JP5954775B2 - de novo carcinogenic pancreatic cancer cell line - Google Patents

Description

  The present invention relates to a de novo carcinogenic human pancreatic cancer model mouse, a de novo carcinogenic pancreatic cancer cell line isolated from the mouse, and a method for screening an anticancer agent specific for pancreatic cancer using the same.

It is estimated that approximately 260,000 people die every year worldwide due to cancer that occurs in the pancreas (Non-Patent Documents 1 and 2). In the United States alone, it is estimated that 36,800 people died in 2010 due to cancer that occurred in the pancreas, and the incidence is known to increase year by year. On the other hand, over the past 30 years, the 5-year survival rate of pancreatic cancer patients has remained at about 5%, and has not changed significantly (Non-Patent Document 3). Since pancreatic cancer is still not suitable for surgical resection and is considered to be less effective with anti-cancer drug treatment, recently developed diagnostic imaging and biomarkers are useful for prognosis of pancreatic cancer. It has been concluded that the problem has not been improved (Non-patent Document 4).
In fact, about 85% of patients diagnosed with pancreatic cancer have advanced stage with invasion of blood vessels in the vicinity of the pancreas, metastasis to surrounding lymph nodes and liver, and progression to the abdominal cavity. There are many cases and resistance to treatment (Non-patent Document 5). In addition, only about 15% of the total is subject to curative surgical resection, and even in cases where surgery is possible, metastatic progression and local recurrence occur early and with high probability. The 5-year survival rate after surgery has been reported to be 10-20% (Non-Patent Document 6), and progression-free at 6 months after diagnosis is less than 15% in patients in advanced stages (non-patent document 6) Patent Documents 5 and 7).
Based on these findings, the improvement of 5-year survival rate includes 1) the development of biomarkers that enable early detection of surgical curative resection and 2) the discovery of molecules responsible for the characteristics of pancreatic cancer. It is clear that the development of a therapeutic drug that targets the therapeutic target is urgently needed (Non-patent Document 3).

  More than 90% of cancers that occur in the pancreas are ductal phenotypes such as gland-forming and mucin-producing pancreatic duct adenocarcinoma (PDAC) Thus, the frequency of diagnosis of acinar cell carcinoma (ACC) showing an acinar cell phenotype is about 1 to 2% of adult pancreatic tumors. The median survival of PDAC undergoing surgery is 24 months, whereas ACC undergoing surgery is 61 months, and ACC has a better prognosis than PDAC (Non-patent Document 8) ). Therefore, cancer that occurs in the pancreas that has a poor prognosis and requires the development of biomarkers that enable early detection refers to pancreatic ductal adenocarcinoma (PDAC).

  In the normal pancreas, 85% of the acinar cells have a complicated three-dimensional structure, and the rest are endocrine components composed of blood vessels and interstitial fibers, and acinar cell secretion. It is composed of a ductal system that transports acinar cell secretions to the duodenum (Non-patent Document 9). If cells with ductal phenotype are proliferating in the pancreas, it is judged as pancreatic intraepithelial neoplasia (PanIN) or pancreatic intraepithelial neoplasia (PanIN). It is difficult to differentiate between ductal cells (DC) that grow sexually and pancreatic ductal adenocarcinoma (PDAC). The main reason is that there are not enough indicators and biomarkers to determine whether cells with ductal phenotype are neoplastic. This is one of the reasons why early detection of pancreatic ductal adenocarcinoma (PDAC) is not achieved.

  Currently, expression of cytokeratin 7 or 19 (CK7, 19), etc. (Non-Patent Documents 10 and 11), cancer-related sugar chains, glycoproteins and mucin core proteins (Non-Patent Documents 11-14), mesothelial cell marker meso Glycosylphosphatidyl inositol-anchored protein expression such as serine (Mesothelin) and prostate stem cell antigen (Prostate stem cell antigen), claudin-4 (Non-patent Document 15), sea urchin The expression of ADAM9 and S-100 (Non-patent Document 16) of the sea urchin fascin homolog is regarded as an indicator and marker molecule for pancreatic ductal adenocarcinoma (PDAC). Increased expression of molecules related to cell cycle regulation such as Cyclin A, Cyclin D1, and D3 is also referred to. However, since these molecules are also expressed in normal ductal cells of the pancreas, they cannot be said to be useful for differentiating neoplastic DCs from non-neoplastic DCs.

Detection of a transcript (mRNA) of a gene encoding a protein that specifically appears in pancreatic ductal adenocarcinoma (PDAC) has also been utilized in the diagnosis of PDAC. In skeltal, CK7, CK17, CK19, Fascin, Pleckstrin and other cytoskeletal proteins (Cytoskeletal protein) mRNA expression is known to increase, topoisomerase IIα (Topoisomerase II-alpha), acute myelogenous leukemia (Acute myelogenous leukemia) increased expression of genes related to DNA transcription such as leukiemia (AML), increased expression of genes related to DNA repair such as complementation D (Ataxia-telangiectasia group D-complementing; ATDC), And it is known that there is an increase in gene expression related to cell cycle regulation represented by p21, Cyclin D1, and D3. Furthermore, HE4 for secretory proteins, Mesothelin, PSCA, CEACAM6, MIC1, Claudin 1, 3, 4, 7, Integrin a6, a10, S-100A4, S-100A6, S-100A10, S-100A11 for cell surface proteins , S-100 calcium-binding protein P, Trop2, and ALG-2 gene expression is known to increase, and its detection is useful for differential diagnosis.
However, since both are molecules that are also expressed in normal ductal cells of the pancreas, it is difficult to set a cutoff that can distinguish neoplastic growth from reactive growth.

