KR102260319B1 - Retinoic acid receptor responder 1(RARRES1) gene knockout animal model and method for its production - Google Patents

Abstract

The present invention provides a targeting vector comprising a part of the RARRES1 gene and sequences used for the production of a conditional knockout animal model, an animal cell for producing a tumorigenic animal model generated using the target vector, It relates to a tumorigenic ^{RARRES1 -/-} animal model obtained using this, a method for manufacturing the same, and a method for screening a cancer therapeutic material using the same. Accordingly, the present inventors have conducted intensive studies to reveal molecular mechanisms for cancer diagnosis and prognosis prediction. As a result, the RAREES1 ^-/- animal model is prone to spontaneous tumors, increases phosphorylation of CDK1 and Cyclin B1, and CDK1-Cyclin B1 By confirming that the activity of the complex was high, it was confirmed that the tumor cell cycle progression was unusually fast due to the decrease in proteolytic capacity. In particular, stem cell proliferation was increased, and mitotic defects were induced and chromosomes were damaged during mitosis. It was confirmed that instability was confirmed that RAREES1 is an important factor in cancer diagnosis, prognosis, and treatment. Furthermore, through the relationship between RARRES1 and CDK1-Cyclin B1 complex, quantitative regulation of CDK1-Cyclin B protein, and increase in stem cell proliferative ability, it is expected to be used in various ways for cancer screening and drug development. do.

Description

RARRES1 gene knockout animal model and manufacturing method thereof {Retinoic acid receptor responder 1 (RARRES1) gene knockout animal model and method for its production}

The present invention relates to a RARRES1 gene knockout animal model, and more particularly, a targeting vector comprising a part of the RARRES1 gene and sequences used for the construction of a conditional knockout animal model, the target vector It relates to an animal cell for producing a tumorigenic animal model generated using the ^{RARRES1 -/-} animal model for tumorigenicity obtained using the same, a production method thereof, and a screening method for a cancer therapeutic material using the same.

A ‘tumor’ refers to a mass that has grown abnormally due to the autonomous overgrowth of body tissues, and can be divided into benign tumors and malignant tumors. While benign tumors have a relatively slow growth rate and do not metastasize, malignant tumors infiltrate into surrounding tissues and grow rapidly and spread or metastasize to various parts of the body, endangering life. Therefore, malignant tumor can be regarded as the same meaning as cancer. Cells, the smallest unit constituting the body, normally divide and grow by the control function of the cells themselves, and when their lifespan is expired or damaged, they die by themselves to maintain the overall balance of numbers. However, if there is a problem with the control function of these cells themselves due to various causes, abnormal cells that should normally die become excessively proliferated. In some cases, they invade surrounding tissues and organs to form a mass, destroy or modify the existing structure. This condition can be defined as cancer.

For the diagnosis of such cancer, X-ray examination often obtains shadows for lung cancer or myeloma by simple imaging and tomography, and for visceral cancer, by observing various shades using a contrast agent, a clue of diagnosis is obtained. In particular, in gastric cancer and colorectal cancer, the double contrast method is used to check the condition of the mucous membrane, thereby discriminating even minute lesions such as early gastric cancer or colorectal cancer, which helps in early detection. As an endoscopy, a rigid endoscope has been used for visceral examination for a long time, but it did not help much in diagnosis due to the limited range of visual observation. Therefore, only the S-colonoscope is currently used. The development of flexible fiberscopes, which can be bent at will, has played a significant role in the diagnosis of various visceral cancers, particularly early diagnosis. Biopsy through endoscopy is also possible, which provides a good clue for confirmation. Currently widely used in clinical practice, there are flexible gastrofiberscope, flexible colonofiberscope, rectal and sigmoidoscope, etc. In addition, peritoneoscope, mediastino scope, bronchoscope ( bronchoscope) are used. Since G.N. Papanicolaou discovered the characteristics of malignant tumor cells in smears of uterine secretions through cell diagnosis, it has brought a breakthrough in group screening and diagnosis of cervical cancer, and especially in early diagnosis. Currently, besides uterine cancer, it is used for secretions such as stomach, lung, prostate, breast, urinary tract, and pancreas, and cell diagnosis by puncture of the thyroid gland and breast is also widely used. Diagnosis of cancer is made by histopathological microscopic examination of tissue pieces obtained through biopsy. There are methods to obtain such tissue pieces through an endoscope or through surgical manipulations such as breast and vagina. Although cancer can be diagnosed through this diagnosis, when cancer is first diagnosed, it is usually in a state that has already metastasized to the surrounding tissues or has distant metastasis. This is very important. Therefore, although it is very important to understand the onset and progression of cancer, research and effective diagnosis of the molecular mechanisms leading to the onset are insufficient.

Meanwhile, the retinoic acid receptor responder 1 (RARRES1) gene was initially identified as the most upregulated gene in skin raft culture by tazarotene, a synthetic retinoid. This gene is only found in a few mammals and is an evolutionarily conserved gene among species. In humans, it is localized at the q arm 25.32 locus on chromosome 3. Alternatively, spliced transcript variants (alternatively spliced transcript variants) encoding two distinct isoforms exist. Subtype 1 (RARRES1-1) consists of 6 exons and encodes 294 proteins, and subtype 2 (RARRES1-2) consists of 5 exons and encodes 228 proteins. The C-terminal and 3'-untranslated region (UTR) were of different conformations between these two subtypes. Expression of RARRES1 in a variety of tumor tissues and cell lines, including prostate, breast, lung, liver, colon, gastric, esophageal, nasopharyngeal, endometrial and head and neck cancers, is often abolished or silenced, mostly due to hypermethylation of the promoter region.

Accordingly, there is a need for effective diagnosis and treatment of cancer, and it is essential to establish an appropriate animal model for the development of biological samples necessary for the study of mechanisms and treatments for cancer. However, studies on the effect of RARRES1 on cancer diagnosis, cancer cell development, and progression or animal models related thereto are still insufficient.

Republic of Korea Registration Publication KR10-1348852

In order to achieve the object of the present invention as described above, the present invention provides a first loxP (locus of X-over P1) site; drug resistance gene region; a gene segment comprising the third exon of the RARRES1 genomic gene; And a second loxP site sequence of the retinoic acid receptor response factor 1 (Retinoic acid receptor responder 1; RARRES1) target vector (targeting vector) for producing a tumorigenic animal model comprising a DNA sequence comprising a transduced tumorigenic animal model production Provided is ^{a tumorigenic RARRES1 +/N} chimeric animal model prepared by injecting an animal cell into a blastocyst.

In addition, the present invention provides a tumorigenic Rarres1 ^+/- animal model prepared by crossing the Rarres1 ^+/N chimeric animal model with an animal expressing cre recombinase.

In addition, the present invention (a) preparing ^{the RARRES1 + / N} chimeric animal model; (b) preparing a RARRES1 ^+/- animal model by crossing the chimeric animal model of step (a); Provides an animal model producing method ^- and (c) the (b) step of RARRES1 ^+/- among the offspring obtained by mating an animal model RARRES1 ^{- / -} tumor formation including the step of selecting an animal model Rarres1 ^{- /.}

In addition, the present invention provides a tumorigenic Rarres1 ^-/- animal model prepared by the above production method.

In addition, the present invention comprises the steps of (a) treating a candidate material in a subject of the tumorigenic RARRES1 ^{-/- animal model;} (b) measuring the phosphorylation degree of cyclin-dependent kinase 1 (Cyclin-dependent kinase 1; CDK1) and cyclin B1 (Cyclin B1) of the subject, measuring the amount or activity of CDK1 and Cyclin B1 protein, Measuring the expression or activity of Mist1 and LGR5, or measuring the activity of SPC (surfactant protein) positive cells; And (c) compared to the candidate substance untreated group, the candidate substance in which the phosphorylation degree of CDK1 and Cyclin B1 is reduced, the amount or activity of the CDK1 and Cyclin B1 protein is reduced, the candidate substance, Mist1 (Muscle, intestine and stomach expression 1) and a candidate substance in which the expression or activity of leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) is reduced, or a candidate substance in which the activity of SPC-positive cells is reduced, is selected as a tumor therapeutic substance. A method for screening a therapeutic substance is provided.

However, the technical task to be achieved by the present invention is not limited to the tasks mentioned above, and other tasks not mentioned can be clearly understood by those of ordinary skill in the art to which the present invention belongs from the following description. There will be.

In order to achieve the object of the present invention as described above, the present invention provides a first loxP (locus of X-over P1) site; drug resistance gene region; a gene segment comprising the third exon of the RARRES1 genomic gene; And a second loxP site sequence of the retinoic acid receptor response factor 1 (Retinoic acid receptor responder 1; RARRES1) target vector (targeting vector) for producing a tumorigenic animal model comprising a DNA sequence comprising a transduced tumorigenic animal model production Provided is ^{a tumorigenic RARRES1 +/N} chimeric animal model prepared by injecting an animal cell into a blastocyst.

In one embodiment of the present invention, in front of the first locus of X-over P1 (loxP) site, splicing acceptor (SA), β-galactosidase (β-galactosidase; βgal), and SV40 polyA It may include a DNA sequence consisting of the sequence of signal (pA).

In another embodiment of the present invention, the drug resistance gene region may be a neomycin resistance gene.

In addition, the present invention provides a tumorigenic Rarres1 ^+/- animal model prepared by crossing the Rarres1 ^+/N chimeric animal model with an animal expressing cre recombinase.

In one embodiment of the present invention, in the animal expressing the cre recombinase, the gene encoding the cre recombinase may be operably linked to the Zona pellucida 3 (Zp3) promoter.

In addition, the present invention (a) preparing ^{the RARRES1 + / N} chimeric animal model; (b) preparing a RARRES1 ^+/- animal model by crossing the chimeric animal model of step (a); Provides an animal model producing method ^- and (c) the (b) step of RARRES1 ^+/- among the offspring obtained by mating an animal model RARRES1 ^{- / -} tumor formation including the step of selecting an animal model Rarres1 ^{- /.}

In one embodiment of the present invention, the animal may be a mouse.

In another embodiment of the present invention, the Rarres1 ^-/- animal model may have a RARRES1 deficiency to induce tumors.

In addition, the present invention provides a tumorigenic Rarres1 ^-/- animal model prepared by the above production method.

In one embodiment of the present invention, the animal model can induce mitotic defects or resist mitotic stress.

In another embodiment of the present invention, the animal model may induce somatic mutation.

In another embodiment of the present invention, in the animal model, one or more genes selected from the group consisting of Ccnd1, Cdkn1a, Cdkn2A, Nanog, Psrc1, and Nup214 in the mitotic cell cycle may be overexpressed.

The present invention comprises the steps of: (a) treating a subject of a tumorigenic RARRES1 ^-/- animal model with a candidate; (b) measuring the phosphorylation degree of cyclin-dependent kinase 1 (Cyclin-dependent kinase 1; CDK1) and cyclin B1 (Cyclin B1) of the subject, measuring the amount or activity of CDK1 and Cyclin B1 protein, Measuring the expression or activity of Mist1 and LGR5, or measuring the activity of SPC (surfactant protein) positive cells; And (c) compared to the candidate substance untreated group, the candidate substance in which the phosphorylation degree of CDK1 and Cyclin B1 is reduced, the amount or activity of the CDK1 and Cyclin B1 protein is reduced, the candidate substance, Mist1 (Muscle, intestine and stomach expression 1) and a candidate substance in which the expression or activity of leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) is reduced, or a candidate substance in which the activity of SPC-positive cells is reduced, is selected as a tumor therapeutic substance. A method for screening a therapeutic substance is provided.

In one embodiment of the present invention, the subject may be isolated from one or more selected from the group consisting of spleen cancer, thymus cancer, liver cancer, lung cancer, kidney cancer, thyroid cancer, small intestine cancer, stomach cancer, uterine cancer, and myeloid leukemia.

In another embodiment of the present invention, the tumor may be one or more selected from the group consisting of spleen cancer, thymus cancer, liver cancer, lung cancer, kidney cancer, thyroid cancer, small intestine cancer, stomach cancer, uterine cancer, and myeloid leukemia.

In another embodiment of the present invention, the Mist1, LGR5 or SPC may be a stem cell marker.

As a result of intensive research to elucidate the molecular mechanism for cancer diagnosis and prognosis prediction, the present inventors found that the RAREES1 ^-/- animal model is prone to spontaneous tumors, increased phosphorylation of CDK1 and Cyclin B1, and increased phosphorylation of CDK1-Cyclin B1 complex. It was confirmed that the activity was high, and it was confirmed that the tumor cell cycle progression was unusually fast due to the decrease in proteolytic capacity. In particular, stem cell proliferation was increased, and also mitotic defects were induced and chromosomes were unstable during mitosis. As a result, it was confirmed that RAREES1 is an important factor in cancer diagnosis, prognosis, and treatment. Furthermore, through the relationship between RARRES1 and CDK1-Cyclin B1 complex, quantitative regulation of CDK1-Cyclin B protein, and increase in stem cell proliferative ability, it is expected to be used in various ways for cancer screening and drug development. do.

