of comparing cancer incidence rates in the normal late mice (Non-Tg, # 1, 5 and 6) and in the transgenic mice (Peli 1-Tg, # 2, 3 and 9). As shown in FIG. 2C, it was confirmed that a higher incidence of cancer was observed in the transgenic mice (Peli 1-Tg) than in the non-Tg mice. In particular, it was confirmed that 43 to 66% of the tumors occurred in the lung, liver, intestine, pancreas, prostate, spleen, thymus and the like in the transgenic mice (Peli 1-Tg) at 15 to 20 weeks of age.

The incidence of cancer in various organs was measured and compared to 38 normal non-Tg mice (# 1) and 49 transgenic mice (Peli 1-Tg, # 9) One).

Comparison of incidence of cancer among organs according to overexpression of Peli 1 gene Organ organization Normal later mouse Transgenic Late Mouse Lymph node
Fire board
spleen
Thymus
Intestine
liver
lungs
Pancreas
prostate 0 (0%)
0 (0%)
0 (0%)
0 (0%)
2 (5.3%)
1 (2.6%)
1 (2.6%)
0 (0%)
0 (0%) 10 (20%)
6 (12%)
9 (18%)
8 (16%)
7 (14%)
19 (39%)
8 (16%)
7 (14%)
1 (2%)

As shown in the above Table 1, it was confirmed that cancer occurred in some organs in the normal posterior mouse, but it was less than 10%, whereas in the transgenic mice, cancer occurred in all organs and its incidence was also more than 10% Respectively. In particular, lymphatic organs such as lymph nodes, fire plates, spleen, and thymus were found to be cancerous only in the transgenic mice.

Example  3: Transgenic mice Carcinoma  Identification

Example  3-1: Histological analysis

The tissues of some organs (thymus, spleen, lymph node, fire plate, liver and lung) were extracted from the above identified organs, treated with 10% formalin and paraffin to fix the tissues to each other, To obtain tissue flakes having a thickness of 3 to 6 mu m, and the obtained tissue flakes were stained with hematoxylin and eosin, and then observed under a microscope (Fig. 3A).

FIG. 3A is a photomicrograph showing the tissues of the thymus, spleen, lymph nodes, fire plates, liver, and lungs of the normal posterior mouse (Non-Tg, # 1) and transgenic mice (Peli 1-Tg, # 9). As shown in FIG. 3A, unlike the organs of the normal posterior mouse composed of a certain tissue, in the organs of the transgenic mice, various types of cells are abnormally high and the infiltration of many immune cells is observed And the structure of the tissue was damaged. In particular, the thymus, spleen and lymph nodes of the transgenic mice showed a histologically similar structure to that of lymphoma.

Example  3-2: Immune cell analysis

The number ratio of B cells and T cells in the spleen, lymph nodes and thymus, which are immunoreactive organs of the transgenic mice, was measured to determine whether the development of cancer and immune function were related in the immunological organs. Specifically, respective tissues were obtained from the spleen, lymph nodes and thymus of normal non-Tg and transgenic mice (Peli 1-Tg), and respective cells were obtained from the obtained tissues. Each of the cells obtained above was subjected to immunostaining using an antibody against B220, an antibody against B220 or an antibody against CD3, which is a T cell marker, and applied to a flow cytometry, The numbers of T cells and B cells were measured and compared with each other by organ (Fig. 3b).

Figure 3b shows the ratio of B cells (B220 ⁺ ) to T cells (CD3 ⁺ ) in the spleen, lymph nodes (LNs) and thymus of normal non-Tg mice and transgenic mice (Peli 1-Tg) And FIG. As shown in FIG. 3B, the proportion of B cells was increased in the immunized organs of transgenic mice (Peli 1-Tg) compared to that of normal non-Tg mice, while the ratio of T cells was decreased. It was confirmed that the immune function was changed by the cancer generated in the immune system. In particular, in the case of Thymus, in which most of the T cells are involved, the B cells were abnormally increased in the transgenic mice, indicating that the immune function of the thymus was greatly changed.

Example  3-3: B cell function analysis

As described above, since it has been confirmed that cancer cells develop in the immune organs in the transgenic mice, and thus the proportion of B cells increases, whether the increased B cells are merely numerically increased or functionally activated .