  Detection of genetic mutation and loss of heterozygosity (LOH) in the region where the gene is found by analyzing clinical cases of pancreatic ductal adenocarcinoma (PDAC) (Non-Patent Document 17) It is effective for. More than 80% of pancreatic ductal adenocarcinoma (PDAC) has a mutation in the K-Ras gene (KRAS) (Non-patent Documents 18 and 19), and 50 to 70% has a p53 mutation (Non-patent Document 20), 85 There are p16 mutations or homozygous deletions (Non-Patent Document 21) in more than 1% of cases. In addition, about 90% of pancreatic cancer cases, the mutation of the tumor suppressor gene MADH4 / DPC4 / SMAD4, which is deficient in the 18q region heterozygous deletion (LOH), was found in about 55% of cases. Exists (Non-Patent Document 22). It is also useful for differentiation from pancreatic acinar cell carcinoma (ACC), and most ACCs do not detect KRAS, p53, p16, or DPC4 gene abnormalities. These things are thought to be related to the fact that the above carcinogenic mechanism is characteristic of pancreatic ductal adenocarcinoma (PDAC) and greatly affects the determination of its biological characteristics (Non-patent Document 23, 24).

Cell cycle regulation and proliferation in both cases of CpG island promoters (promoter CpG islands), which are unmethylated regions with high cytosine / guanine base ratio in tumor suppressor genes, or miRNAs expression Causes abnormalities in metastasis and apoptosis. Therefore, detecting these changes in tumor cells is considered useful for the diagnosis of pancreatic ductal adenocarcinoma (PDAC).
In pancreatic ductal adenocarcinoma (PDAC), methylation occurs in CpG island promoters of tumor suppressor genes. This suppresses the expression of the tumor suppressor gene as well as the loss of function of the tumor suppressor gene due to gene mutation or heterozygous deletion (LOH) (Non-patent Document 17). In addition to methylation, the mechanism of silencing tumor suppressor genes is micro RNAs that are small non-coding RNAs (RNAs) with a small 17-25 gene length (nts). The involvement of (miRNAs) has also been clarified. miRNAs control the activity of genes encoding proteins. In pancreatic ductal adenocarcinoma (PDAC), it has been reported that miRNAs whose expression is increased are involved in translational inhibition of STAT3 and SP1, and miRNAs whose expression is decreased are involved in the decrease in degradation of E2F3, SMAD7, and KRAS ( Non-Patent Documents 25 and 26).

Detection of genes that are upregulated in relation to pancreatic ductal adenocarcinoma (PDAC) identifies potentially existing pancreatic cancer patients and enables their early detection, so further exploration has been vigorously attempted. (Non-Patent Documents 27 to 30). However, since it is difficult to specifically collect and compare target duct cells (DC) to be compared with pancreatic ductal adenocarcinoma (PDAC), search has been made in comparison with acinar cells. Is the current situation. Therefore, it is difficult to define the difference between pancreatic ductal cells (DC) and pancreatic ductal adenocarcinoma (PDAC) by the difference in the expression of marker molecules.

  In order to find molecules with different expression in cancer cells and cell populations that are the origin of cancer, it is necessary to sort each population as accurately as possible. This is because cancer tissue is composed of stromal tissue composed of cancer cells and blood vessels and fiber tissues that support their growth, and stromal tissue is a component common to cancer tissue and normal tissue. It is. In order to sort out a specific cell population, techniques called fluorescent color cell sorting (FACS) and laser capture microdissection (LCM) are used. FACS is effective for the separation of hematological tumor cells and the cells that are their origin, and LCM is effective for the separation of solid tumor cells and the cells that are their origin.

  However, it is not always possible to make a comparative study of cancer cells and cell populations that are the origin of cancer by simply sorting cancer tissue and stromal tissue. This is because, due to the complexity of histocytologic construction, it may be difficult to separate and take out specific cells that are the origin of cancer even using LCM. The pancreas DC corresponds to this.

  Due to the complexity of the histological structure of the pancreas, it is difficult to conduct an omics study in which only the cells that make up the duct system are sorted by LCM and compared with pancreatic ductal adenocarcinoma (PDAC). For this reason, in most studies, pancreatic tissue is used as it is without special sorting, and compared with pancreatic cancer tissue without considering acinar cells, which account for 85%. Therefore, the current examination results are effectively the comparison results of the expression profiles of acinar cells and ductal adenocarcinoma.

In order to sort cells of the duct system, the human main pancreatic duct epithelium is immortalized and cultured and used. By using the HPDE-16E7 cell line HPDE (Non-patent Document 31), which was obtained exclusively from the main pancreatic ductal epithelium, important information on the mechanism and diagnosis of pancreatic carcinogenesis was revealed. (Non-Patent Documents 32-34).
Cell immortalization is usually performed by introducing a gene that causes immortalization into non-neoplastic cells, so that non-neoplastic cells can be expanded indefinitely in culture. An operation for creating an immortalized state by introducing an immortalized gene after removing a target cell from a living body requires high technology and time. Depending on the cell type, it may be difficult to introduce a foreign gene, and immortalization by gene introduction may not be possible.
When referring to the above HPDE, it must be pointed out that it is clearly a cell derived from the main pancreatic duct and is different from a ductal cell (DC) to be compared with pancreatic ductal adenocarcinoma (PDAC). Same as HPDE, tumors thought to be derived from relatively thick pancreatic duct epithelial cells such as the main pancreatic duct, carcinoma of distal bile ducts adenocarcinoma (CABD), cancer of ampulla of cancer Vater (CAAV) has a histology similar to that of pancreatic ductal adenocarcinoma (PDAC), but has a better prognosis than PDAC and is more responsive to surgical resection, chemotherapy, and radiation therapy. As the differential diagnosis of these tumors is very important (Non-patent Document 35), the biological characteristics are quite different at the site of development, so it is not in the epithelial cells of the main pancreatic duct but in the lobule. • Should be compared to DCs derived from the interlobular pancreatic duct.