1A is a diagram illustrating a strategy for generating a target RARRES1 allele.
Figure 1b is a diagram confirming the Rarres1 gene deficiency by extracting RNA from the Rarres1 knockout embryo.
Figure 1c is a diagram confirming the deletion of Rarres1 exon 3 by extracting DNA from a Rarres1 knockout embryo.
1D is a diagram confirming the genotypes of wild-type and Rarres1-deficient mice by PCR genotyping analysis of the tail genomic DNA of the mouse according to the method of Examples 1-6.
1E shows cDNA prepared from RNA of RARRES1 ^+/+ , RARRES1 ^+/- , and RARRES1 ^−/- mouse embryo fibroblasts (MEFs), exon 1 (sense) exon 6 (antisense), and specific It is a diagram confirming the expression of RARRES1 gene in different genotypes through RT-PCR according to the method of Example 1-1 using oligonucleotides.
Figure 1f is a diagram showing the results of analyzing RARRES1 expression through Western blot (Western blot) in whole embryos at 13.5 days (E13.5) after embryonic development.
1g is a diagram showing the results of LacZ staining according to Examples 1-8 in heterozygous embryos at 11.5 to 14.5 days (E11.5 to E14.5) after embryonic origin.
2 is a diagram showing the results of analyzing the phenotype of leukocytes isolated from bone marrow, spleen, and peripheral blood of wild-type, RARRES ^-/- mice of the same age (18 months old).
3A is a diagram showing Kaplan-Meier cancer-free survival curves of wild-type, RARRES1 ^+/- , and RARRES1 ^{-/- mice of the same age.}
3B is a diagram showing the results of gross morphology and histological analysis of spontaneous tumors through hematoxylin and eosin staining in RARRES1 heterozygous and homozygous mice.
Figure 3c is a diagram showing the histological form of a tumor naturally occurring in the stomach, liver, lung, and thyroid gland ^{of RARRES1 -/- mice.}
Figure 3d is a diagram showing the results of immunohistochemical staining for a specific marker (CD3) of lymphomas multiplied in various organs ^{of RARRES1 -/- mice.}
Figure 3e is a diagram showing the results of immunohistochemical staining for a specific marker (myeloperoxidase; MPO) to determine the specific gravity of myeloid cells in the bone marrow and spleen of wild-type mice and RARRES1 ^{-/- mice.
4 shows RARRES1 +/+} , RARRES1 ^+/- , and RARRES1 ^−/- at 19 to 21.5 months of age. It is a diagram confirming the shape and relative weight of the spleen, liver, and kidney of the mouse.
Figure 5 is a wild-type (Wild type; WT) and knockout (Knockout; KO) mice according to Examples 1-9 by ^{[ 18 F] FDG} Glucose uptake was monitored through PET/CT imaging.
6 is a diagram showing the results of immunoblotting according to Examples 1-3 with the indicated antibody of a cell lysate derived from wild-type or RARRES1-deficient MEF.
Figure 7a is a diagram ^{showing the growth curve of each genotype obtained by seeding 2x10 4} MEF cells for each genotype and counting at the indicated time.
Figure 7b is a diagram showing the results of flow cytometry analysis according to Examples 1-5 of WT and KO MEF.
Figure 7c is a diagram showing the results of Western blot analysis for the indicated antibodies and WT and KO MEF cells treated as described in Figure 7b.
Figure 7d shows RARRES1 ^+/+ and RARRES1 ^-/- It is a diagram showing the results of flow cytometry analysis of MEFs according to Examples 1-5.
Figure 7e shows RARRES1 ^+/+ and RARRES1 ^−/- treated as described in Figure 7d above. A diagram confirming the expression and phosphorylation of CDK1 and Cylcin B1 proteins in MEF cells.
Figure 8a is a diagram confirming that an error occurred during mitosis in ^{RARRES1 -/- MEF.}
FIG. 8b shows wild-type (WT) or RARRES1 ^-/- according to Examples 1-7 on the 13.5 day (E 13.5) day after embryonic in vitro culture. A diagram confirming the results of staining embryonic fibroblasts with antibodies to α-tubulin (red), phalloidin (green), and DAPI (blue).
Figure 8c is a diagram showing representative photos and quantification of errors appearing in cell division in WT and KO fibroblasts according to Examples 1-7.
FIG. 8d is a diagram showing the quantification of micronuclei cells showing H2AX staining among all micronuclei cells in WT and KO MEFs counterstained with an antibody against H2AX (red) and DAPI (blue) in FIG. 8a.
Figure 9a is a diagram showing the results of flow cytometry analysis according to Examples 1-5 of WT and KO MEFs treated with 50 and 100ng/ml nocodazole or DMSO for 48 hours.
Figure 9b is a diagram showing the quantification of the sub-G1 DNA content through the PI-stained cells treated in Figure 9a.
Figure 9c is a diagram showing the measurement result of the total number of cells in the WT and KO MEF treated in Figure 9a.
Figure 9d is a diagram showing the results of Western blot analysis for PARP and Caspase3 proteins in MEF treated with nocodazole (50 and 100 ng / ml) for 40 hours.
Figure 10a is a diagram showing the results of histopathological analysis through liver and lung sections of normal or cancerous mice excised from WT or RARRES1 KO mice stained with phospho-Cyclin B1-Ser126, and phospho-CDK1-T161. .
Figure 10b shows immunohistochemistry for markers indicating cell proliferation (ki67), CDK1, and phosphorylated Rb protein in continuous sectioned cross-sections of lungs of wild-type mice and RARRES1 ^+/- , ^{RARRES1 −/-} mice of the same age (18 months of age). is the result of staining with
Figure 11a shows the results of immunohistochemical staining comparing the cell proliferative activity (positive to Ki67) of type 2 alveolar cells (positive for SP-C) in the lungs of wild-type mice and RARRES1 ^-/- mice of the same age (18 months of age); is a diagram showing
11B is a diagram showing the results of immunohistochemical staining for Mist1 and LGR5, which are known organ-specific stem cell markers, in the stomachs of wild-type mice and stomach-specific RARRES1 deficient mice.
11c is a diagram showing the results of immunohistochemical staining for Mist1 and CDK1 in the stomach of wild-type mice and RARRES1 ^+/- , RARRES1 ^−/- mice of the same age (18 months old).
11d is a diagram showing the results of a spheroid formation assay confirming how the characteristics of stem cells are changed when the CDK1 activity inhibitor, RO3306, is treated in epithelial cells isolated from wild-type mice and RARRES1 ^{-/- mouse embryos.}
Figure 11e is a diagram showing numerically the result of Figure 11d spheroid formation assay.
11f is a view showing the results of confirming the organoid formation for confirming the stem cell characteristics of cells isolated from the stomach of wild-type mice and RARRES1 ^{-/- mice.}
Figure 11g is a diagram showing the preparation for RNQ sequencing of lungs at 19 days (E19) and 10 months and 18 months after embryonic in vitro culture of wild-type mice and RARRES1 ^{-/- mice.}
12 is a diagram showing the copy number variant (CNV) analysis results for each tumor sample.
13A is a diagram showing differentially expressed genes and up-regulated genes involved in WNT signaling and mitotic pathways.
13B is a diagram showing pathway activity estimated from pathways as it was confirmed that WT exhibited the opposite activity activated in KO tumors when compared to KO in the unfolded protein response (UPR).
13c is a diagram showing the mRNA expression state of Hspa8 in UPR and high binding affinity due to the IgG experiment.
13D is a diagram showing the results of gene set enrichment analysis using differentially expressed genes.
14a is a road for examining the characteristics of the genome for lung adenocarcinoma expressed in humans, and is a diagram showing CDK1 mRNA, protein expression and correlation status.
14B is a diagram showing RARRES1 mRNA expression for each subtype.
14c is a diagram showing a low RARRRES1 group C1 showing down-regulation in the cell cycle pathway.
14D is a diagram illustrating the estimation of lung cell abundance using a mouse data set and human lung adenocarcinoma.
14e is a diagram analyzing gene expression characteristics for 6 subtypes of human lung cancer (TCGA LUAD).
15 is a diagram schematically illustrating a mechanism for inducing cancer in mice lacking the Rarres1 gene. In the absence of RARRES1, abnormal activation of Cdk1, a cell division regulatory protein, causes abnormalities in the overall cell cycle, indicating that cancer may occur.

As a result of intensive research to elucidate molecular mechanisms for cancer diagnosis and prognosis prediction, the present inventors confirmed ^{that spontaneous tumors are susceptible to spontaneous tumors in the RAREES1 -/-} animal model, increased phosphorylation of CDK1 and Cyclin B1, and CDK1-Cyclin By confirming the high activity of the B1 complex, it was confirmed that the tumor cell cycle progression was unusually fast, and it was confirmed that mitotic defects and chromosomes were unstable during mitosis. It was confirmed that it is an important factor and completed the present invention based on this.

Hereinafter, the present invention will be described in detail.

In one embodiment of the present invention, in order to determine the physiological function of RARRES1 in vivo, conditional RARRES1 knockout (KO) mice were induced (see Example 2).

In another embodiment of the present invention, in order to determine whether tumors spontaneously form in RARRES1 knockout (KO) mice, in crossbred RARRES1 heterozygous mice (C57BL/6), RARRES1 ^{+ / +} (n = 30 ), RARRES1 ^+/- (n=57), and RARRES1 ^−/- (n=63) mice were formed and observed up to 22 months of age. As a result, RARRES1 ^+/- and RARRES1 ^−/- mice were more spontaneously tumor- dependent ^{than RARRES1 +/+ mice.} was found to be more predisposed, and RARRES1 ^+/− and RARRES1 ^−/− mice differ ^{from RARRES1 +/+} mice, including spleen, thymus, liver, lung, kidney, thyroid, small intestine, stomach, endometrium and eye. It was confirmed that other types of tumors occurred (FIG. 3), and the size of organs including the spleen, liver, and kidneys gradually increased according to the genotype in 19-month-old mice (FIG. 4). In addition, according to Examples 1-9 Through PET/CT imaging, it was confirmed that tumors were developed much earlier in RARRES1 KO mice than in wild type (WT) mice ( FIG. 5 ), and it was confirmed that tumors could be formed only by deletion of the RAREES1 gene ( see Example 4).

In another embodiment of the present invention, in order to confirm that CDK1-cyclin B1 complex formation and its activation promote tumorigenesis in RARRES1 KO mice, wild-type embryos at day 13.5 for in vitro culture And RARRES1-deficient MEFs according to Examples 1-7 were prepared and confirmed by western blot. As a result, phosphorylation at threonine 14 and 161 residues of CDK1 was enhanced in KO MEFs, but CDK1 expression was not changed in other MEF cells. In addition, Rb protein was increased in KO MEFs and Separase, another substrate of CDK1, was cleaved in KO cells, confirming that CDK1-Cyclin B1 activity appeared in deletion of RARRES1 (Fig. 6). In addition, in order to determine whether the increase in CDK1-Cyclin B1 activity can affect the cell growth of MEF cells, MEF cells were seeded in a 6-well plate and the number of cells was counted daily for 5 days. As a result, RARRES1 ^--- MEFs were confirmed to grow rapidly (Fig. 7a), RARRES1 ^-/- In order to evaluate whether the improved tumor cell proliferation observed in MEFs is due to the altered cell cycle progression, a cell synchronization experiment according to Example 1-2 was performed from G1 to G2 / M phase of the cell cycle RARRES1 ^-/- It could be confirmed that the cells progressed faster (Fig. 7b), and the expression of Cyclin E in WT cells peaked at 30 h after serum stimulation and decreased rapidly, whereas in KO cells, Cyclin E peaked at 30 h and slightly It was confirmed that there was a decrease (Fig. 7c). And, through the nocodazole-releasing method together with FACS and Western blot analysis, it was confirmed that the termination of mitosis in RARRES1-deficient cells was faster in RARRES1-deficient cells than in WT cells ( FIG. 7d ), and in KO cells, CDK1 The phosphorylation of the substrate, Rb, was increased from 6 hours to 18 hours after release ( FIG. 7e ). Taken together, it was confirmed that CDK1-Cyclin B1 activity was high in RARRES1 KO MEFs, when compared to WT MEFs. It was confirmed that the timing of cell cycle progression was inadequate and rapid (see Example 5).

In another embodiment of the present invention, in order to determine whether a mitotic defect occurs in RARRES1-deficient cells, several types of mitotic errors were observed,

During mitosis of RARRES1 KO MEFs, it was confirmed that chromosome misalignment and non-segregation of chromosomes occurred in KO cells ( FIGS. 8a and 8b ). To determine whether knockdown of RARRES1 would affect chromosome stability, colcemid (colcemide) according to Examples 1-11 of RARRES1 ^+/+ and RARRES1 ^−/- MEFs cultured according to Examples 1-7 Analysis of metaphase spreads revealed that more than 20% of RARRES1 ^-/- MEFs in metaphase showed significant aneuploidy or ploidy, and histone variant H2AX (H2AX foci formation) in both micronuclei and primary nuclei of RARRES1-deficient cells. Significant DNA damage was confirmed when measured by damage-dependent phosphorylation of , and it was confirmed that the reason for the increase in chromosomal non-segregation is related to the development of DNA damage foci and aneuploidy in RARRES1-deleted cells. (See Example 6).

In another embodiment of the present invention, in order to determine how a drug inducing mitotic stress such as nocodazole in RARRES1 KO MEFs affects RARRES1 KO MEFs, MEF cells are treated with nocodazole or DMSO and As a result of performing flow cytometry and cell counting according to Examples 1-5, apoptosis induced by nocodazole was significantly reduced in a dose-dependent manner in RARRES1-deleted MEFs ( FIGS. 9a and b ), and nocodazole treatment did not reduce the number of cells compared to WT MEF by (Fig. 9c), and consistent with these results, it was confirmed that the cleavage of PARP and Caspase3 was reduced in KO cells compared to WT cells (Fig. 9d), loss of RARRES1 It was confirmed that it induces resistance to nocodazole treatment through (see Example 7).

In another embodiment of the present invention, according to Examples 1-12 for phospho-Cyclin B1-Ser126 and phospho-CDK1-T161 in liver cancer and lung cancer in RARRES1 KO mice. As a result of immunohistochemistry, tumor sections from RARRES1-deficient mice were strongly stained with phospho-Cyclin B1-S126 and phosph-CDK1-T161. ), it was confirmed that the activity of Cdk1-Cyclin B1 in RARRES1 deficient mice was associated with tumor development (see Example 8).