Specifically, spleen cells were obtained from the spleen of normal non-Tg and transgenic mice (Peli 1-Tg), and anti-CD40 antibody and anti-IgM antibody were added to the spleen cells obtained for 24 hours The spleen cells were stimulated and the stimulated splenocytes were stained with anti-CD86 antibody specific for B cells and anti-MHC class II antibody, and analyzed for flow cytometry (FIG. 3C).

FIG. 3c is a flow cytometry graph showing the results of stimulating splenocyte-derived B cells of normal non-Tg and transgenic mice (Peli 1-Tg) to be. As shown in FIG. 3C, the expression of CD86 and MHC class II, which are B cell specific markers, in the splenocytes of transgenic mice (Peli 1-Tg) is markedly increased compared with that of normal non-Tg spleen cells Respectively. Thus, the splenocytes of transgenic mice (Peli 1-Tg) not only increased the proportion of B cells but also increased the activity of the B cells.

Example  3-4: Immunohistochemical analysis of immune-related organs

Immunohistochemical staining of the thymus and spleen, which are immunoreactive organs of the transgenic mice, was performed to confirm the onset of the lymphoma. Specifically, thymocytes and spleen tissues of normal non-Tg and transgenic mice (Peli 1-Tg) were subjected to hematoxylin / eosin (H & E) staining, antibody to CD3, An antibody against IgG of rat combined with biotin-conjugated primary antibody such as an antibody against B220 cell marker, an antibody against CD20 which is a B cell lymphoma marker, or an antibody against CD23 which is an early B cell lymphoma marker, Immunostaining was performed using a secondary antibody, which is an antibody against IgG. The sections of immunostained tissues were reacted with avidin-biotin-horseradish peroxidase complex (Vectastain Elite ABC kit; Vector Laboratories) and the activity of peroxidase was determined using 3,3'-diaminobenzidine substrate kit (Vector Laboratories) The tissue sections were stained with Harris Hematoxylin (BBC Biochemical) and images were stained using a microscope (AxioCam digital microscope camera and the AxioVision image processing software (Carl Zeiss)) (FIG. 3D).

Figure 3D shows the thymic and spleen tissues of normal non-Tg and transgenic mice (Peli 1-Tg) stained with hematoxylin / eosin (H & E) staining and immunocyte markers (CD3, B220, CD20 or CD23 ). ≪ / RTI > As shown in FIG. 3D, the distribution of B cells was increased in the thymus and spleen tissues of the transgenic mice (Peli 1-Tg), and the germinal center structure of the spleen was expressed in the non- Tg), and it was confirmed to be lost in transgenic mice (Peli 1-Tg). In addition, CD20, a B cell lymphoma marker, was significantly increased in the thymus and spleen tissues of transgenic mice (Peli 1-Tg) rather than in normal non-Tg mice, and transgenic mice (Peli 1-Tg ) Were found to be lymphoma in the thymus and spleen tissues.

Example  3-5: Nonimmune organ  Immunohistochemical analysis

Immunohistochemical staining was performed on liver and lungs of the non-immunological related organs of the transgenic late-stage mice to determine whether or not the lymphoma was developed. Specifically, hepatocyte / eosin (H & E) staining, liver cell and T cell marker antibody to CD3 were examined in liver tissues and lung tissues of normal non-Tg and transgenic mice (Peli 1-Tg) Immunostaining was performed using an antibody against B220, a cell marker, an antibody against Ki67, an antibody against CD20, which is a B cell lymphoma marker, or an antibody against CD23, an early B cell lymphoma marker ).

Figure 3e shows the liver and lung tissues of normal non-Tg and transgenic mice (Peli 1-Tg) stained with hematoxylin / eosin (H & E) staining and various markers (CD3, B220, Ki67, ≪ / RTI > CD23) using an antibody. As shown in FIG. 3E, it was confirmed that liver and lung tissues of the transgenic mice (Peli 1-Tg) were immunostained with antibodies against B220, a B cell marker, (infilteration), and it was confirmed that the antibody against Ki67, which is a cell proliferation marker, immunostained. Thus, it was found that B cells infiltrated into liver tissue and lung tissue actively proliferated. In addition, it was confirmed that liver tissue was immunostained with an antibody against CD20, a B cell lymphoma marker, so that the liver tissue was found to have lymphoma.