  In recent years, genetically engineered mouse models (GEM) that carry genetic abnormalities found in clinical specimens of pancreatic cancer have been created and analyzed, and it has become clear that human pancreatic cancer can be reproduced. By using this mouse, elucidation of the process at the molecular level leading to the development of pancreatic ductal adenocarcinoma (PDAC) has progressed, and the PanIN-PDAC linear pathway has been clarified (Non-patent Document 36). ~ 38). Furthermore, the mouse model is being developed for the development of new diagnostic methods (Non-Patent Documents 39 and 40).

Immortalized cells can also be systematically produced using genetically modified mice.
In the preparation of HPDE, E6 and E7 antigens are introduced to produce immortalized cells, but cells can also be immortalized by introducing SV40 large T antigen (TAg). TAg binds to Rb and Rb-related proteins and p53 to inhibit its function and suppress senescence, so it has been attempted to establish immortalized cells by creating mice that express the same molecule. .
In addition, SV40 tsA 58 large T antigen (tsA58TAg) (Non-patent Documents 41 and 42) derived from a virus isolated as thermolabile can immortalize the introduced cells at 33 ° C., but 39 ° C. Then, due to senescence, cell division is stopped within 24 hours after culturing (Non-patent Document 43).
Therefore, even if expressed in mice, it is unlikely to cause developmental abnormalities. Therefore, hematopoietic progenitor (hemopoietic progenitor) is derived from a genetically modified mouse model (GEM) in which tsA58TAg is introduced under the ubiquitously activated H2Kb promoter. cells), hematopoietic stromal cells (Non-patent Documents 44 and 45), mesenchymal stem cells present in tendons (Non-patent Document 46), germ cells (gonad cells) ), Ck19-positive biliary epithelial cells (Non-patent document 47), gastric mucosal parietal cells (Non-patent document 48), lung Clara cells (Non-patent document 48), vascular endothelium of various tissues Cell lines derived from nervous system cells such as cells (Non-patent Documents 49 and 50), enteric neuronal cells (Non-patent Document 51) and glial cells (Non-patent Document 51) are established. Has been analyzed.

  Recently, the present inventors have crossed with a mouse that expresses the Cre enzyme, thereby removing the transcription repressing sequence of loxP-βGalactosidase-poly A-loxP by Cre-mediated recombination, Transgenic mice that express tsA58TAg (loxP-βgeo-poly A-loxP-SV40 mice carrying the tsA58 large T antigen-poly A structure transgene, mouse strain name is LBL-tsA58TAg Tg mouse or T26 mouse) (FIG. 1) (Non-Patent Document 52). By crossing the same mouse with a Tie2-Cre mouse that expresses the Cre enzyme in the vascular endothelium, tsA58TAg is expressed specifically in the vascular endothelium and lymphatic endothelium. By culturing below, an immortalized vascular / lymphatic endothelial cell line has been successfully established (Non-patent Document 52).

The preparation and analysis of GEM that expresses TAg has been made early, and tsA58TAg expression in the pancreas has been shown to form tumors derived from islet cells and acinar cells. Yes.
In rat insulin II promoter (RIP) -Tag transgenic mice, it has been reported that hyperplasia and dysplasia of endocrine cells occur in 4-5w (Non-patent Document 53). , 54), pTet-on / pTRE-SV40Tag double-transgenic mice model has been reported to form endocrine tumors by TAg expressed in the brain and pancreas (Non-patent Document 55). ). On the other hand, in mouse elastase SV40-Tag (non-patent document 56) and pancreatic amylase Amy-2.2-SV40 (non-patent document 57) in which TAg is expressed in acinar cells, acinar cell carcinoma (ACC) is detected. It is known to form.