Accordingly, the present invention provides a first loxP (locus of X-over P1) site; drug resistance gene region; a gene segment comprising the third exon of the RARRES1 genomic gene; And a second loxP site sequence of the retinoic acid receptor response factor 1 (Retinoic acid receptor responder 1; RARRES1) target vector (targeting vector) for producing a tumorigenic animal model comprising a DNA sequence comprising a transduced tumorigenic animal model production Provided is ^{a tumorigenic RARRES1 +/N} chimeric animal model prepared by injecting an animal cell into a blastocyst.

In front of the first locus of X-over P1 (loxP) site, splicing acceptor (SA), β-galactosidase (β-gal), and SV40 polyA signal (pA) in the order of It may further include a DNA sequence consisting of, but is not limited thereto, and the drug resistance gene region may be a neomycin resistance gene, but is not limited thereto.

All or part of the RARRES1 gene in the target vector is "floxed" according to the DNA sequence in mammals other than humans, particularly mice. The floxed RARRES1 can be abolished by deletion, inversion, etc. in a manner that allows expression of Cre recombinase in transgenic animals. In addition, transgenic animals expressing the CRE recombinase tissue-specifically can knockout (KO) RARRES1 tissue-specifically.

The Cre-loxP system using the Cre recombinase and loxP is a gene knockout system that inserts a loxP site into a target genome of interest, and expresses Cre recombinase to induce mutations in a target gene. It is a known method. When a target gene is floxed, two loxP sites are inserted at or around an intron site at a predetermined interval, and recombination occurs between the loxP sites in the presence of the Cre enzyme, between the two loxP sites. gene is deleted or inverted. The first and second loxP sites herein may flank all or part of the RARRES1 gene, resulting in deletion of all or part of RARRES1 activity in the presence of Cre. In the absence of Cre, floxed RARRES1 is not affected.

The full-length E. coli β-galactosidase (βgal) gene having an intron containing the SV40-derived splicing donor/splice acceptor (SA), a polyadenylation signal (pA), and a eukaryotic translation initiation signal pCMVβ containing β-galactosidase is a mammalian reporter vector designed to express β-galactosidase in mammalian cells of the human cytomegalovirus precursor gene promoter. pCMVβ expresses high levels of β-galactosidase and can be used as a reference plasmid when transfecting other reporter gene constructs, transfection using standard assays or staining to assay β-galactosidase activity. It can be used to optimize the protocol. Alternatively, the β-galactosidase gene has a Not I site at each end to insert another gene into the pCMVβ vector backbone for expression in mammalian cells, or insert a β-galactosidase fragment into another expression vector. It can be resected using, but is not limited to.

Since the neomycin resistance gene cannot survive in the environment treated with neomycin without the gene, the neomycin resistance gene has a characteristic of filtering out cells into which the carrier is not inserted, but is not limited thereto.

The animal cells may be preferably embryonic stem cells (ES cells), and the embryonic stem cells may be generally obtained from preimplantation embryos cultured in vitro. ES cells can be cultured using methods known in the art. Introduction of the target vector into embryonic stem cells can be performed by a method known in the art. For example, pronuclear microinjection, retrovirus mediated gene transfer, gene targeting, electroporation, semen mediated gene transfer, calcium phosphate/DNA co-precipitation and microfluidic and microinjection. After transfection of the embryonic stem cells, only cells that have undergone homologous recombination are selected by culturing in a selective medium containing a specific antibiotic to select resistant embryonic stem cell clones. In one embodiment according to the present invention, loxP is recombinated and exon 3 between them is deleted, so that RARRES1 is knocked out. Transgenic animals can be prepared for expression of Cre recombinase. In this case, Cre recombinase can be expressed in all cells of the transgenic animal or only in cells of a specific tissue.

In the present invention, a chimeric animal model may be provided by injecting the animal cells into the gastrulation and then implanting them into a surrogate mother, but is not limited thereto.

In addition, as another aspect of the present invention, it is possible to provide a tumorigenic RARRES1 ^+/- animal model prepared by crossing a RARRES1 ^+/N chimeric animal model and an animal expressing cre recombinase, wherein the cre recombinase is expressed. In animals that do, the gene encoding cre recombinase may be operably linked to the Zona pellucida 3 (Zp3) promoter, but is not limited thereto.

As used herein, the term "Zona pellucida 3", also known as Zona pellucida sperm-binding protein 3, zona pellucida glycoprotein 3, or sperm receptor, is a ZP module-containing protein encoded by the ZP3 gene in humans. ZP3 is, but is not limited to, a receptor for zona pellucida that binds sperm at the beginning of fertilization.

In another aspect of the present invention, (a) preparing ^{the RARRES1 + / N} chimeric animal model; (b) preparing a RARRES1 ^+/- animal model by crossing the chimeric animal model of step (a); Provides an animal model producing method ^- and (c) the (b) step of RARRES1 ^+/- among the offspring obtained by mating an animal model RARRES1 ^{- / -} tumor formation including the step of selecting an animal model Rarres1 ^{- /.}

In addition, as another aspect of the present invention, there is provided a tumor-forming RARRES1 ^-/- animal model prepared by the above production method.

The animal model can induce tumors due to RARRES1 deficiency, can induce mitotic defects or resist mitotic stress, can induce somatic mutations, and can induce Ccnd1, Cdkn1a, Cdkn2A, Nanog, Genes such as Psrc1 or Nup214 may be overexpressed, but are not limited thereto.

In addition, the animal model may be prepared using a mammal other than a human, and the mammal other than a human may be a monkey, a rat, a mouse, a rabbit, a dog, a primate, etc., preferably a Muridae animal. may, but is not limited to.

In addition, as another aspect of the present invention, the present invention provides a method comprising: (a) treating a subject of a tumorigenic RARRES1 ^-/- animal model with a candidate substance; (b) measuring the phosphorylation degree of cyclin-dependent kinase 1 (Cyclin-dependent kinase 1; CDK1) and cyclin B1 (Cyclin B1) of the subject, measuring the amount or activity of CDK1 and Cyclin B1 protein, Measuring the expression or activity of Mist1 and LGR5, or measuring the activity of SPC (surfactant protein) positive cells; And (c) compared to the candidate substance untreated group, the candidate substance in which the phosphorylation degree of CDK1 and Cyclin B1 is reduced, the amount or activity of the CDK1 and Cyclin B1 protein is reduced, the candidate substance, Mist1 (Muscle, intestine and stomach expression 1) and selecting a candidate substance in which the expression or activity of leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) is reduced or a candidate substance in which the activity of SPC (surfactant protein)-positive cells is reduced as a tumor therapeutic substance. It provides a screening method of a tumor therapeutic material comprising a.

In addition, in the present invention, (a) treating a candidate substance in vitro in vitro; (b) measuring the degree of binding of the subject's cyclin-dependent kinase 1 (Cyclin-dependent kinase 1; CDK1) and cyclin B1 (Cyclin B1); and (c) selecting a candidate material having a reduced degree of binding between CDK1 and Cyclin B1 as a cancer treatment material compared to the candidate material untreated group.

Measuring the degree of binding between the tissue retinoic acid receptor responder 1 (RARRES1) and CDK1 or Cyclin B1 in the step (b); and selecting, as a cancer treatment material, a candidate in which the degree of binding of CDK1 and Cyclin B1 is reduced in step (c) and the degree of binding of RARRES1 and CDK1 or Cyclin B1 is increased, as a cancer treatment material, but is not limited thereto. and, eventually, in step (c), RARRES1 binds to CDK1 or Cyclin B1 to inhibit CDK1-Cyclin B1 complex formation, but is not limited thereto.

In addition, the step (b) is a polymerase chain reaction (PCR), microarray (microarray), northern blotting (northern blotting), western blotting (western blotting), enzyme immunoassay (ELISA), immunoprecipitation (immunoprecipitation) ), immunohistochemistry, or immunofluorescence, but is not limited thereto.

In addition, the subject is characterized in that it is isolated from one or more selected from the group consisting of spleen cancer, thymus cancer, liver cancer, lung cancer, kidney cancer, thyroid cancer, small intestine cancer, stomach cancer, uterine cancer, and myeloid leukemia, but is not limited thereto, The subject may be one or more selected from the group consisting of tissues, cells, whole blood, blood, saliva, sputum, cerebrospinal fluid, and urine, but is not limited thereto.

In addition, the decrease in the degree of binding between CDK1 and Cyclin B1 is characterized in that phosphorylation of the 126th serine of the Cyclin B1 protein is inhibited, but is not limited thereto. It is characterized in that it binds to CDK1 inactivated at the C-terminal site including amino acid 294, but is not limited thereto.

In addition, the candidate substance is an unknown substance used in screening to measure the degree of phosphorylation, an unknown substance used to measure the amounts or activity of CDK1 and Cyclin B1 proteins, Mist1 and LGR5 To measure the expression or activity Used in screening to measure an unknown substance used, an unknown substance used to measure the activity of SPC (surfactant protein)-positive cells, or the binding degree of CDK1 and Cyclin B1 or RARRES1 and CDK1 or Cyclin B1 It means an unknown substance, preferably one or more selected from the group consisting of a compound, a microbial culture medium or extract, a natural product extract, a nucleic acid, and a peptide, but is not limited thereto, and the nucleic acid is an aptamer. , LNA (locked nucleic acid), PNA (peptide nucleic acid), and characterized in that at least one selected from the group consisting of morpholino (morpholino), but is not limited thereto.

In addition, measuring the degree of phosphorylation of the subject, measuring the amounts or activity of CDK1 and Cyclin B1 proteins, measuring the expression or activity of Mist1 and LGR5, measuring the activity of SPC (surfactant protein) positive cells, or measuring the activity of the subject The degree of binding of CDK1 and Cyclin B1 or the degree of binding of RARRES1 and CDK1 or Cyclin B1 was measured by polymerase chain reaction (PCR), microarray, northern blotting, western blotting, Enzyme immunoassay (ELISA), immunoprecipitation (immunoprecipitation), immunohistochemistry (immunohistochemistry), or characterized in that the measurement using the immunofluorescence (immunofluorescence) method, but is not limited thereto.

In addition, the tumor may be selected from the group consisting of spleen cancer, thymus cancer, liver cancer, lung cancer, kidney cancer, thyroid cancer, small intestine cancer, stomach cancer, uterine cancer, and myeloid leukemia, but is not limited thereto.

In addition, the Mist1, LGR5, or SPC may be a stem cell marker, preferably a stem cell marker isolated from a mouse. In addition, the Mist1, LGR5, or SPC can be isolated from various organs of a mouse, preferably, Mist1 or LGR5 is a stem cell marker isolated from the mouse stomach, and SPC is a stem cell marker isolated from the mouse lung. not limited

Hereinafter, preferred examples are presented to help the understanding of the present invention. However, the following examples are only provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

[Example]

Example 1. Experimental preparation and experimental method

1-1. RT-PCR

Total RNA was extracted from murine embryonic fibroblasts using TRIZOL reagent (Invitrogen) according to the manufacturer's instructions, and 1 ug of total RNA was converted to cDNA with Superscript® III reverse transcriptase (Invitrogen). The primers used were: Rarres1-F (GCGCTGCACTTCTTCAACTT), Rarres1-R (GCCATAGCTGATGCTTCCAT). Gapdh-F (TGCACCACCAACTGCTTA), Gapdh-R (GGATGCAGGGATGATGTTC), the above primers yielded PCR products of predicted sizes of 653 bp and 177 bp.

1-2. Cell synchronizations (blood iron starvation and nocodazole release)

In order to synchronize the mouse embryonic fibroblasts (MEFs) to the G0 phase, the cells were washed 4 times with PBS and then cultured in a medium containing 0.1% fetal bovine serum (FBS) for 72 hours. Thereafter, the normal medium containing 10% FBS was changed to start the cell cycle, and then the cells were harvested at a specific time.

Nocodazole release was performed to monitor the completion of cell division. MEFs grown to 30-40% were cultured in a medium containing 80 ng/ml nocodazole for 12 hours, then the cells were washed 4 times with PBS, changed to a normal medium, and allowed to exit the cell division for a specific time. cells were harvested in

1-3. Immunoblotting

The cells were lysed in lysis buffer (20 mM Tris, pH7.4, 5 mM EDTA, 10 mM Na4P2O7, 100 mM NaF, 1% NP-40, 1 mM PMSF, 0.2% protease inhibitor cocktail and phosphatase inhibitor). The protein concentration of the cell lysate was measured using the Pierce BCA Protein Assay kit (Pierce, Rockford, USA). The protein lysates were then resuspended in loading buffer, boiled for 5 minutes, followed by SDS-PAGE and immunoblotting with the indicated antibodies. Protein expression was detected by chemiluminescence using SuperSignal West Pico Chemiluminescent Substrate (Pierce).