Therefore, it can be understood from the results of FIGS. 3A to 3E that the overexpression of Peli 1 gene can induce the development of B cell lymphoma.

Example  4: Peli  1 < / RTI > On cancer development  Effect analysis

From the results of Example 3 above, it was found that overexpression of Peli 1 gene can induce the development of B cell lymphoma. Therefore, the effect of inhibiting expression of Peli 1 gene on cancer development was examined.

Example  4-1: Peli  1 < / RTI > On the carcinogenic mechanism  Effect analysis

Example  4-1-1: Peli  1 < / RTI >

A vector obtained by substituting the puromycin gene for the 4th accessory of the gDNA of the gDNA containing the Peli 1 gene was prepared and the vector was introduced into embryonic fibroblast (MEF) to express the Peli 1 gene This inhibited transformed cell line (Peli 1 MEF) was prepared (Fig. 4A).

FIG. 4A shows the structure of a vector obtained by substituting a puromycin gene for the 4th accessory of the gDNA of the gDNA containing the Peli 1 gene and the structure of the transformed cell line (Peli 1 MEF ) Is produced.

GDNA was obtained from the transformed cell line (Peli 1 MEF) in which the expression of the prepared Peli 1 gene was inhibited, and subjected to PCR amplification to firstly select a transformed cell line in which a puromycin gene was detected. GDNA was obtained from the transformant cell line selected first and PCR amplification was performed to detect the 4th accession of gDNA containing Peli 1 gene. One of the two Peli 1 genes contained in the allele gene was a puromycin gene (+/-) or a transformed cell line (- / -) in which two Peli 1 genes contained in the allele gene were all substituted with the puromycin gene were obtained.

Example  4-1-2: Peli  1 < / RTI > On the carcinogenic mechanism  Effect analysis

The expression level of the p53 gene, which is known as a cancer suppressor gene, and the p21 gene, downstream of the p53 gene, in the transformed cell line (Peli 1 MEF) inhibited by expression of the Peli 1 gene prepared in Example 4-1-1 The changes were confirmed by Western blot analysis (Fig. 4B). At this time, wild type embryonic fibroblast (MEF) was used as a control group and HSP60 was used as an internal control group.

FIG. 4B is a graph showing changes in the level of the protein expressed from the p53 gene and the p53 gene, which are cancer suppressor genes expressed in the wild-type embryonic fibroblast (MEF) and the transformed embryonic fibroblast (Peli 1 koMEF) (+/-) represents wild-type embryonic fibroblast (MEF), and (+/-) indicates that one of two Peli 1 genes contained in the allele is substituted with a puromycin gene (- / -) represents transfected fibroblasts (MEFs) in which all two Peli 1 genes contained in the allele are replaced with puromycin genes. As shown in FIG. 4B, it was confirmed that the expression level of p53 protein and p21 protein increased as the expression of Peli 1 gene was suppressed.

Therefore, it was found that the inhibition of the expression of Peli 1 gene improves the expression of cancer inhibitory protein, which is not directly related to the expression of Peli 1 gene.

Example  4-2: Peli  1 gene is suppressed Cancer model  Mouse Creation and Effect Verification

(Peli 1-Tg) produced by the method of Example 1-1 and a cancer model mouse (Eμ-Myc Tg) in which a Burkitt lymphoma (B-cell lymphoma) , And a cancer model mouse (Peli 1 KO; Eμ-Myc Tg) in which Peli 1 protein was not expressed was prepared. The incidence and mortality rates of Burkett's lymphoma were compared with those of a control model mouse model (Eμ-Myc Tg) and a model mouse model (Peli 1 KO; Eμ-Myc Tg) (Fig. 4C).