Pancreatic and duodenal homeobox 1 (Pdx1) or pancreas transcription factor 1 subunit alpha (Ptf1a) promoter-controlled mice expressed in the foregut and pancreas Cre-mediated recombination is caused to cause the always-activated Kras Kras ^G12D and Kras ^G12V to be expressed specifically in the pancreas, or lack of Ink4a / Arf gene, Trp53, and Smad4. A mouse to be lost has been created so far. As a result of these analyses, mice that express Kras ^G12D (Non-patent Documents 58 and 59) or Kras ^G12V (Non-patent Document 60) specifically in the pancreas form PanIN lesions in the first year of life. In addition to the deletion of Ink4a / Arf gene (Non-patent Documents 61 and 62), suppression of Trp53 gene function (Non-patent Document 63), lack of tumor suppressor genes Madh4 / Dpc4 / Smad4 (Non-patent documents 64 and 65), Pten deletion (Non-patent document 67), and Rb1 gene deletion (Non-patent document 68) accelerate the progression of PanIN lesions and promote the carcinogenic process. In addition to the expression of Kras ^G12D , it suppresses the activation of c-Src, which is known to be activated in Tgfbr (Non-patent Document 69) that triggers Smad4 activation and pancreatic ductal adenocarcinoma (PDAC) It has been shown that carcinogenesis is promoted in mice lacking c-Src kinase, whereas carcinogenesis is suppressed in mice lacking Rac1 (Non-patent Document 70). All of these showed a model of carcinogenesis that developed from PanIN to PDAC due to abnormalities in the ARF / MDM2 / p53 pathway and the INK4a / Rb / E2F pathway.
Based on the above, together with a model mouse that is a carcinogenic model that accurately reflects the characteristics of human pancreatic ductal adenocarcinoma (PDAC), it is highly desired to provide a de novo carcinogenic pancreatic cancer cell line as an in vitro analysis tool. It was. At that time, in order to obtain an accurate carcinogenesis model, an excellent control system is required. Therefore, it has been desired to provide a control derived from the same strain at the same time.

Jemal, A., et al., (2011) CA. Cancer J. Clin., 61, 2: 69-90 Parkin, D.M., et al., (2001) Int J Cancer, 94: 153-6 Siegel, R., et al., (2011) CA Cancer J Clin, 61, 4: 212-36 Cengel, K.A., (2004) Cancer Biol Ther, 3, 2: 165-6 Li, D., et al., (2004) Lancet, 363, 9414: 1049-57 American Gastroenterological Association, (1999) Gastroenterology, 117, 6: 1463-84 Burris, H.A., 3rd, et al., (1997) J Clin Oncol, 15, 6: 2403-13 Matos, J.M., et al, (2009) J Gastrointest Surg, 13, 8: 1495-502 Akao, S., et al., (1986) Gastroenterology, 90, 3: 661-8 Goldstein, N.S. and D. Bassi, (2001) Am J Clin Pathol, 115, 5: 695-702 Lee, M.J., et al. (2003) Mod Pathol, 16, 5: 403-10 Monges, G.M., et al., (1999) Am J Clin Pathol, 112, 5: 635-40 Andrianifahanana, M., et al., (2001) Clin Cancer Res, 7, 12: 4033-40 Kim, G.E., et al., (2002) Gastroenterology, 123, 4: 1052-60 Nichols, L.S., R. Ashfaq, and C.A. Iacobuzio-Donahue: Am J Clin Pathol: 121: 2: 226-30: 2004 Swierczynski, S.L., et al .: Hum Pathol: 35: 3: 357-66: 2004 Rozenblum, E., et al .: Cancer Res: 57: 9: 1731-4: 1997 Almoguera, C., et al .: Cell: 53: 4: 549-54: 1988 Hruban, R.H., et al .: Am J Pathol: 143: 2: 545-54: 1993 Redston, M.S., et al .: Cancer Res: 54: 11: 3025-33: 1994 Caldas, C., et al .: Nat Genet: 8: 1: 27-32: 1994 Wilentz, R.E., et al .: Am J Pathol: 156: 1: 37-43: 2000 Abraham, S.C., et al .: Am J Pathol: 161: 5: 1735-42: 2002 Moore, P.S., et al .: Mol Cancer: 2: 7: 2003 Emmrich, S. and B.M.Putzer: Cell Cycle: 9: 13: 2555-67: 2010 Rachagani, S., S. Kumar, and S.K. Batra: Cancer Lett: 292: 1: 8-16: 2010 Ryu, B., et al .: Cancer Res: 62: 3: 819-26: 2002 Crnogorac-Jurcevic, T., et al .: Oncogene: 21: 29: 4587-94 :: 2002 Dennis, J.L., et al .: Cancer Res: 62: 21: 5999-6005: 2002 Gr, tzmann, R., et al: Virchows Arch: 443: 4: 508-17: 2003 Furukawa, T., et al .: Am J Pathol: 148: 6: 1763-70: 1996 Liu, N., et al .: Am J Pathol: 153: 1: 263-9 .: 1998 Wang, H., et al .: Cancer Res: 69: 3: 1063-70: 2009 Matsuo, Y., et al .: Mol Cancer Res: 7: 6: 799-808: 2009 Rosewicz, S. and B. Wiedenmann ,: Lancet: 349: 9050: 485-9: 1997 Doetschman, T .: Gastroenterology: 140: 2: 380-385 e2.: 2011 Morris, J.P.t., S.C. Wang, and M. Hebrok ,: Nat Rev Cancer: 10: 10: 683-95: 2010 Rooman, I. and F.X. Real: Gut (Epub): 2011 Eser, S., et al .: Proc Natl Acad Sci U S A: 108: 24: 9945-50: 2011 LaConti, J.J., et al .: PLoS One: 6: 6: e20687: 2011 Petit, C.A., M. Gardes, and J. Feunteun, Virology, 1983. 127 (1): p. 74-82 Rassoulzadegan, M., et al .: Proc Natl Acad Sci U S A: 80: 14: 4354-8: 1983 Jat, P.S. and P.A. Sharp ,: Mol Cell Biol: 9: 4 Page: 1672-81: 1989 Cairns, L.A., et al .: EMBO J: 13: 19: 4577-86: 1994 Oostendorp, R.A., et al .: J Cell Sci: 115: Pt 10: 2099-108: 2002 Salingcarnboriboon, R., et al.:Exp Cell Res: 287: 2: 289-300: 2003 Paradis, K., et al .: Gastroenterology: 109: 4: 1308-15: 1995 Stewart, L.A., I.R. van Driel, and P.A. Gleeson ,: Eur J Cell Biol: 81: 5: 281-93: 2002 Terasaki, T., et al .: Drug Discov Today: 8: 20: 944-54: 2003 Langley, R.R., et al .: Cancer Res: 63: 11: 2971-6: 2003 Anitha, M., et al .: Gastroenterology: 134: 5: 1424-35: 2008 Yamaguchi, T., et al .: FEBS J: 275: 9: 1988-98: 2008 Herzig, M., M. Novatchkova, and G. Christofori ,: Biol Chem: 380: 2: 203-11: 1999 Casanovas, O., et al .: Oncogene: 24: 44: 6597-604: 2005 Shen, Q., et al.,: J Biosci Bioeng: 104: 1: 14-21: 2007 Ornitz, D.M., et al .: Science: 238: 4824: 188-93: 1987 Ceci, J.D., et al .: Oncogene: 6: 2: 323-32: 1991 Jackson, E.L., et al., Genes Dev, 2001. 15 (24): p. 3243-8. Hingorani, S.R., et al., Cancer Cell, 2003. 4 (6): p. 437-50. Guerra, C., et al .: Cancer Cell: 4: 2: 111-20: 2003 Aguirre, A.J., et al .: Genes Dev: 17: 24: 3112-26: 2003 Bardeesy, N., et al .: Proc Natl Acad Sci U S A: 103: 15: 5947-52: 2006 Hingorani, S.R., et al .: Cancer Cell: 7: 5: 469-83: 2005 Bardeesy, N., et al .: Genes & Development: 20: 22: 3130-3146: 2006 Izeradjene, K., et al .: Cancer Cell: 11: 3: 229-43: 2007 Kojima, K., et al .: Cancer Res: 67: 17: 8121-30: 2007 Hill, R., et al .: Cancer Res: 70: 18: 7114-24: 2010 Carriere, C., et al .: Gastroenterology (Epub): 2011 Ijichi, H., et al .: Genes Dev: 20: 22: 3147-60: 2006 Shields, D.J., et al .: Oncogene: 30: 18: 2123-34: 2011