1-4. Antibodies and Reagents

The following main antibodies were used for immunoblotting according to Examples 1-3 and for indirect immunofluorescence according to Examples 1-10: a mouse monoclonal antibody against Cdc2/cdk1 (Santa cruz, sc-54,1/1000) ), cyclin B1 (Cell signaling, #4135, 1/2000), cyclin D1 (Santa cruz, sc-246, 1/1000), Rb (Cell signaling, #9309, 1/2000), histone H3-phospho-Ser 10 (abcam, ab14955, 1/500), β-actin, and α-tubulin (Sigma-Aldrich, 1/5000), γH2AX (Millipore, #05-636, 1/500). Rabbit polyclonal antibody Cdk1-phospho-Thr 14 (Abgent, AP7517d, 1/500) as well as Cdk1-phospho-Thr 161 (Cell signaling, #9114, 1/1000, phosphor-(ser) CDKs substrate (Cell signaling, #2324, 1/1000), cyclin B1 (Cell signaling, #4138,1/1000), cyclin B1-phospho-ser 126 (abcam, ab55184,1/1000), cyclin B1-phospho-ser 128 (Santa Cruz, sc-130591, 1/1000), cyclin B1-phospho-ser 133 (Cell signaling, #4133, 1/1000), cyclin B1-phospho-ser 147 (Cell signaling, #4131, 1/1000), separase (Abcam) , ab3762, 1/1000), cyclin E (Santa cruz, sc-481, 1/1000), Rb-phospho-Ser 807,811 (Cell signaling, #9308, 1/1000), PARP (Cell signaling, #9542, 1) /1000), caspase3 (Cell signaling, #9602, 1/1000), Ki-67 (Abcam, ab15580, 1/3000) and goat antibody to RARRES1 (R&D systems, AF4255, 1/1000). Alexa Flour 488 phalloidin (Invitrogen) was used to stain F-actin Horseradish peroxidase-tagged secondary antibodies (Jackson Immuno Research Laboratories, Inc., West Grove, USA) were used. Other reagents used in this study were obtained from Sigma-Aldrich (St. Louis, USA) unless otherwise specified. wore it All reagents were used according to the manufacturer's recommended protocol.

1-5. Flow cytometry

For cell cycle or sub-G1 DNA content analysis, cells were fixed in ice-cold 80% ethanol and stored at 4 °C. It was stained with PBS containing 50 μg/ml propidium iodide (PI) and 100 μg ml RNase A at 37° C. for 30 minutes. DNA content was analyzed by FACS Calibur cytometry and results were analyzed with Cellquest software (Becton-Dickinson Immunocytometry Systems, San Diego, CA) and Modfit LT 3.3 software. A minimum of 10000 cells per sample was analyzed.

1-6. Generation of RARRES1 knockout mice

ES cell clones (F07 and A05) for RARRES1 knockout mice were obtained through the Knockout Mouse Project (KOMP). Beta-galactosidase (β-galactosidase; βgal) and a neomycin-resistant (neomycin-resistant; neo) selection cassette were inserted into intron 2 of the RARRES1 gene in mice. Chimeras were generated through blastocyst injection of ES cells, and embryonic stem chimeras were backcrossed to C57BL/6 strain to obtain mice heterozygous for RARRES1. All mice were used from a C57BL/6 genetic background and were housed in a pathogen-free barrier environment and maintained on a normal diet. Genotyping of embryos and mice was performed using primers KO-1F (CTGGGTTCTAGCCAGTTTACAGTT), Ex3-R (ACTCAGCTTTGGGTAGCATTAGTC), F4 (CAGTTGGTCTGGTGTCAAAAATAA), KO-2R (CTCAGGTTCTAGACTTCCCTGAAA) and the primers were 593 bp (wild-type knockout allele) and 478 bp (knockout allele) Allele) of the predicted size of the PCR product was calculated.

1-7. Culture of Mouse embryo fibroblasts and epithelial cells

Mouse embryonic fibroblasts (MEFs) and mouse embryonic epithelial cells (MEEs) were derived from 13.5-day-old embryos as previously described. That is, after removing the head and internal organs, the embryos were washed with phosphate-buffered saline (PBS), chopped, and treated with trypsin/EDTA to make single cells. Embryonic fibroblasts (MEFs) were resuspended in DMEM containing 10% FBS, 2 mM L-glutamine, 0.1 mM MEM nonessential amino acids, 55 uM beta-mercaptoethanol, 100 IU/ml penicillin, and 100 ug/ml streptomysin. Cell media and reagents were obtained from GIBCO (Paisley, England). Embryonic epithelial cells (MEEs) contain 1% FBS, 1 mg Insulin, 1 mg Hydrocortisone, 12.5 μg EGF, 10 mg Ascorbic acid, 10 mg Transferin, 14.1 mg Phosphoethanolamine, Na selenite, 1 μg Cholera toxin, 6.5 μg Triiodo thyronine, 35 It was cultured in D-MEM/F-12 medium containing mg Bovine pituitary extract, Ethanolamine, 50 IU/ml penicillin, and 50 ug/ml streptomysin. Incubated in a humid chamber at 37° C. 5% CO _{2 .} After embryo dissociation, plating was considered passage 0. All experiments were performed using cells within passage 5 from 3 different batches. MEF and MEEs genotypes were confirmed by genotyping PCR. A minimum of three independently generated cell lines per genotype were used.

1-8. M. LacZ staining of mouse embryos

To visualize the expression of RARRES1, we analyzed the expression of LacZ from the knockout allele. Embryos aged 11.5 (E11.5) to 14.5 (E14.5) days were fixed overnight on ice with 2% paraformaldehyde, washed with PBS, and staining solution (1 mg/ml X-gal [5] -bromo-4-chloro-3-indolyl-β-D-galactopyranoside], 1 mM potassium ferricyanide, 1 mM potassium ferrocyanide in washing buffer) overnight. Embryos were washed three times (20 min each) with washing buffer (2 mM MgCl ₂ , 0.01% deoxycholate, 0.02% NP-40, 0.1M sodium phosphate, pH 7.3), and re-fixed overnight at 4 °C with 2% paraformaldehyde. It was then dehydrated with 70% ethanol.

1-9. PET/CT imaging

All rats were fasted (water only) for at least 6 hours for PET/CT scans. After intravenous injection of 18F-fluorodeoxyglucose (FDG) (370 MBq), axial raw data were obtained on a PET scanner. Acquisition time was about 20 minutes. Axial images were reconstructed with a Shepp-Logan filter (cutoff frequency, 0.35 cycles per pixel) and rearranged in coronal and sagittal planes. The spatial resolution was 6.1 ± 6.1 ± 4.3 mm.

1-10. Immunofluorescence

MEFs were fixed in PBS/3.7% paraformaldehyde for 10 min at room temperature (RT), permeabilized in 0.2% PBS/Triton X-100 for 5 min, and blocked in PBS/3% BSA for 30 min at room temperature. Samples were incubated overnight at 4°C with primary antibody. Then, it was washed with 0.05% Tween-20/PBS (3 times), the secondary antibody was incubated at RT for 2 hours, and then washed with PBS after DAPI (0.5ug/ml) for 5 minutes at RT. Coverslips were mounted on glass slides using Prolong Gold antifade reagent (Invitrogen) and observed with a confocal microscope (Zeiss 510 Meta, Carl Zeiss). For quantification of gamma-H2AX positive cells, at least 100 cells per each MEF line were analyzed.

1-11. Metaphase spreading

To prepare metaphase diffusion from MEFs and analyze them through aneuploidy, MEFs from passage 5 were treated with 0.1ug/ml Colcemide (GIBCO BRL) at 37°C for 4-5 hours. After trypsinization, the cells were centrifuged at 800 rpm for 10 minutes, resuspended in 5 ml of 0.075M KCl, and incubated at 37° C. for 20 minutes. They were fixed in freshly prepared Carnoy's solution (methanol : glacial acetic acid = 3 : 1), and then resuspended in an appropriate amount of Carnoy's solution. The cell suspension was dropped onto a microscope/glass slide and air dried. Chromosomes were visualized with DAPI (0.5ug/ml) staining and analyzed under a confocal microscope (Zeiss 510 Meta, Carl Zeiss) using a 100X objective.

1-12. Immunohistochemistry (IHC)

Mice were sacrificed and their organs placed in buffered formalin (10%), fixed for ~24 h, and embedded in paraffin. Paraffin-embedded tissue blocks were cut to a thickness of 4 μm, and sections were dried at 56°C for 1 hour. Immunohistochemical staining was performed using an automated staining apparatus Discovery XT (Ventana Medical System, Inc. Tucson Medical System, Inc., Tucson Arizona, USA) as follows. Sections were deparaffinized and rehydrated with EZ prep (Ventana) and washed with reaction buffer (Ventana). For Phopho-cyclin B1-Ser126, anti-phospho-Cdk1-Thr 161 and anti-Ki67, antigens were recovered by heat treatment at 90° C. for 30 minutes in pH 6.0 citrate buffer (Ribo CC, Ventana).

1-13. Next-generation sequencing analysis for whole genome sequencing and RNA-Seq

WGS data were generated from the genomic DNA of each sample, which was amplified and sequenced using a HiSeq 2500. Low-quality readings were reduced using Trimmomatic v.0.36, and the reduced readings were aligned to mm10 using BWA 0.7.13. Co-cleaning and post-alignment quality control were performed using GATK 3.5 and Picard 2.7.1. MuTect2 was used to identify somatic mutations for matched pairs of tumor and normal samples. In the absence of a matching normal sample, variant calls were made using each of the three non-matching normal samples as controls, and variants called at least twice were selected. All somatic mutations were filtered out to exclude germline variants called wild-type embryos and annotated using ANNOVAR 2016.2.1. Following GATK best practices, somatic copy number variants (CNVs) were detected when all normal samples were used as normal panels. Somatic Structural Variants (SV) were called using Manta v1.3.2. For uncomparable tumor samples, SV calls were performed on each of the three uncomparable normal samples as controls, and crossover points were selected. In addition, RNA-Seq low resolution reads were trimmed and aligned to mm10 and RefSeq genetic model references using STAR 2.5.2b. Gene expression profiles were quantified using RSEM 1.3.0. The following comparisons were tested to identify genes that changed significantly over time in the knockout group compared to the wildtype group.

1. 3 time points in the knockout group (with 18 month old tumor samples), 2. 3 time points in the knockout group (with 18 month old normal samples), 3. 3 time points in the wild type group, 4. 18 months old tumor and normal samples.

The R package 'EBSeqHMM' was used to identify differentially expressed genes in the first three tests. A fourth test was performed using the R package 'limma'. Among the significant genes in the first test, genes that were not differentially expressed in the fourth test were filtered out, but the results were statistically significant in the second and third tests. Gene set enrichment analysis (GSEA) was performed on selected gene sets using DAVID 6.7, and pathway activity was evaluated using gene set variation analysis (GSVA) with reference to the MSigDB HALLMARK pathway gene set.

We also integrated the results of differential expression analysis via protein-protein interaction (PPI) networks. First, a PPI network was constructed by adding experimentally validated protein interactions to the mouse BioGRID network. Based on the total p-values calculated with the adjusted p-values in the first and fourth tests, an optimal subnetwork was derived from the PPI network using the R package "BioNet". Again, GSEA was performed on genes in the optimal subnetwork.

1-14. Additional Functional Study Using TCGA Lung Adenocarcinoma and Comparison with Lung Single Cell Profiles

Somatic mutations in RARRES1 from TCGA lung adenocarcinoma, CNVs, gene expression profiles and related pathways were investigated. We induced somatic mutations and CNV in TCGA level3 data. Next, the pathway activity for each cluster was estimated using GSVA, and the gene expression of key markers was investigated from the gene expression profile. In addition, the protein and mRNA status of Cdk1 binding to RARRES1 was investigated from comparison of RPPA and gene expression. The abundance status of normal and interstitial lung cells was estimated from single-cell mouse atlas (scMouse) lung tissue results, defining 24 normal lung cells and their marker genes (Supple 1). Cell deconvolution was attempted using mouse gene expression profiles in 24 lung cells using MCP-counters referencing marker genes. Equivalent estimates were made in the TCGA lung adenocarcinoma expression profile and the mean cell abundance for each subtype defined in the TCGA study was summarized.

1-15. Cell proliferation

Growth was confirmed using MEFs derived from 13.5-day-old fetuses. After placing 20000 cells/well in a 6-well plate, the number of cells was counted every day for 5 days from the next day. To investigate the cell survival response to the cell division inhibitor nocodazole, cells were grown by treatment with 50 and 100 ng/ml nocodazole for 48 hours. Thereafter, the cells were detached by treatment with trypsin/EDTA, and the number of cells was measured.

1-16. Live cell imaging

The day before, the retro virus capable of expressing the H2B-separase sensor was infected in the embryonic epithelial cells laid on the lab-tekⅡ chamber. After 3 days, fluorescence images were taken by inserting a total of three z-stacks at intervals of 5 minutes using a microscope (Carl Zeiss, Germany) in a humidified incubator condition at 37 °C and 5% CO2 inside the video microscope platform. After merging the z-stacks of the captured images, the fluorescence intensity in the chromosome for each image was measured using Zen 2 pro blue software.

Example 2. Generation of RARRES1 knockout mice

To determine the in vivo physiological function of RARRES1, conditional RARRES1 knockout mice were derived from mouse embryonic stem (ES) cell clones (F07 and A05) for the knock out mouse project (KOMP). A target construct containing a splicing acceptor (SA), β-galactosidase (βgal), and an SV40 polyA signal (pA) followed by a floxed neomycin-resistance cassette controlled by the β-actin promoter ( floxed neomycin-resistance cassette (neo)) was inserted into intron 2 of the mouse RARRES1 gene. In addition, the third loxP is inserted into intron 3, and as a result, exon 3 is surrounded by two loxP sequences that are recognized and removed by cre recombinase (Fig. 1a). A correctly targeted ES clone was injected into the blastocyst to obtain RARRES1 ^{+/N progeny.} RARRES1 ^+/- mice were created by mating RARRES1 ^+/N males with transgenic females expressing cre recombinase in the germline, which is regulated by the mouse zona pellucida 3 (Zp3) promoter. In addition, the coverage of RARRES1 confirmed by sequencing of RARRES1 RNA-seq in order to confirm the generation of a knockout document of Rarres1 is shown in a table ( FIG. 1b ). In addition, it was confirmed through whole genome sequencing analysis that full RARRES1 mRNA expression was not normally achieved due to deletion of the DNA exon3 sequence (FIG. 1c). As a result, it was confirmed that the RNA-seq coverage of the corresponding nucleotide sequence became 0.