4C is a graph showing the incidence and mortality rates of buckling lymphoma of a cancer model mouse (Eμ-Myc Tg) and a cancer model mouse (Peli 1 KO; Eμ-Myc Tg) in which Peli 1 protein is not expressed. As shown in FIG. 4C, a male model mouse (Eμ-Myc Tg) showed a bucket lymphoma phenotype at the age of 9 weeks of age, and died at the age of 12 weeks, whereas Peli 1 protein It was confirmed that the expression model of Burkett's lymphoma did not appear even after 12 weeks of age in the unexpressed cancer model mice (Peli 1 KO; Eμ-Myc Tg). The survival rate of the cancer model mouse (Eμ-Myc Tg) was found to be about 50% at the end of the breeding, but the survival rate of the cancer model mouse (Peli 1 KO; Eμ-Myc Tg) About 80%.

Thus, inhibition of the expression of Peli 1 gene revealed that the incidence of cancer associated with the Peli 1 gene and the mortality due to the cancer were reduced.

Taken together, the results of Examples 4-1 and 4-2 show that inhibiting the expression of Peli 1 gene promotes the expression of the p53 or p21 gene, which is a cancer suppressor gene, to lower the incidence of cancer and the mortality due to cancer Therefore, it can be used for prevention or treatment of effective cancer.

Example  5: Peli  1 Transgenic Mouse with Overexpression

As shown in the results of Examples 1 to 4, B-cell lymphoma is highly likely to develop in a transgenic mouse capable of overexpressing the Peli 1 gene, and thus the transgenic mouse capable of overexpressing the Peli 1 gene is a cancer- Can be used as model animals, particularly B-cell lymphoma onset model animals. However, since the transgenic mice develop B-cell lymphoma at an almost constant time, it is inconvenient to use the transgenic mouse to carry out the drug experiment, there was. Thus, an improved transgenic mouse was constructed to control the overexpression time of the Peli 1 gene and verified.

Example  5-1: Peli  1 Transgenic Mouse with Overexpression

To construct a transgenic mouse (doxycycline inducible Peli 1 transgenic mice) in which overexpression of the Peli 1 gene was induced by the administration of doxycycline, a transformation vector in which the Myc-epitope was bound to the Peli 1 gene was prepared (Fig. 5A).

5A is a schematic diagram showing the construction of a transformation vector for producing doxycycline inducible Peli 1 transgenic mice in which overexpression of Peli 1 gene is induced by administration of doxycycline.

Transgenic embryos obtained above were microinjected into mouse embryos to obtain transgenic embryos. The obtained transgenic embryos were implanted into the uterus of a surrogate mouse (C57BL / 6J) . Tissue samples of the tail were obtained from the first generation late-born mice, and gDNA was obtained from the tissue samples obtained. Using the obtained gDNA as a template, PCR was carried out using the following primers to select a transgenic mouse into which the Peli 1 gene was introduced (FIG. 5B). At this time, the PCR was performed four times in total at 95 ° C for 30 seconds, 55 ° C for 30 seconds, and 72 ° C for 1 minute, and then 34 times for 95 ° C for 30 seconds, 58 ° C for 1 minute and 72 ° C for 1 minute

Primers for Peli 1 transfection confirmation

Forward primer (cmv TetO-1): 5'-AAGTGAAAGTCGAGCTCG-3 '(SEQ ID NO: 4)

Reverse primer (Peli 1 1/3 length): 5'-TGATATCGTGTGCTGGTCTTTG -3 '(SEQ ID NO: 5)

5B is an electrophoresis image showing a result of selecting transgenic mice in which overexpression of the Peli 1 gene is induced. Negative indicates a negative control mouse to which no expression vector is introduced, and Positive indicates an expression vector containing no Peli 1 gene , And # 1 to # 3 represent the first-generation late-stage mice. As shown in FIG. 5B, Peli 1 was expressed in the # 3 postnatal mouse (female), and this was selected.

The selected first-generation late-stage mice and rtTA (reverse tetracycline transactivator) mice were crossed to give birth to seven second-generation late-stage mice, to obtain the tail tissue of the second-generation late-stage mice, Respectively. Using the obtained gDNA as a template, PCR was carried out using the following primers, and second generation late-stage mice in which Peli 1 and rtTA were simultaneously expressed were selected from the second generation late-stage mice (Fig. 5C). As a control, PCR products amplified using primers of SEQ ID NOS: 6, 7 and 8 were amplified in the following conditions: 95 ° C. for 30 seconds, 65 ° C. for 1 minute, 72 ° C. for 1 minute Min for 34 times.