Conventionally, multiple human pancreatic cancer cell lines have been established, and as a cell line derived from pancreatic ductal adenocarcinoma (PDAC), there were PDAC-derived cell lines and PDAC model mice generated in the PanIN-PDAC linear pathway, Not only was there no suitable control, but even when the cell line was transplanted into nude mice or the like, carcinogenesis and cancer cell growth rate were extremely slow. Therefore, it was insufficient as a model mouse and model cell line used for anticancer drug sensitivity screening for pancreatic cancer.
In the present invention, a pancreas cancer model mouse having rapid carcinogenicity and cancer cell proliferating ability and a pancreatic duct epithelial cell mouse that is a suitable control derived from the same parents are simultaneously provided by the mating method, and the pancreas that is carcinogenic in de novo The objective is to establish a cancer cell line and a pancreatic duct epithelial cell control line simultaneously.

The present inventors produced a mouse that specifically expressed tsA58TAg in the pancreas by mating an LBL-tsA58TAg mouse and a mouse that expresses the Cre enzyme under the control of the Pdx1 promoter (Pdx1-Cre mouse). We succeeded in producing de novo type pancreatic ductal adenocarcinoma (PDAC) by simultaneously expressing Kras ^G12D and tsA58TAg in the pancreas. A "rapidly carcinogenic pancreatic cancer cell line" was established from cells collected from the mouse pancreas. In addition, a pancreatic duct epithelial cell mouse and a “pancreatic duct epithelial cell line” derived from the same parent were established. When a "rapidly carcinogenic pancreatic cancer cell line" was transplanted subcutaneously in the back of nude mice, carcinogenesis was confirmed in de novo.
The “YamaPaCa Clone 6.2” strain among the pancreatic cancer cell clones obtained in the present invention and the “DC Clone 19.12” strain among the control pancreatic duct epithelial cell clones were deposited with the Patent Organisms of the National Institute of Advanced Industrial Science and Technology. Deposited at the center on March 16, 2012 and accepted as receipt numbers FERM AP-22231 and FERM AP-22230, respectively.
Obtaining the above knowledge, the present invention has been completed.