Progeny ^{were produced through PCR genotyping of RARRES1 +/+} , RARRES1 ^+/- , and tail genomic DNA of RARRES1 ^−/- MEFs ( FIG. 1d ) and RT-PCR according to the method of Example 1-1 ( FIG. 1e ). One RARRES1 ^+/- mice were validated by cross-analysis. At 13.5 days of embryonicity, Western blot analysis of whole embryo lysates showed that there was no decreased protein expression level in ^{RARRES1 +/-} compared to RARRES1 ^+/+ , but the level of RARRES1 ^{protein was low in RARRES1 −/-} embryos. appeared (Fig. 1f). Due to the fusion of RARRES1 with βgal, the expression of RARRES1 in RARRES1 ^+/- embryos can be easily monitored through LacZ staining according to Examples 1-8. As shown in Fig. 1g, RARRES1 showed limited expression in whole embryos from 11.5 to 14.5 days after embryogenesis, and was mainly stained with LacZ according to Examples 1-8 around the forelimbs and hindlimbs, and the eyes of the embryos. was lightly dyed.

In addition, to investigate the effect of RARRES1 knockout on embryonic lethality, we cross-analyzed the genotypes of the heterozygotes and embryos at day 13.5 and investigated them in infant mice. As shown in Table 1, 82 embryos and 165 neonatal mice were present at the expected Mendelian ratio. RARRES1 heterozygous and homozygous mice were normal in appearance and health. Thus, this targeted knockout of RARRES1 did not affect survival during embryonic development.

Next, we tested whether RARRES1-deficient mice suffered from infertility by crossing RARRES1 ^-/- females with RARRES1 ^+/- males or RARRES1 ^-/- males with RARRES1 ^{+/- females.} Regardless of sex, if KO mice have fertility abnormalities, offspring cannot be born. Neonatal animals survived as predicted according to Mendel's genetic laws, and were apparently normal and healthy, suggesting that RARRES1-deficient mice had normal fertility (Table 2).

Example 3. Identification of non-lymphoid hematopoietic tumors in RARRES1 knockout mice

To determine the possibility of non-lymphoid neoplasia in Rarres1 knockout mice (KO mice), bone marrow, spleen, and terminal blood cells were extracted from the genotypes of Rarres1 ^+/+ and Rarres1 ^-/-. This was reacted with the following antibodies in FACS buffer (1×PBS with 0.1% bovine calf serum and 0.05% sodium azide) at 4°C for 30 minutes- eFluor 450-conjugated anti-mouse hematopoietic lineage antibody cocktail [CD3 (17A2), CD45R (RA3-6B2), CD11b (M1/70), TER-119 (TER-119), Gr-1 (RB6-8C5)] (eBioscience, San Diego, CA, USA), eFluor 450-conjugated anti-Ly-6G (RB6-8C5, eBioscience), eFluor 450-conjugated anti-CD3ε (145-2C11, eBioscience), Alexa Fluor 488-conjugated anti-CD8α (53-6.7, eBioscience), phycoerythrin-cyanine7 (PE-Cy7)-conjugated anti-CD11b (M1/70, eBioscience) , allophycocyanin-eFluor 780 (APC-eFluor 780)-conjugated anti-CD11c (N418, eBioscience), APC-eFluor 780-conjugated anti-CD4 (GK1.5, eBioscience), Alexa Fluor 488-conjugated anti-Sca-1 (D7, Biolegend, San Diego, CA, USA), PE-conjugated anti-CD45R (RA3-6B2, BioLegend), PE-conjugated anti-CD25 (PC61, BioLegend), Alexa Fluor 647-conjugated anti-CD117 (2B8, BioLegend), Alexa Fluor 647-conjugated anti-Ly-6c (HK1.4, BioLegend), PE-Cy7 conjugated anti-CD16/32 (93, BioLegend), PE-Cy7 conjugated anti-CD19 (6D5, BioLegend), Brilliant Violet 650 (BV650) -conjugated anti-CD11b (M1/70, BioLegend), BV650-conjugated anti-IA/IE (M5/114.15.2, BioLegend), BV650-conjugated anti-CD44 (IM7, BioLegend).

For intracellular staining, it was fixed with 4% paraformaldehyde for 20 minutes at room temperature. After fixation, it was washed with 1×PBS, cell permeability was secured with 0.5% Triton X-100, and stained with Alexa Fluor 647-conjugated anti-FOXP3 (MF-14, BioLegend) at room temperature for 1 hour. The stained cells were analyzed for their fluorescence values with BD LSRFortessa (BD Bioscience, San Jose, CA, USA), and the results were analyzed using FlowJo software (TreeStar, Ashland, OR, USA). Through this, c-Kit positive staining, Sca-1 tone staining cell group, known as the progenitor layer of myeloid cells, increased in the knockout document group, and CMP (common myeloid progenitor) and GMP (granulocyte, monocyte progenitor) cells increased in the knockout spleen. did. In actual terminal blood, an increase in the number of myeloid cells was observed ( FIG. 2 ).

Example 4. RARRES1 Knockout (KO) Mice Predisposed to Spontaneous Tumors

To determine whether tumors form spontaneously in RARRES1 KO mice, in outbred RARRES1 heterozygous mice (C57BL/6), RARRES1 ^+/+ (n=30), RARRES1 ^+/- (n=57), and RARRES1 ^−/- (n=63) mice were formed and observed up to 22 months of age. An autopsy was performed immediately after the animals died during the course of the experiment, or they were sacrificed by carbon dioxide asphyxiation and an autopsy was performed to find the tumor of this mouse.

After autopsy, the parenchymal organs of each mouse were immediately fixed in 10% neutral formalin, and the fixed tissues were made into paraffin blocks and sections, and then subjected to conventional hematoxylin/eosin staining and histopathological examination. In the histopathologic examination of each organ, an abnormal neoplasm distinct from the normal histological structure was defined as a tumor, and a neoplasm that compresses the surrounding normal tissue and does not show invasiveness or metastasis was defined as a benign tumor, invasive or A neoplasm showing metastasis was diagnosed as a malignant tumor.

As a result, we found that RARRES1 ^+/− and RARRES1 ^−/− mice were more prone to spontaneous tumors than RARRES1 ^{+/+ mice.} In addition, RARRES1 ^+/- and RARRES1 ^{- / -} mice caused the spleen, thymus, liver, lung, kidney, thyroid, intestine, stomach, endometrial and snow RARRES1 ^{+ / +} mice with a variety of different types of tumors, including It was confirmed that (Table 3). RARRES1 ^-/- mice developed solid tumors in various major organs such as liver, lung, and gastric tumors, and solid tumors from the thyroid gland showed metastasis to the liver (Table 3 and FIGS. 3A, 3B and 3C). . In addition, unlike RARRES1 ^+/+ mice, RARRES1 ^-/- mice developed a more advanced form of T-lymphocyte-derived lymphoma that had spread to various organs, such as the thymus, kidney, and bladder, resulting in immunity to CD3, a T-lymphocyte marker. It was confirmed through histochemical staining (Table 3 and FIG. 3D). In addition, it was confirmed by immunohistochemical staining for myeloperoxidase, a myeloid cell marker, that myeloid leukemia associated with bone marrow and spleen occurred more frequently in RARRES1 ^{-/- mice (Figure 3e).} Meanwhile, in this regard, the size of organs including the spleen, liver, and kidney gradually increased according to the genotype in 19-month-old mice (FIG. 4).

^{Also, RARRES1 + / + (n =} 6), RARRES1 +/- (n = 6), and RARRES1 ^-/- (n = 6) according to Example 1-9 for the mouse population Tumor growth was monitored using PET/CT, and identical mice from 6 to 15 months of age were observed over two months. according to examples 1-9 [ ¹⁸ F] When FDG PET/CT imaging was used, it was not possible to distinguish the degree of mouse FDG (fludeoxyglucose) absorption between KO and WT mice until 10 months of age. Compared with wild-type mice, the intensity of FDG uptake in RARRES1-deficient mice was increased mainly around the spinal cord. At 14.5 months, the intensity of FDG uptake in the liver of RARRES1 KO mice gradually improved (2/6 (33%)) when compared to WT mice. according to examples 1-9 PET/CT imaging showed tumor initiation much earlier in RARRES1 KO mice than in WT mice (Fig. 5).

Collectively, we established a causal relationship between knockdown of RARRES1 expression and cancer development, confirming that RARRES1 deletion alone is sufficient to induce tumorigenesis.

Example 5. Cell cycle progression through fine tyning regulatin of CDK1-Cyclin B1 activity in RARRES1 knockout (KO) mice

We found that RARRES1 inhibited CDK1-cyclin B1 activity in mitosis, and that RARRES1 KO mice increased tumorigenesis. In addition, Cyclin B1 transgenic mice were shown to be highly resistant to tumors. Through this, to test whether activation of CDK1-cyclin B1 regulated by direct binding of RARRES1 for each component promotes tumorigenesis in RARRES1 KO mice, wild-type and wild-type and RARRES1 deficient MEFs were prepared according to Examples 1-7, and western blot was performed. As expected, phosphorylation at residues 14 and 161 of threonine of CDK1 was enhanced in KO MEFs, but CDK1 expression was not altered in other MEF cells. In addition, Cyclin B1 phosphorylation (serine 126, 128, 133 and 147) was more accumulated in RARRES1-deficient MEFs compared to their WT counterparts. CDK activity was measured using an antibody against phosphorylated CDK substrate that detects phospho-serine in the (K/R)(S*)PX(K/R) motif, a sequence of the CDK substrate, which is elevated in deficient MEFs. did. Rb, phosphorylated and dephosphorylated at G1, and phosphorylated from S to M of the cell cycle, is a substrate for CDK1 and is phosphorylated by the active CDK1-cyclin B1 complex in mitosis. Rb protein was increased and phosphorylated in KO MEFs. Separase, another substrate of CDK1, is cleaved in KO cells, indicating that CDK1-Cyclin B1 activity appears due to the lack of RARRES1 (FIG. 6).

Next, it was investigated whether the increase in CDK1-Cyclin B1 activity could affect the cell growth of MEF cells. ^{MEF cells were seeded three times at a density of 2×10 4} cells per 6-well plate cell and cell counts were counted daily for 5 days. When compared to RARRES1 ^+/+ cells, RARRES1 ^-/- MEFs grew faster (Fig. 7a).

RARRES1 ^--- To evaluate whether the improved tumor cell proliferation observed in MEFs is due to altered cell cycle progression, G1 to G2/M phase and G2/M to G1 transition using cell synchronization experiments according to Example 1-2 studied Serum stimulation (10% FBS) for up to 42 hours followed by serum starvation (0.1% FBS) for 72 hours was observed in RARRES1 ^{−/- compared to WT cells from the G1 to G2/M phases of the cell cycle.} cells progressed faster and persisted in the G2/M phase much longer (Fig. 7b). Expression of Cyclin D, found to bind and activate CDK4/6 during G1 to prepare for DNA synthesis after expression during G1, peaked in fresh medium from serum starvation of WT cells 24 h after release, although this protein Expression was down-regulated during the overall duration of KO cells. Activation of the CDK4/6-cyclin D complex inactivates Rb retinoblastoma in the G1 phase by multiple phosphorylation called 'hypo-phosphorylation'. Rb phosphorylation peaked in both cell lines within 27 hours after serum stimulation. However, in cells lacking RARRES1, phosphorylation was maintained up to 42 h. Rb binds to E2F transcription factor in early G1 phase, and hypo-phosphorylated Rb induces release of E2F leading to expression of cyclin E, which binds and activates CDK2, thereby hypo-phosphorylating Rb protein leads to activation of the CDK2-cyclin E complex, which inactivates Cyclin E expression in WT cells peaked at 30 h after serum stimulation and decreased rapidly, but Cyclin E peaked at 30 h and decreased slightly in KO cells ( FIGS. 7c and 7d ). Using a nocodazole-releasing method combined with FACS and western blot analysis, it was found that the termination of mitosis was faster in RARRES1-deficient cells compared to WT cells (FIG. 7e). These cell cycle differences were accompanied by differences in the activation of CDK1 and Cyclin B1. KO cells enhanced the active phosphorylation of CDK1 (Threonine 161) and Cyclin B1 (Serine 126, 133, and 147). High CDK1 activity leads to the segregation of sister chromatids by regulating the activity of the dissociation enzyme, but does not complete cytoplasmic division. Expression of separation enzyme was cleaved by the activated increased throughout the time, and after 18 hours, the phosphorylation of a CDK1 substrate in KO cell Rb is was enhanced at 6 hours and 18 hours after release (Fig. 7f), which RARRES1 ^{- //} indicates that the cells have increased CDK1-Cyclin B1 activity. From these results, it was confirmed that the cell cycle progression was inappropriate and rapid in RARRES1 KO MEFs compared to WT MEFs, as the CDK1-Cyclin B1 complex activity was high.

Example 6. Loss of RARRES1 leading to mitotic defects and chromosomal instability

We investigated whether mitotic defects occur in RARRES1-deficient cells, confirming high activity of the CDK1-Cyclin B1 complex and unregulated cell cycle progression. Several types of mitotic errors were observed, including misalignment chromosomes, chromatin bridges, and lagging chromosomes (Fig. 8a). according to examples 1-10 As a result of immunofluorescence analysis, it was confirmed that chromosome alignment was mismatched and chromosome non-separation occurred in KO cells during mitosis of RARRES1 KO MEFs ( FIGS. 8b and 8c ).