Primers for rtTA transformation confirmation

Forward primer (R26): 5'-AAAGTCGCTCTGAGTTGTTAT -3 '(SEQ ID NO: 6)

Reverse primer (R26): 5'-GGAGCGGGAGAAATGGATATG -3 '(SEQ ID NO: 7)

Reverse primer (rtTA): 5'-GCGAAGAGTTTGTCCTCAACC -3 '(SEQ ID NO: 8)

FIG. 5C is an electrophoresis image showing the result of selecting rtTA model animals. In FIG. 5C, (- / -) represents a control group in which no DNA is inserted, Normal represents a negative control mouse to which no expression vector is introduced, ) Represents a transgenic mouse in which one R26 gene of the allele gene is replaced with the rtTA gene, and (+ / +) represents a transgenic mouse in which both R26 genes of the allele gene are replaced with the rtTA gene, Represents a later mouse. In the lower part, 600 bp represents the size of the PCR amplification product of the gene including the R26 gene as it is, and 300 bp represents the size of the PCR amplification product in which the R26 gene is substituted with the rtTA gene.

As shown in FIG. 5C, it was confirmed that the second generation late-stage mice in which Peli 1 and rtTA are simultaneously expressed among the seven second-generation second-generation mice born were # 4, # 2, # 4 and # 7, 1 and rtTA at the same time were defined as transgenic mice in which the overexpression of Peli 1 gene was induced by the administration of doxycycline.

Example  5-2: Peli  Verification of transgenic mice capable of overexpressing 1 gene

(Peli 1 / rtTA) in which the overexpression of the Peli 1 gene was induced upon administration of the doxycycline prepared in Example 5-1 was either not administered or administered with the doxycycline, Brain, Lung, Heart, Thymus, Stomach, Small Intestine, Epididymis, Colon, Kidney, Skin, Tissue samples of Testis, Prostate, Pancreas and Liver were obtained, and western blot analysis was performed using these tissue samples and antibodies against Peli 1, and Peli 1 protein were measured and compared (Figure 5d and Table 2). At this time, rtTA (reverse tetracycline transactivator) mouse was used as a control mouse and? -Actin was used as an internal control.

FIG. 5D shows the expression levels of Peli 1 protein according to the administration of doxycycline in each organ tissue of a control mouse (- / rtTA) and a transgenic mouse (Peli 1 / rtTA) in which overexpression of Peli 1 gene is induced upon administration of doxycycline Western blot analysis photograph showing the result.

Relative expression level of Peli 1 protein in each organs of transgenic mice in which overexpression of Peli 1 gene is induced upon administration of doxycycline Organ organization Detection of Peli 1 protein brain
lungs
Heart
Thymus
top
Intestine
Irregular
Leader
kidney
skin
testicle
prostate
Pancreas
liver -
++
+
+++
+++
-
+++
+++
+++
+
-
+++
+++
+++

As shown in FIG. 5D and Table 2, when doxycycline is administered to a transgenic mouse (Peli 1 / rtTA) in which overexpression of Peli 1 gene is induced upon administration of doxycycline, And Peli 1 overexpression in liver and liver, whereas expression of Peli 1 gene was detected in lung, heart and skin, and expression of Peli 1 gene was not detected in brain, small intestine and testis.

Thus, it was found that the transgenic mouse prepared above was a mouse in which Peli 1 was overexpressed by the administration of doxycycline.