That is, the present invention is as follows.
[1] From a group of offspring mice produced by crossing a mouse having “tsA58Tag” at a position where the immortalization switch is turned on by the action of Cre and a mouse having the Cre gene, which holds “Kras ^G12D ” A pair of model mice for analysis of human pancreatic cancer, which comprises selected transgenic mice of the following (a) and (b):
(A) a human pancreatic cancer model mouse having rapid carcinogenicity and retaining “Kras ^G12D ” and “tsA58Tag”;
(B) The control model mouse that holds only “tsA58Tag” without holding “Kras ^G12D ”.
[2] The human pancreatic cancer model mouse in (a) is LBL-tsA58TAg / +; Kras ^{LSL-G12D / +} ; Pdx-1-Cre / + mouse, and the control model mouse in (b) is LBL-tsA58TAg / +; The pair of human pancreatic cancer analysis model mice according to [1], which is a Pdx-1-Cre / + mouse.
[3] The following cells (c) and (d) established from the pancreas of the human pancreatic cancer model mouse (a) of [1] or [2] and the control model mouse of (b): A cultured cell line pair for in vitro analysis of human pancreatic cancer, characterized by comprising a strain;
(C) a human pancreatic cancer model cell line having rapid carcinogenicity and retaining “Kras ^G12D ” and “tsA58Tag”;
(D) A pancreatic duct epithelial cell line that retains only “tsA58Tag” without retaining “Kras ^G12D ”.
[4] The human pancreatic cancer strain of (c) is LBL-tsA58TAg / +; Kras ^{LSL-G12D / +} ; Pdx-1-Cre / + mouse-derived FERM AP-22231 strain, and (d) pancreatic duct epithelium The cell line is composed of the human pancreatic cancer model cell line and pancreatic duct epithelial cell line described in [3] above, which is a FERM AP-22230 strain derived from LBL-tsA58TAg / +; Pdx-1-Cre / + mouse A pair of cultured cell lines for in vitro analysis of human pancreatic cancer.
[5] FERM AP-22231 strain which is a human pancreatic cancer model cell line having rapid carcinogenicity.
[6] FERM AP-22230 strain, which is a pancreatic duct epithelial cell line that serves as a control for a human pancreatic cancer model cell line having rapid carcinogenicity.
[7] The human pancreatic cancer model cell line having rapid carcinogenicity of (c) and the pancreatic duct epithelial cell line of (d) described in [3] or [5], respectively, A pair of model non-human animals for in vivo analysis of human pancreatic cancer, comprising the model non-human animals of (e) and (f);
(E) a human pancreatic cancer model non-human animal having rapid carcinogenicity and retaining “Kras ^G12D ” and “tsA58Tag”;
(F) ^Pancreatic duct epithelial cell model non-human animal that does not retain “Kras ^G12D ” but retains only “tsA58Tag”.
[8] The human pancreatic cancer analysis model mouse pair according to [1] or [2], or the human pancreatic cancer in vivo analysis model non-human animal pair according to claim 7, A method for analyzing or evaluating in vivo the specific action of a test substance on pancreatic cancer.
[9] The human pancreatic cancer analysis model mouse pair according to [1] or [2], or the human pancreatic cancer in vivo analysis model non-human animal pair according to claim 7, A method for screening an anticancer agent specific for human pancreatic cancer.
[10] For the model mouse pairs (a) and (b) below, which were born from the same parents consisting of a mouse having “Kras ^G12D ” and “tsA58Tag” and a mouse having the Cre gene, respectively. A method for analyzing or evaluating a specific action of a test substance on pancreatic cancer, comprising a step of administering an amount of the test substance;
(A) a human pancreatic cancer model mouse having rapid carcinogenicity and retaining “Kras ^G12D ” and “tsA58Tag”;
(B) The control model mouse that holds only “tsA58Tag” without holding “Kras ^G12D ”.
[11] The human pancreatic cancer model mouse in (a) is LBL-tsA58TAg / +; Kras ^{LSL-G12D / +} ; Pdx-1-Cre / + mouse, and the control model mouse in (b) is LBL-tsA58TAg / +; The method for analysis or evaluation according to [10] above, which is a Pdx-1-Cre / + mouse.
[12] A screening method for a human pancreatic cancer-specific anticancer agent, which comprises using the cultured cell line pair for human pancreatic cancer in vitro analysis according to [3] or [4].
[13] A method for producing a pancreatic cancer model non-human animal, comprising planting FERM AP-22231 strain, which is a human pancreatic cancer model cell line having rapid carcinogenicity, into a non-human animal.

In the present invention, the resulting pancreas-specific expression of Kras ^G12D and tsA58TAg simultaneously, a transgenic mouse in which a de novo type pancreatic ductal adenocarcinoma (PDAC) is produced is a suitable human pancreatic cancer model mouse alone, In the present invention, since the mating method is used, transgenic mice expressing only tsA58Tag and having normal pancreatic ductal epithelial cells can be obtained simultaneously from the same parents, the latter being an excellent pancreatic ductal adenocarcinoma (PDAC) control.
In addition, pancreatic ductal adenocarcinoma (PDAC) strains and pancreatic duct epithelial cell lines that develop in de novo from both mice are ideal samples for in vitro analysis of pancreatic ductal adenocarcinoma (PDAC) when used in pairs. It becomes. In particular, it can be used for anticancer drug sensitivity screening for pancreatic cancer.