To determine whether knockdown of RARRES1 would affect chromosome stability, as it has recently been shown that chromosomal segregation errors in mitosis cause chromosomal aberrations, including aneuploidies (numerical) and structural abnormalities (translocations and deletions), Colse RARRES1 ^+/+ and RARRES1 ^−/- according to example 1-7 according to example 1-7 cultured with colcemide according to example 1-11 in MEFs Metaphase spreads were analyzed and chromosome numbers determined. At passage 5 (P5), RARRES1 ^+/+ MEFs had an almost normal karyotype, but more than 20% of RARRES1 ^−/- MEFs in metaphase showed significant aneuploidy or ploidy (Table 4).

Structural abnormalities of MEF cells were investigated. RARRES1-deficient cells showed significant DNA damage as measured by damage-dependent phosphorylation of the histone variant H2AX (gamma-H2AX foci formation) in both micronuclei and primary nuclei. In comparison, gamma-H2AX incorporation was hardly found in WT MEFs ( FIGS. 8c and 8d ). Taken together, it was confirmed that chromosomal non-separation can be increased by the occurrence of DNA damage foci and aneuploidy in RARRES1 deletion cells.

Example 7. Nocodazole Resistance in RARRES1 Knockout (KO) Cells

We determined how drugs that induce mitotic stress, such as nocodazole, affect RARRES1 KO MEFs. MEF cells were treated with nocodazole (50 and 100 ng/ml) or DMSO for 48 hours, and flow cytometry and cell counting were performed according to Examples 1-5. As a result, apoptosis induced by nocodazole was significantly decreased in a dose-dependent manner in RARRES1-deficient MEFs ( FIGS. 9a and 9b ), and nocodazole treatment did not decrease the number of cells than WT MEFs ( FIG. 9c ). . Consistent with these results, cleavage of PARP and Caspase3 was decreased in KO cells compared to WT cells ( FIG. 9d ), confirming that loss of RARRES1 induced resistance to nocodazole treatment.

Example 8. Increased phosphorylation of CDK1 and Cyclin B1 in solid tumors of RARRES1 deficient mice

According to Examples 1-12 for phospho-Cyclin B1-Ser126 and phospho-CDK1-T161 in liver cancer and lung cancer in RARRES1 KO mice Immunohistochemistry (IHC) was performed. The liver and lung sections of WT mice were negatively stained with these two antibodies, but tumor sections of RARRES1-deficient mice were strongly stained with phospho-Cyclin B1-S126 and phosph-CDK1-T161. It showed negative staining (Fig. 10a). These IHC results suggest that the activity of Cdk1-Cyclin B1 in RARRES1-deficient mice is associated with tumorigenesis. On the other hand, RARRES1 ^+/- compared to the same age (18 months of age), subjected to immunohistochemical staining for the marker Ki67, CDK1, phosphorylated Rb protein representing the activation of the cell cycle in the mouse lung resulting in, RARRES1 ^{+ / +,} In RARRES1 ^-/- mice, a large number of cells positive for the above three markers were observed ( FIG. 10b ).

Example 9. RARRES1 contributing to organ-blast cell homeostasis

In lung tissue obtained from mice of the same age (18 months of age), the type II pneumocyte marker surfactant protein C (SPC), which is known as progenitor cells, respectively, and Ki67 indicating cell cycle activation were simultaneously used by fluorescence. As a result of immunohistochemical staining, cell cycle-activated type 2 cells were more frequently observed in RARRES1 ^-/- than in RARRES1 ^{+/+ (FIG. 11a).} Stomach-specific stem cell markers Mist1, LGR5 (3 months old) or gastric tissue obtained from a 3-month-old mouse (Fig. 11b) deficient in stomach-specific RARRES1 and an 18-month-old mouse (Fig. 11c) deficient in RARRES1 systemically As a result of fluorescence-immunohistochemical staining with Mist1 and CDK1 (18 months old), a large number of gastric-specific stem cells were observed in both RARRES1 systemically deficient mice and gastric-specifically deficient mice compared to control mice ( 11b and 11c).

For the spheroid-formation assay, as mentioned above, the advanced DMEM/F12 medium used for culturing embryonic epithelial cells was used, and 2000 or 5000 cells per well in a 24-well plate were suspended in 300ul of medium. and was busy (seeding). The size and number of formed spheres were measured while adding 300ul of medium once every 3-4 days. In addition, it was confirmed how the sphere formation was changed by treating the embryonic epithelial cells obtained from control mice and mice lacking RARRES1 with RO3306, which inhibits CDK1 activity. As a result, the sphere formation of embryonic epithelial cells obtained from RARRES1 -deficient mice was more active in the group not treated with RO3306 compared to the control mice. On the other hand, in the group treated with RO3306 to inhibit CDK1 activity, overall sphere formation was significantly reduced, confirming that CDK1 activity is an important factor in sphere formation. In addition, while embryonic epithelial cells of control mice was not nearly not formed, obtain, embryo epithelial cells derived from the RARRES1 deficient mice shows that sikindaneun because they form a phrase to a lack of RARRES1 increase of CDK1 activity maintaining stem cell function could be (Figs. 11d, 11e).

In the case of gastric organoids of mice, after taking out the stomach of the mouse, separate the fundus and pylorus of the stomach, put them in 8mM EDTA solution, respectively, and cut them (chopping) at 4℃ for 1 hour incubated during Thereafter, through centrifugation and filtering, the cells were separated into single cells. Then, after suspension (suspension) with matrigel (matrigel) was dispensed in a 48 well plate (seeding). After incubation at 37°C for 5-10 minutes to harden matrigel, the medium containing advanced DMEM/F12 media was added to the growth factor around the matrix. filled it Thereafter, the medium and growth factors were replaced and maintained once every 2-3 days. As a result, it was confirmed that gastric organoids obtained from mice lacking RARRES1 in the stomach were formed faster than organoids obtained from control mice (see FIG. 11f ).

Example 10. Somatic transformation analysis using whole genome sequencing

As shown in Table 5, all non-silent somatic mutations were revealed. In particular, the mouse BRAF p.V637E mutation matched the target of Vemurafenib human BRAF p.V600E mutant. That means that the RARRES1 KO mouse model induces targetable somatic mutations. Bcl9l nonsynonymous (p.S898T) and Gnas (p.N964delinsNG) nonframeshift insertions were found in cancer-associated genes. All somatic mutations are shown in Table 6.

Sample Chr Start End Ref Alt Gene. ExonicFunc AAChange refGene refGene refGene 6T chr6 39627783 39627783 A T Braf nonsynonymous SNV exon18:c.T1910A:p.V637E 4T chr2 174345439 174345439 - CGG Gnas nonframeshift insertion exon8:c.2891_2892insCGG:p.N964delinsNG 5T chr9 44507447 44507447 T A Bcl9l nonsynonymous SNV exon7:c.T2692A:p.S898T

Cell Marker Alveolar bipotent progenitor Krt8,Emp2,Aqp5,Sftpd,Sftpa1 Alveolar macrophages Ear2,Ear1,Cd68,Marco,Siglecf,Chil3,Pclaf,Marco,Siglecf,Ccna2 AT1 Cell Ager,Igfbp2,Hopx,Clic5,Pdpn AT2 Cell Sftpc,Sftpa1,Sftpb,Sfta2,Dram1 B Cell Cd79a,Ms4a1,Cd79b,Ighd,Cd19 Basophil Ccl4,Ccl3,Il6,Cd69,Cd200r3 Cilated cell Ccdc153,Tmem212,1110017D15Rik,Foxj1,Ccdc17 Clara Cell Scgb1a1,Aldh1a1,Cyp2f2,Scgb3a1,Hp Conventional dendritic cells Gngt2,Lst1,Plac8,Itgb2,Cd68,Cd209a,Itgax,Cd74,H2-Eb1,Itgam,Fscn1,Ccl22,Nudt17,H2-M2,Syngr2 Dendritic cells Naaa,Irf8,Cd74,Itgax,Itgae dividing cells Cdc20, Ube2c, Stmn1, Pclaf, Tubb5 Dividing dendritic cells Cd74,H2-Aa,H2-Ab1,Naaa,Ccnb2 Dividing T cells Thy1,Cd8b1,Cdk1,Cd3g,Cd3d Endothelial cells Eng,Kdr,Flt1,Cdh5,Pecam1,Car4,Kdr,Flt1,Cdh5,Pecam1,Vwf,Kdr,Flt1,Cdh5,Pecam1 Eosinophil granulocytes G0s2,Clec4d,S100a9,S100a8,Cd14 Ig-producing B cells Jchain,Igha,Igkc,Ighm,Igkv2-109 Interstitial macrophages C1qc,C1qa,Pf4,Cd74,Adgre1 Monocyte progenitor cell Elane,Mpo,Ctsg,Prtn3,Ms4a3 Neutrophil granulocytes Ngp,S100a9,S100a8,Cd177,Ly6g NK Cells Nkg7,Klra8,Klra4,Klrb1c,Klra13-ps Nuocyte Cxcr6,Icos,Thy1,S100a4,Il7r Plasmacytoid dendritic cells Ms4a6c,Plac8,Bst2,Irf7,Irf5 stromal cell Dcn,Col3a1,Fgf10,Tcf21,Hoxa5,Inmt,Gsn,Fgf10,Tcf21,Hoxa5,Acta2,Myl9,Fgf10,Tcf21,Hoxa5 T Cell Trbc2,Cd8b1,Cd3d,Cd3g,Thy1

Somatic copy number variants (CNVs) and structural variants (SVs) in all mouse tumor samples (n = 5) occurred at low frequency, considering genome stable cancer. 12 , no segmentation-level amplified or deleted CNVs were present in the tumor samples. Meanwhile, as shown in Table 7, 64 somatic structural variants (SVs) were identified in the tumor samples. Deletions (n = 27) and translocations (n = 32) occurred most frequently. In particular, there is a Cdkn1a deletion around the chr17 region: 27975841-29991317 in the KO4T sample.