<110> SUNGKYUNKWAN UNIVERSITY Foundation for Corporate Collaboration <120> Animal model for lymphoma and uses thereof <130> KPA140926-KR <160> 8 <170> Kopatentin 2.0 <210> 1 <211> 1257 <212> DNA <213> Artificial Sequence <220> <223> Peli 1 polynucleotide <400> 1 atgttttctc ctgatcaaga aaatcatcca tctaaagcac cagtaaaata tggtgaactc 60 attgtcttag ggtataatgg gtctctccca aatggcgata gaggaaggag gaaaagtagg 120 tttgctttgt ttaaaagacc taaggcaaat ggggtgaagc ccagcactgt gcatattgct 180 tgtactcctc aggctgcaaa ggcaataagc aacaaagacc agcatagcat atcatatact 240 ttatctcggg cccagactgt ggtggttgaa tatactcatg acagcaacac cgatatgttt 300 cagattggcc ggtcgactga aagccccatt gattttgtag taactgacac ggttcctgga 360 agtcaaagta attctgatac acagtcagta caaagcacta tatcaagatt tgcctgcaga 420 atcatatgtg aacggaatcc tccctttaca gcacggattt atgctgcagg atttgactca 480 tcaaaaaaca tctttcttgg ggagaaggct gccaaatgga agacatcaga tggacagatg 540 gatggcttga ccactaatgg tgttcttgtg atgcatccac gcaatgggtt cacagaagac 600 tccaagcctg gaatatggag agaaatatcg gtgtgtggaa atgtatttag cctacgtgaa 660 accagatcgg ctcagcagag aggaaaaatg gtggaaattg aaaccaatca gttacaagat 720 ggctcgttaa ttgacctctg tggtgcaaca ttgttatggc gtactgcaga aggcctttcc 780 cacactccta ccgtgaagca tttagaagct ttaagacagg aaatcaatgc agcacgacct 840 cagtgccctg tagggttcaa cacactagca tttcctagta tgaagaggaa agacgttgta 900 gatgaaaaac aaccatgggt atatctaaac tgcggccatg tacatggcta tcataactgg 960 ggaaacaaag aagaacgtga tggaaaagat cgtgaatgtc ctatgtgtag gtctgttggt 1020 ccctatgttc ctctgtggct tggatgtgaa gctggatttt atgtggacgc cggccctcca 1080 acccatgcgt ttagcccgtg tgggcatgtg tgttcagaaa agacaactgc ctattggtcc 1140 cagatcccac ttcctcatgg tactcatact tttcatgcag cctgtccctt ttgtgcacat 1200 cagttggctg gtgaacaagg ctacatcaga cttatttttc aaggacctct agactaa 1257 <210> 2 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 gcaataagca acaaag 16 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 atgagccaaa tcctgcag 18 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 aagtgaaagt cgagctcg 18 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 tgatatcgtg tgctggtctt tg 22 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 aaagtcgctc tgagttgtta t 21 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 ggagcgggag aaatggatat g 21 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 gcgaagagtt tgtcctcaac c 21

Claims (12)

A lymphoma model animal in which the Peli 1 (pellino homolog 1) gene is overexpressed.
The method according to claim 1,
Wherein the Peli 1 gene is a polynucleotide of SEQ ID NO: 1.
The method according to claim 1,
Wherein the model animal is a mouse.
(a) introducing an expression vector comprising a Peli 1 (pellino homolog 1) gene into a fertilized egg of a host animal; And
(b) implanting the introduced embryo into a uterus of a surrogate mother and giving birth to a progeny animal, wherein the Peli 1 gene is overexpressed.
5. The method of claim 4,
Wherein the Peli 1 gene is a polynucleotide of SEQ ID NO: 1.
5. The method of claim 4,
Wherein said expression vector has the cleavage map shown in Fig. 1A.
5. The method of claim 4,
Wherein said host animal is a mouse.
(a) introducing an expression vector comprising a Peli 1 (pellino homolog 1) gene into a fertilized egg of a host animal;
(b) implanting the introduced embryos into the uterus of a surrogate mother and giving birth to the first-generation descendant animal; And
(c) a step of crossing the obtained first-generation late-stage animal with a rtTA (reverse tetracycline transactivator) model animal to obtain a second-generation late-stage animal, wherein the method is a method of producing a model animal capable of developing lymphoma by administration of a doxycycline .
9. The method of claim 8,
Wherein said expression vector has a cleavage map as shown in Fig. 5A.
9. The method of claim 8,
Wherein said host animal is a mouse.
A lymphoma model animal produced by the method of claim 8, wherein the Peli 1 gene is overexpressed upon administration of the doxycycline and the Peli 1 gene is not overexpressed when the doxycycline is infected.
(a) administering a lymphoma treatment candidate agent to the lymphoma model animal of claims 1 or 11; And
(b) confirming the prognosis while raising the lymphoma model animal to which the candidate substance has been administered.

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