Establishment of a cell line from a genetically modified mouse First, a mouse (A) having the LBL-tsA58TAg locus and the genetic locus X to be studied simultaneously (A) and a mouse having a tissue-specific promoter-Cre locus (B) are crossed. To do. A mouse having two LBL-tsA58TAg loci and a tissue-specific promoter-Cre locus (C) contained in the mouse offspring born by the mating, and the LBL-tsA58TAg locus and the tissue-specific promoter- In the mouse (D) having three modified loci X in addition to the Cre locus, tsA58TAg is expressed in the tissues in which Cre / loxP gene recombination was caused, respectively. Next, the tissue (which is to be cultured) is collected. Thereafter, tissue dispersibility treatment is performed, followed by culture at 33 ° C. in an appropriate medium, thereby obtaining immortalized cells that can be cultured. Breeding of pancreatic cancer model mice by mating LBL-tsA58TAg / +; Kras ^{LSL-G12D / +} mice (A) and Pdx1-Cre / + mice (B) are crossed, and Cre / loxP gene recombination occurs in the pancreas, ^Mice that express Kras ^{G12D in the} pancreas (H), mice that express tsA58TAg (I), mice that express both Kras ^G12D and tsA58TAg (J), and other mice (CG) are born. Of these, pancreatic cancer develops rapidly in mice (J). In mouse (I), no significant change in histopathology is observed before 3 weeks of age. Using the ability of tsA58TAg expressed in these two types of mice (I, J), cells are immortalized and in vitro biological comparative analysis is performed. Anatomical photo of mice by genotype (16 days of age) Genital type mice (16 days of age) detachment photos (A to D), laparotomy photos (E to H), stomach, duodenum, pancreas and spleen were collected and formalin collected Photos after fixing (I to L). In mice (D, H, L) in which Kras ^G12D and tsA58TAg are expressed in the pancreas, retention of bloody ascites is confirmed (D). Furthermore, the pancreas of the same mouse is significantly larger than the pancreas of other mice, and hard and denatured PDAC is confirmed (H, L). At the same time, mice expressing Kras ^G12D (B, F, J) or tsA58TAg (C, G, K) alone in the pancreas are control mice (A, E, I, for example Pdx1-Cre / There is no significant change in appearance compared to (the mouse is displayed). Survival curves of mice by various genotypes (A) Survival curves of four main groups created by the rapid pancreatic cancer system of the present invention. Mice that express both Kras ^G12D and tsA58TAg in the pancreas (LBL-tsA58TAg / +; Kras ^{LSL-G12D / +} ; Pdx1-Cre / +) generally die within 3 weeks due to the rapid development of pancreatic cancer. Mice that express tsA58TAg alone in the pancreas (LBL-tsA58TAg / +; Pdx1-Cre / +) generally die within 200 days. Mice (Kras ^{LSL-G12D / +} ; Pdx1-Cre / +) in which Kras ^G12D is expressed alone in the pancreas rarely die during the 200-day rearing period, as in the Control group. Compared to these previous reports shown in (FIG. 4B) and (FIG. 4C), mice expressing both Kras ^G12D and tsA58TAg in this study (A LBL-tsA58TAg / +; Kras ^{LSL-G12D / +} ; In Pdx1-Cre / +), the speed of progression of pancreatic cancer and the shortening of survival time are remarkable. Survival curves (Hingorani, 2005, Cancer Cell) between mice expressing Kras ^G12D and / or Trp53 ^R172H (Trp53 mutant), or both of which are expressed in the pancreas and another mouse reported by another group. Survival curves (Siveke, 2007, Cancer Cell) between mice expressing either Kras ^G12D and / or TGFα, or both, in the pancreas, and comparative mice. Pancreatic cancer cell immortalized culture system The figure shows a control from a mouse that expresses tsA58TAg alone in the pancreas (left) and a rapid pancreatic cancer mouse that expresses both tsA58TAg and Kras ^{G12D in} the pancreas (right). The procedure for obtaining immortalized pancreatic ductal epithelial cells and immortalized pancreatic cancer cells is shown. First, pancreas is collected from each of these two types of mice. Thereafter, the cells are dispersed and then cultured at 33 ° C. Immediately after the start of culturing, a certain number of tsA58TAg-negative cells are contained, but almost all of them become tsA58TAg-positive by continuing the passage operation for 3 to 4 weeks in the passage operation. Thereafter, the cloning operation is started by the limiting dilution method using a 96-well culture plate. Colonies can be confirmed in about 1 to 2 weeks. Each cloned population is screened with a pancreatic ductal epithelial marker gene (such as CK19) and cells positive for the marker are used for subsequent in vitro analyses. 3D culture in collagen of an immortalized culture system for pancreatic cancer cells. Immortalized pancreatic ductal epithelial cell lines derived from LBL-tsA58TAg / +; Pdx1-Cre / + mice (e.g. DC Clone 19 (a)) and LBL-tsA58TAg / +; Kras ^{LSL-G12D / +} ; Pdx1-Cre / + When a PDAC cell line derived from a mouse (eg, YamaPaCa Clone 6 (b)) is three-dimensionally cultured in a collagen gel, a lumen-like structure close to that in a living body is formed. Expression of various genes in cloned cells (A) Immunostaining of pancreatic duct epithelial markers Ck8 and Ck19 in cloned mouse-derived control immortalized pancreatic duct epithelial cells and immortalized pancreatic cancer cells. (B) Normal pancreatic duct epithelial cell clone: DC Clone 1, 15, 19, pancreatic cancer cell clone: YamaPaCa Clone 6, 8, 25 The collected gene was analyzed by real-time PCR, and the expression levels of various genes were analyzed by real-time PCR. Tacstd2 and SerpinB2, which are known to be strongly expressed in pancreatic cancer, were confirmed to have a statistically significant increase in expression on the pancreatic cancer clone side (P <0.05). Similarly, Itgb6, known as a pancreatic cancer marker, tended to increase on the pancreatic cancer side, although there was some variation. In addition to these pancreatic cancer markers, the expression of IL-24, which is known to be induced by oncogenic Kras, was examined. It was confirmed that the activation of Kras signal caused by the expression of Kras ^G12D was also caused as ^{expected in} the pancreatic cancer clones. In addition, a clone with high Dhh expression (YamaPaCa Clone 6) that induces a hedgehog signal known to cause fibrosis in pancreatic cancer was also confirmed. Pancreatic cancer cell clone subcutaneous transplantation experiment to nude mice-1 In order to investigate the transplantability of pancreatic cancer clones to individual mice, transplantation of pancreatic cancer clones and control normal pancreatic duct epithelial clones into nude mice subcutaneously Went. YamaPaCa Clone 6, 8, and 25 were selected as pancreatic cancer clones to be transplanted. DC Clone 15 and 19 were selected as control side pancreatic duct epithelial clones. (A) 1 × 10 ⁶ cells were transplanted subcutaneously, and the volume of the tumor was measured every 7 days (the volume as a rectangular parallelepiped by measuring the length, width, and height of the tumor). In the pancreatic cancer clone group, subcutaneous transplantation was confirmed in all 3 transplanted clones (YamaPaCa Clone 6, 8, 25), but no subcutaneous colonization was observed in the control pancreatic duct epithelial clone (DC Clone 19). (Only DC Clone 19 is shown in the graph, but DC Clone 1 and 15 are not established subcutaneously as DC Clone 19 is.) Therefore, pancreatic cancer cells established by this system are acceptable. It was confirmed that it was a transplanted strain. Subcutaneous transplantation experiment of pancreatic cancer cell clones to nude mice-2 (B) A state of colonization of a subcutaneous tumor at 35 days after transplantation. It can be confirmed that the skin is raised by the transplanted pancreatic cancer cells (b to d). Control DC Clone 19 does not settle (a).