SAMPLE CHROM1 POS1 CHROM2 POS2 SVCLASS GENE CANCERGENE INFO FORMAT TUMOR T18_KO2T chr12 111538547 chr12 111539406 Deletion Eif5 . END=111539406;SVTYPE=DEL;SVLEN=-859;CIGAR=1M859D;CIPOS=0,1;HOMLEN=1;HOMSEQ=G;SOMATIC;SOMATICSCORE=115 PR:SR 42,15:42,19 T18_KO2T chr12 111539685 chr12 111539798 Deletion Eif5 . END=111539798;SVTYPE=DEL;SVLEN=-113;CIGAR=1M113D;CIPOS=0,4;HOMLEN=4;HOMSEQ=AGGT;SOMATIC;SOMATICSCORE=71 PR:SR 10,0:51,20 T18_KO2T chr12 111539882 chr12 111540129 Deletion Eif5 . END=111540129;SVTYPE=DEL;SVLEN=-247;CIGAR=1M247D;SOMATIC;SOMATICSCORE=87 PR:SR 46,12:44,25 T18_KO2T chr12 111540571 chr12 111541704 Deletion Snora28, Eif5 . END=111541704;SVTYPE=DEL;SVLEN=-1133;CIPOS=0,1;CIEND=0,1;HOMLEN=1;HOMSEQ=G;SOMATIC;SOMATICSCORE=49 PR:SR 54,4:56,9 T18_KO2T chr12 111541844 chr12 111542150 Deletion Eif5 . END=111542150;SVTYPE=DEL;SVLEN=-306;CIGAR=1M306D;CIPOS=0,2;HOMLEN=2;HOMSEQ=GG;SOMATIC;SOMATICSCORE=85 PR:SR 41,1:39,19 T18_KO2T chr12 111542841 chr12 111543099 Deletion Eif5 . END=111543099;SVTYPE=DEL;SVLEN=-258;CIGAR=1M258D;SOMATIC;SOMATICSCORE=65 PR:SR 52,3:62,18 T18_KO2T chr12 111543262 chr12 111543524 Deletion Eif5 . END=111543524;SVTYPE=DEL;SVLEN=-262;CIGAR=1M262D;CIPOS=0,3;HOMLEN=3;HOMSEQ=AGG;SOMATIC;SOMATICSCORE=65 PR:SR 48,0:57,18 T18_KO2T chr12 111543659 chr12 111544526 Deletion Eif5 . END=111544526;SVTYPE=DEL;SVLEN=-867;CIGAR=1M867D;CIPOS=0,4;HOMLEN=4;HOMSEQ=AGGT;SOMATIC;SOMATICSCORE=96 PR:SR 59,4:53,27 T18_KO2T chr5 45985059 chr5 45986594 Deletion . END=45986594;SVTYPE=DEL;SVLEN=-1535;CIPOS=0,8;CIEND=0,8;HOMLEN=8;HOMSEQ=AGGAAGCT;SOMATIC;SOMATICSCORE=115 PR:SR 27,20:20,15 T18_KO2T chr5 121093327 chr5 151725086 Inversion . END=151725086;SVTYPE=INV;SVLEN=30631759;INV3;SOMATIC;SOMATICSCORE=33 PR:SR 51,2:50,2 T18_KO2T chr7 34328736 chr7 34329386 Deletion . END=34329386;SVTYPE=DEL;SVLEN=-650;CIGAR=1M650D;CIPOS=0,40;HOMLEN=40;HOMSEQ=TCTCTTCTCTTCTCTTCTCTTCTCTTCTCTTCTCTTCTCT;SOMATIC;SOMATICSCORE=39 PR:SR 23,0:21,9 T18_KO2T chr7 55643415 chr12 111538242 translocation Eif5 . SVTYPE=BND;MATEID=MantaBND:7051:10:11:0:0:0:1;SOMATIC;SOMATICSCORE=223;BND_DEPTH=41;MATE_BND_DEPTH=46 PR:SR 18,20:26,30 T18_KO2T chr7 55643426 chr12 111546484 translocation Eif5 . SVTYPE=BND;MATEID=MantaBND:7051:9:11:0:0:0:0;IMPRECISE;CIPOS=-448,448;SOMATIC;SOMATICSCORE=101;BND_DEPTH=41;MATE_BND_DEPTH=38 PR 39,19 T18_KO2T chr7 55643556 chr12 111539298 translocation Eif5 . SVTYPE=BND;MATEID=MantaBND:7051:1:11:0:0:0:1;IMPRECISE;CIPOS=-285,286;SOMATIC;SOMATICSCORE=51;BND_DEPTH=45;MATE_BND_DEPTH=44 PR 44,6 T18_KO4T chr17 16978502 chr17 17046447 Deletion . END=17046447;SVTYPE=DEL;SVLEN=-67945;IMPRECISE;CIPOS=-273,273;CIEND=-319,319;SOMATIC;SOMATICSCORE=61 PR 32,7 T18_KO4T chr17 27975841 chr17 29991317 Deletion Trp53cor1,Mir6969,Stk38,Clps,Cdkn1a,Fkbp5,Pim1,Ppard,Rpl10a,Srsf3,4930539E08Rik,Srpk1,Tcp11,Tead3,Tulp1,Anks1,Zfp523,Slc26a8,Mapk13,Mapk13,Brp6,Cpne5,Brpgd3,Cpne5 Clpsl2,Lhfpl5,Tbc1d22b,Pnpla1,Rab44,Mtch1,Rnf8,Kctd20,Armc12,Ccdc167,Ppil1,1810013A23Rik,Pxt1,Tmem217,Fance,Tbc1d22bos,Pi16,Cmtr1,1700030A11Rik,Mdga1 Cdkn1a,Pim1,Srsf3,Fance END=29991317;SVTYPE=DEL;SVLEN=-2015476;CIPOS=0,5;CIEND=0,5;HOMLEN=5;HOMSEQ=TCCCA;SOMATIC;SOMATICSCORE=21 PR:SR 58,2:43,2 T18_KO4T chr3 95073759 chr10 81306111 translocation Pip5k1a,Pip5k1c . SVTYPE=BND;MATEID=MantaBND:1714:0:1:0:0:0:1;IMPRECISE;CIPOS=-242,242;SOMATIC;SOMATICSCORE=18;BND_DEPTH=39;MATE_BND_DEPTH=38 PR 58,6 T18_KO4T chr6 90596933 chr6 90638559 Tandem-duplication Aldh1l1,Slc41a3 . END=90638559;SVTYPE=DUP;SVLEN=41626;CIPOS=0,1;CIEND=0,1;HOMLEN=1;HOMSEQ=C;SOMATIC;SOMATICSCORE=95 PR:SR 50,13:50,12 T18_KO4T chr6 122323813 chr6 122325457 Deletion Phc1 . END=122325457;SVTYPE=DEL;SVLEN=-1644;CIPOS=0,2;CIEND=0,2;HOMLEN=2;HOMSEQ=CT;SOMATIC;SOMATICSCORE=36 PR:SR 42,2:39,8 T18_KO4T chr7 16475870 chr7 16475933 Deletion Npas1 . END=16475933;SVTYPE=DEL;SVLEN=-63;CIGAR=1M63D;CIPOS=0,8;HOMLEN=8;HOMSEQ=GCCCGCGC;SOMATIC;SOMATICSCORE=74 PR:SR 1,0:15,13 T18_KO4T chr7 108528278 chr5 34913634 translocation . SVTYPE=BND;MATEID=MantaBND:31880:0:3:0:0:0:0;IMPRECISE;CIPOS=-261,261;SOMATIC;SOMATICSCORE=58;BND_DEPTH=50;MATE_BND_DEPTH=40 PR 37,7 T18_KO4T chr7 119308417 chr13 84011843 translocation . SVTYPE=BND;MATEID=MantaBND:9143:0:1:0:0:0:1;IMPRECISE;CIPOS=-281,281;SOMATIC;SOMATICSCORE=30;BND_DEPTH=39;MATE_BND_DEPTH=19 PR 62,6 T18_KO5T chr11 104535053 chr11 104536427 Deletion Cdc27 . END=104536427;SVTYPE=DEL;SVLEN=-1374;IMPRECISE;CIPOS=-186,186;CIEND=-185,186;SOMATIC;SOMATICSCORE=11 PR 45,4 T18_KO5T chr12 9077711 chr12 9184781 Tandem-duplication . END=9184781;SVTYPE=DUP;SVLEN=107070;CIPOS=0,2;CIEND=0,2;HOMLEN=2;HOMSEQ=GC;SOMATIC;SOMATICSCORE=92 PR:SR 49,11:42,9 T18_KO5T chr12 111538547 chr12 111539406 Deletion Eif5 . END=111539406;SVTYPE=DEL;SVLEN=-859;CIGAR=1M859D;CIPOS=0,1;HOMLEN=1;HOMSEQ=G;SOMATIC;SOMATICSCORE=55 PR:SR 38,7:25,5 T18_KO5T chr12 111539882 chr12 111540129 Deletion Eif5 . END=111540129;SVTYPE=DEL;SVLEN=-247;CIGAR=1M247D;SOMATIC;SOMATICSCORE=31 PR:SR 34,4:39,4 T18_KO5T chr12 111542841 chr12 111543099 Deletion Eif5 . END=111543099;SVTYPE=DEL;SVLEN=-258;CIGAR=1M258D;SOMATIC;SOMATICSCORE=45 PR:SR 34,0:32,6 T18_KO5T chr12 111543659 chr12 111544526 Deletion Eif5 . END=111544526;SVTYPE=DEL;SVLEN=-867;CIGAR=1M867D;CIPOS=0,4;HOMLEN=4;HOMSEQ=AGGT;SOMATIC;SOMATICSCORE=61 PR:SR 44,3:36,8 T18_KO5T chr14 95923489 chr14 95924636 Deletion . END=95924636;SVTYPE=DEL;SVLEN=-1147;CIPOS=0,2;CIEND=0,2;HOMLEN=2;HOMSEQ=TT;SOMATIC;SOMATICSCORE=46 PR:SR 19,2:11,5 T18_KO5T chr2 169539577 chr2 169540156 Inversion . END=169540156;SVTYPE=INV;SVLEN=579;CIPOS=0,11;CIEND=-11,0;HOMLEN=11;HOMSEQ=ACACACACACC;INV5;SOMATIC;SOMATICSCORE=16 PR:SR 44,2:33,2 T18_KO5T chr5 75143783 chr5 75145463 Deletion GM19583 . END=75145463;SVTYPE=DEL;SVLEN=-1680;CIPOS=0,2;CIEND=0,2;HOMLEN=2;HOMSEQ=TA;SOMATIC;SOMATICSCORE=172 PR:SR 11,14:8,12 T18_KO5T chr6 143203342 chr1 40661555 translocation Etnk1 Etnk1 SVTYPE=BND;MATEID=MantaBND:20826:0:1:0:0:0:0;SOMATIC;SOMATICSCORE=28;BND_DEPTH=34;MATE_BND_DEPTH=43 PR:SR 49,2:43,2 T18_KO5T chr7 55643330 chr12 111546658 translocation Eif5 . SVTYPE=BND;MATEID=MantaBND:7387:2:3:0:0:0:0;IMPRECISE;CIPOS=-352,353;SOMATIC;SOMATICSCORE=84;BND_DEPTH=44;MATE_BND_DEPTH=36 PR 38,17 T18_KO5T chr7 55643415 chr12 111538242 translocation Eif5 . SVTYPE=BND;MATEID=MantaBND:7387:0:2:0:0:0:0;SOMATIC;SOMATICSCORE=108;BND_DEPTH=41;MATE_BND_DEPTH=46 PR:SR 23,11:21,8 T18_KO5T chr7 108528280 chr5 34913636 translocation . SVTYPE=BND;MATEID=MantaBND:32855:0:2:0:0:0:0;IMPRECISE;CIPOS=-258,259;SOMATIC;SOMATICSCORE=20;BND_DEPTH=50;MATE_BND_DEPTH=40 PR 45,5 T18_KO6T chr1 42137470 chr1 42137678 Deletion . END=42137678;SVTYPE=DEL;SVLEN=-208;CIGAR=1M208D;CIPOS=0,30;HOMLEN=30;HOMSEQ=CAGCAGAGTTGCCCAACACCCGCAAGGG;SOMATIC;SOMATICSCORE=35 PR:SR 18,3:27,7 T18_KO6T chr1 127908344 chr1 127908502 Deletion Rab3gap1 . END=127908502;SVTYPE=DEL;SVLEN=-158;CIGAR=1M158D;CIPOS=0,9;HOMLEN=9;HOMSEQ=CACACACAC;SOMATIC;SOMATICSCORE=13 PR:SR 17,0:33,5 T18_KO6T chr1 171070172 chr1 171077780 Deletion . END=171077780;SVTYPE=DEL;SVLEN=-7608;IMPRECISE;CIPOS=-537,537;CIEND=-332,333;SOMATIC;SOMATICSCORE=19 PR 247,11 T18_KO6T chr15 30984772 chr15 30986359 Tandem-duplication ctnnd2 . END=30986359;SVTYPE=DUP;SVLEN=1587;IMPRECISE;CIPOS=-266,266;CIEND=-384,385;SOMATIC;SOMATICSCORE=10 PR 71,5 T18_KO6T chr18 9575535 chr14 13358092 translocation Synpr . SVTYPE=BND;MATEID=MantaBND:11510:0:1:0:0:0:0;SOMATIC;SOMATICSCORE=32;BND_DEPTH=49;MATE_BND_DEPTH=44 PR:SR 92,2:83,2 T18_KO6T chr19 42469585 chr12 97249299 translocation Gm38437 . SVTYPE=BND;MATEID=MantaBND:7967:0:2:0:0:0:1;IMPRECISE;CIPOS=-281,281;SOMATIC;SOMATICSCORE=20;BND_DEPTH=44;MATE_BND_DEPTH=66 PR 84,7 T18_KO6T chr3 7000070 chr11 91724710 translocation . SVTYPE=BND;MATEID=MantaBND:5232:0:1:0:0:0:1;IMPRECISE;CIPOS=-257,258;SOMATIC;SOMATICSCORE=21;BND_DEPTH=50;MATE_BND_DEPTH=34 PR 81,10 T18_KO6T chr3 109622539 chr13 20487851 translocation Vav3,Elmo1 Vav3 SVTYPE=BND;MATEID=MantaBND:8855:0:6:0:0:0:1;IMPRECISE;CIPOS=-258,258;SOMATIC;SOMATICSCORE=10;BND_DEPTH=49;MATE_BND_DEPTH=38 PR 48,6 T18_KO6T chr3 120703425 chr14 70715950 translocation 6530403H02Rik,Xpo7 Xpo7 SVTYPE=BND;MATEID=MantaBND:12704:4:8:0:0:0:0;IMPRECISE;CIPOS=-236,237;SOMATIC;SOMATICSCORE=10;BND_DEPTH=49;MATE_BND_DEPTH=41 PR 85,5 T18_KO6T chr4 25544294 chr13 102567888 translocation . SVTYPE=BND;MATEID=MantaBND:10932:0:1:0:0:0:0;SOMATIC;SOMATICSCORE=25;BND_DEPTH=56;MATE_BND_DEPTH=44 PR:SR 71,2:77,2 T18_KO6T chr4 125060132 chr12 97601280 translocation Dnali1 . SVTYPE=BND;MATEID=MantaBND:7966:0:1:0:0:0:0;CIPOS=0,1;HOMLEN=1;HOMSEQ=G;SOMATIC;SOMATICSCORE=16;BND_DEPTH=44;MATE_BND_DEPTH=52 PR:SR 55,2:52,2 T18_KO6T chr5 33111812 chr1 126334627 translocation Slc5a1,Nckap5 . SVTYPE=BND;MATEID=MantaBND:25541:0:2:0:0:0:1;IMPRECISE;CIPOS=-250,251;SOMATIC;SOMATICSCORE=10;BND_DEPTH=35;MATE_BND_DEPTH=48 PR 84,5 T18_KO6T chr5 81150341 chr5 81150535 Deletion Adgrl3 . END=81150535;SVTYPE=DEL;SVLEN=-194;CIGAR=1M194D;CIPOS=0,8;HOMLEN=8;HOMSEQ=TGTGTGTG;SOMATIC;SOMATICSCORE=10 PR:SR 27,2:34,4 T18_KO6T chr5 128099724 chr1 141912421 translocation Tmem132d . SVTYPE=BND;MATEID=MantaBND:9003:0:1:0:0:0:0;SOMATIC;SOMATICSCORE=11;BND_DEPTH=54;MATE_BND_DEPTH=42 PR:SR 83,2:64,2 T18_KO6T chr6 90775745 chr13 65106495 translocation Iqsec1, Mfsd14b . SVTYPE=BND;MATEID=MantaBND:9855:0:2:0:0:0:0;IMPRECISE;CIPOS=-229,230;SOMATIC;SOMATICSCORE=38;BND_DEPTH=66;MATE_BND_DEPTH=45 PR 76,6 T18_KO6T chr6 94897007 chr6 94897062 Deletion . END=94897062;SVTYPE=DEL;SVLEN=-55;CIGAR=1M1I55D;SOMATIC;SOMATICSCORE=13 PR:SR 2,0:18,8 T18_KO6T chr7 81108995 chr3 62201316 translocation . SVTYPE=BND;MATEID=MantaBND:1:7082:12215:0:0:0:1;IMPRECISE;CIPOS=-271,272;SOMATIC;SOMATICSCORE=23;BND_DEPTH=50;MATE_BND_DEPTH=70 PR 49,6 T18_KO6T chr8 4479310 chr7 54004741 translocation . SVTYPE=BND;MATEID=MantaBND:8256:9:10:0:0:0:0;IMPRECISE;CIPOS=-253,253;SOMATIC;SOMATICSCORE=11;BND_DEPTH=41;MATE_BND_DEPTH=53 PR 69,5 T18_KO6T chr8 57572028 chr14 63280308 translocation Galnt7 . SVTYPE=BND;MATEID=MantaBND:1:8069:8070:0:1:0:1;SOMATIC;SOMATICSCORE=50;BND_DEPTH=47;MATE_BND_DEPTH=84 PR:SR 43,6:47,6 T18_KO6T chr9 60802071 chr18 35854024 translocation Uaca,Cxxc5 . SVTYPE=BND;MATEID=MantaBND:8011:3:6:0:0:0:1;IMPRECISE;CIPOS=-244,244;SOMATIC;SOMATICSCORE=26;BND_DEPTH=39;MATE_BND_DEPTH=44 PR 86,10 T18_KO6T chrX 18640983 chr18 33772125 translocation Gm14345,Gm14346,Gm10921 . SVTYPE=BND;MATEID=MantaBND:1:41100:41101:0:0:0:0;IMPRECISE;CIPOS=-270,271;SOMATIC;SOMATICSCORE=14;BND_DEPTH=29;MATE_BND_DEPTH=44 PR 65,6 T18_KO6T chrX 75637613 chr9 52949211 translocation . SVTYPE=BND;MATEID=MantaBND:23533:1:2:0:0:0:1;IMPRECISE;CIPOS=-323,324;SOMATIC;SOMATICSCORE=20;BND_DEPTH=28;MATE_BND_DEPTH=60 PR 57,8 T18_KO7T chr17 50143150 chr14 12743313 translocation Rftn1, Cadps . SVTYPE=BND;MATEID=MantaBND:13894:0:1:0:0:0:1;CIPOS=0,1;HOMLEN=1;HOMSEQ=G;SOMATIC;SOMATICSCORE=29;BND_DEPTH=55;MATE_BND_DEPTH=48 PR:SR 70,2:61,2 T18_KO7T chr18 25838191 chr12 117721570 translocation Rapgef5 . SVTYPE=BND;MATEID=MantaBND:10175:2:5:0:0:0:1;IMPRECISE;CIPOS=-249,249;EVENT=MantaBND:10175:2:5:0:0:0:0;SOMATIC;SOMATICSCORE =0;JUNCTION_SOMATICSCORE=10;BND_DEPTH=55;MATE_BND_DEPTH=39 PR 54,5 T18_KO7T chr18 25838370 chr12 117721238 translocation Rapgef5 . SVTYPE=BND;MATEID=MantaBND:10175:2:5:1:0:0:1;CIPOS=0,1;HOMLEN=1;HOMSEQ=G;SVINSLEN=85;SVINSSEQ=TGAATACTCACCACAGAAGAAGAATAAAGCCCTTEVENT=MANtaBATTCAGTCTTAAGGAGAACTGGCTCCAGCACACAGAGAGNDGACTCCAGCACAC; 2:5:0:0:0:0;SOMATIC;SOMATICSCORE=0;JUNCTION_SOMATICSCORE=0;BND_DEPTH=50;MATE_BND_DEPTH=69 PR:SR 47,11:35,10 T18_KO7T chr19 17533464 chr16 27971791 translocation pcsk5 pcsk5 SVTYPE=BND;MATEID=MantaBND:19884:0:1:0:0:0:1;CIPOS=0,1;HOMLEN=1;HOMSEQ=T;SOMATIC;SOMATICSCORE=25;BND_DEPTH=50;MATE_BND_DEPTH=62 PR:SR 77,2:76,2 T18_KO7T chr4 154211152 chr15 97566353 translocation Megf6 . SVTYPE=BND;MATEID=MantaBND:2:9296:10229:1:0:0:0;IMPRECISE;CIPOS=-252,253;EVENT=MantaBND:2:9296:10229:0:0:0:0;SOMATIC;SOMATICSCORE =24;JUNCTION_SOMATICSCORE=0;BND_DEPTH=31;MATE_BND_DEPTH=47 PR 73,2 T18_KO7T chr4 154211357 chr15 97566889 translocation Megf6 . SVTYPE=BND;MATEID=MantaBND:2:9296:10229:0:0:0:1;IMPRECISE;CIPOS=-391,391;EVENT=MantaBND:2:9296:10229:0:0:0:0;SOMATIC;SOMATICSCORE =24;JUNCTION_SOMATICSCORE=2;BND_DEPTH=69;MATE_BND_DEPTH=41 PR 39,4 T18_KO7T chr7 97887440 chr7 97887904 Deletion Pak1 Pak1 END=97887904;SVTYPE=DEL;SVLEN=-464;CIGAR=1M464D;CIPOS=0,7;HOMLEN=7;HOMSEQ=CTGGCCT;SOMATIC;SOMATICSCORE=16 PR:SR 64,0:51,6

Example 11. Gene expression profile and function investigation

Gene expression profile analysis showed consistent traits within each case. When comparing tumor and normal samples (FDR <1.0e ⁻⁰⁵ ) from KO tumor mice, differentially expressed genes were selected, depending on the time factor (FDR <1.0e ^{−04 ) described in the method after gene removal.} . In addition, 1720 genes were selected as the DEG set by selecting the subnetwork genes. As a result of gene set enrichment analysis (GSEA), we identified pathways involved in canonical Wnt signaling, cell cycle and mitosis (Fig. 13a), and further unfolded protein response (UPR) was It has been shown to act in the opposite pathway between KO and WT. Relatively, UPR genes Ciar, Eif2s1, Hspa5, Hspa8 and Hsp90b1 were downregulated in KO normals than in WT, but were highly expressed in KO tumors (Fig. 13b). In addition, as shown in FIG. 13c , Hspa8 was identified as a binding protein with RARRES1 from the IgG evidence. At Wnt signaling or mitotic cell cycle, Ccnd1, Cdkn1a, Cdkn2A, Nanog, Psrc1 and Nup214 were highly expressed in KO mouse tumor samples ( FIG. 13D ).

Cdk1 mRNA exhibited a fold change XXX between KO tumors and KO normals. However, from the comparison of TCGA LUAD RNA-Seq and RPPA, a low correlation (XXX) between mRNA expression and protein abundance was confirmed. Otherwise, it was found that the strong binding potential of Cdk1-RARRES1 (Fig. 13c) was demonstrated through IgG experiments.

Example 12. RARRES1 status in TCGA lung adenocarcinoma and lung cell overburden deconvolution

RARRES1 status was investigated in both DNA and RNA evidence of TCGA human lung adenocarcinoma (TCGA LUAD, n = 230). As shown in Fig. 14a, the Rarres1 mutant was amplified by 1.3% CNV, and there was no somatic mutation. In addition, as shown in FIG. 14b , there was a clear difference in Rarres1 mRNA expression among the six TCGA LUAD subtype clusters. The C1 group belongs to low expression of Rarres1 (log2 fold change = -1.52, T-test P-value = 5.58e ^-08 , Figure 14b and Figure 14c).

Gene expression profiles were used to estimate the presence of 24 known lung cells. The proportion of most cells was consistent with WT and KO status. However, alveolar type 2 (AT2) cells appeared extremely abundant only in KO tumor samples, but at low concentrations in all other conditions (fold change 2.8 between KO normal and KO tumors, FIG. 14d ). In addition, in human TCGA LUAD, the C1 group had a higher proportion of AT2 cells (fold change 2.7, FIG. 14d).

In addition, as shown in Fig. 14e, through the upper right graph, it was found that RARRES1 showed the lowest expression in the C1 group among human lung cancer subtypes. Among the subtypes of human lung cancer, AT2 cells and alveolar bipotent progenitor were the highest in group C1. Through the histogram at the bottom right, the quantitative overall distribution histogram of AT2 cells, progenitor cells of the lung, group C1 among human lung cancer subtypes It was found that this was the highest.

The above description of the present invention is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

<110> National Cancer Center <120> Retinoic acid receptor responder 1 (RARRES1) gene knockout animal model and method for its production <130> MP18-188 <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Rarres1-F <400> 1 gcgctgcact tcttcaactt 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Rarres1-R <400> 2 gccatagctg atgcttccat 20 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Gapdh-F <400> 3 tgcaccacca actgctta 18 <210> 4 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Gapdh-R <400> 4 ggatgcaggg atgatgttc 19 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> KO-1F <400> 5 ctgggttcta gccagtttac agtt 24 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Ex3-R <400> 6 actcagcttt gggtagcatt agtc 24 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> F4 <400> 7 cagttggtct ggtgtcaaaa ataa 24 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> KO-2R <400> 8 ctcaggttct agacttccct gaaa 24

Claims (18)

a first locus of X-over P1 (loxP) site; drug resistance gene region; a gene segment comprising the third exon of the RARRES1 genomic gene; And a second loxP site retinoic acid receptor response factor 1 (Retinoic acid receptor responder 1; RARRES1) target vector (targeting vector) for making a tumorigenic animal model other than humans comprising a DNA sequence consisting of a transduced human Non-human tumorigenic RARRES1 ^+/N chimeric animal model prepared by injecting animal cells into the blastocyst for production of non-tumorigenic animal models.
According to claim 1,
In front of the first locus of X-over P1 (loxP) site, splicing acceptor (SA), β-galactosidase (β-galactosidase; βgal), and SV40 polyA signal (pA) in the order A non-human tumorigenic RARRES1 ^+/N chimeric animal model, characterized in that it comprises a DNA sequence consisting of
According to claim 1,
The drug resistance gene region is a neomycin resistance gene, characterized in that the non-human tumorigenic RARRES1 ^{+ / N} chimeric animal model.
A non- human tumorigenic RARRES1 ^+/- animal model prepared by crossing the non-human tumorigenic RARRES1 ^+/N chimeric animal model of claim 1 and an animal other than a human expressing cre recombinase.
5. The method of claim 4,
In animals other than humans expressing the cre recombinase, the gene encoding the cre recombinase is operably linked to the Zona pellucida 3 (Zp3) promoter, non-human tumorigenic RARRES1 ^+/- animal model.
A method for preparing a non ^{-human tumorigenic RARRES1 -/-} animal model comprising the following steps.
(a) preparing a non-human tumorigenic RARRES1 ^+/N chimeric animal model of claim 1;
(b) preparing a RARRES1 ^+/- animal model excluding humans by crossing the chimeric animal model of step (a); and
(c) selecting a non ^{-human RARRES1 −/−} animal model from among the progeny obtained by crossing the non-human RARRES1 ^+/- animal model of step (b).
7. The method of claim 6,
The production method, characterized in that the animal is a mouse.
7. The method of claim 6,
The oncogenic RARRES1 ^-/- animal model except for the human is characterized in that the tumor is induced by the deletion of RARRES1, the manufacturing method.
7. The method of claim 6,
The tumor is characterized in that at least one selected from the group consisting of spleen cancer, thymus cancer, liver cancer, lung cancer, kidney cancer, thyroid cancer, small intestine cancer, stomach cancer, uterine cancer, and myeloid leukemia, a manufacturing method.
A tumorigenic RARRES1 excluding humans prepared by the method of claim 6 ^-/- animal model.
11. The method of claim 10,
The animal model is characterized in that for inducing mitotic defects or for resistance to mitotic stress, non-human tumorigenic RARRES1 ^-/- animal model.
11. The method of claim 10,
The animal model is characterized in that the induction of somatic mutation, non-human tumorigenic RARRES1 ^-/- animal model.
11. The method of claim 10,
The animal model is characterized in that at least one gene selected from the group consisting of Ccnd1, Cdkn1a, Cdkn2A, Nanog, Psrc1, and Nup214 in the mitotic cell cycle is overexpressed, non-human tumorigenic RARRES1 ^-/- animal model.
11. The method of claim 10,
Wherein the tumor is a non-colorectal cancer, thymus cancer, liver cancer, lung cancer, kidney cancer, thyroid cancer, small intestine cancer, stomach cancer, cervical cancer, and tumors other than the human, characterized in that the induction in the one or more selected from the group consisting of myeloid leukemia formed RARRES1 ^{- //} Animal model.
A method for screening a tumor therapeutic agent, comprising the steps of:
(a) treating a candidate material in a subject of a non-human tumorigenic RARRES1 ^{-/- animal model;}
(b) measuring the degree of phosphorylation of the subject's cyclin-dependent kinase 1 (Cyclin-dependent kinase 1; CDK1) and cyclin B1 (Cyclin B1);
Measuring the amount or activity of CDK1 and Cyclin B1 protein,
measuring the expression or activity of Mist1 and LGR5, or
measuring the activity of SPC (surfactant protein) positive cells; and
(c) compared to the untreated group,
Candidate substances that decrease the degree of phosphorylation of CDK1 and Cyclin B1;
Candidate substances that decrease the amount or activity of CDK1 and Cyclin B1 proteins;
Candidate substances in which the expression or activity of Mist1 (Muscle, intestine and stomach expression 1) and LGR5 (Leucine-rich repeat-containing G-protein coupled receptor 5) is reduced, or
A step of selecting a candidate material that reduces the activity of SPC-positive cells as a tumor treatment material.
16. The method of claim 15,
The screening method, characterized in that the subject is isolated from one or more selected from the group consisting of spleen cancer, thymus cancer, liver cancer, lung cancer, kidney cancer, thyroid cancer, small intestine cancer, stomach cancer, uterine cancer, and myeloid leukemia.
16. The method of claim 15,
The tumor is spleen cancer, thymus cancer, liver cancer, lung cancer, kidney cancer, thyroid cancer, small intestine cancer, stomach cancer, uterine cancer, and myeloid leukemia, characterized in that at least one selected from the group consisting of, a screening method.
16. The method of claim 15,
The Mist1, LGR5, or SPC is a stem cell marker, characterized in that, screening method.

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