1. Human pancreatic cancer model mouse in the present invention
(1-1) Presence of modified loci specific to human pancreatic cancer
Many human pancreatic cancers are known to have mutations in the glycine, the 12th amino acid of the Kras gene. Since this site is responsible for the enzyme activity that converts GTP with the ability to activate Kras itself by binding to Kras to GDP that makes Kras inactive, Kras with a mutation in the same amino acid is GTP Is suppressed from changing to GDP. In other words, this Kras mutant functions as a constantly activated form that is always bound to GTP. As a result, activation of the Ras signal cascade is considered to be the basis for the development of pancreatic cancer. In the present invention, simply “pancreatic cancer” refers to “pancreatic ductal adenocarcinoma (PDAC)” that accounts for 90% or more of pancreatic cancer. It may be accurately described as “pancreatic ductal adenocarcinoma (PDAC)”, such as when it is necessary to distinguish it from “pancreatic acinar cell carcinoma (ACC)” that occurs in acinar cells that constitute 85% of normal pancreatic tissue is there.
So far, Kras (Kras ^G12D ), a glycine (G), the 12th amino acid of Kras, has been mutated to aspartic acid (D), one of the amino acid mutations found in human pancreatic cancer. In mice that have been genetically modified to be expressed in the pancreas, pancreatic cancer-like degeneration occurs, and additional mutations such as Trp53, a tumor suppressor gene, promote the development of pancreatic cancer It has been made clear.

(1-2) Mating method using Cre expression system to genetically modified mice and establishment of cell lines from the mice
In the present invention, in establishing a human pancreatic cancer model mouse and a human pancreatic cancer cell line, a “mating method using a Cre expression system” and a method for establishing a cell line from the mouse were used. Hereinafter, the method will be briefly described with reference to FIG. The following mice (A) and (B) are prepared.
(A) LBL-tsA58TAg mouse (FIG. 1A): A mouse having the LBL-tsA58TAg locus, and another name for the mouse is CAG-Betageo-TsA58T Tg. The 26th strain is sometimes referred to as the T26 mouse. The registered name on the strain's Mouse Genome Informatics database is “Tg (CAG-Bgeo, -tsA58T) T26Ichi”. Expression of SV40 tsA58 large T antigen (TAg) can be induced by Cre / loxP gene recombination.
(B) Mice possessing a site-specific Cre expression locus (FIG. 1B): A mouse capable of causing Cre / loxP gene recombination by a specific promoter sequence.
(C) Mice possessing a modified locus X (FIG. 1A): As an example, a modified locus that produces the phenotype being studied is denoted as X (disease model mouse). However, the number of loci for inducing phenotype is not necessarily one, and it may be composed of a plurality of loci. If phenotype activation is independent of Cre / loxP gene recombination, it may be present in the same mouse together with the site-specific Cre expression locus, but Cre / loxP gene recombination activates the disease phenotype. In the preparatory stage prior to the final mating, the Cre expression loci are not held in the same individual.
In the present invention, the target disease is pancreatic cancer, and the modified locus X corresponds to “Kras ^G12D ” in which a mutation is introduced into the 12th glycine of the Kras gene, and is possessed by the mouse (a). Yes.

Such a mouse is prepared in advance, and both mice are mated (crossing of FIGS. 1A and B). Among the mice born by the crossing of these mice, mice possessing only two LBL-tsA58TAg locus and tissue-specific Cre expression locus (FIG. 1C), LBL-tsA58TAg locus and tissue-specific Cre expression In addition to the gene locus, a mouse carrying three modified gene loci X is created (FIG. 1D).
In the mouse of FIG. 1C, expression of tsA58TAg is induced at the site where Cre / loxP gene recombination has occurred. In one mouse (Fig. 1D), in addition to tsA58TAg, phenotypes expressed by modified gene locus X are also expressed (various types of modifications such as when specific proteins are expressed or when specific genes are deleted) Applicable to genetic loci).

From both mice (FIG. 1C) and (FIG. 1D), a tissue in which Cre / loxP gene recombination has occurred and contains the desired cells to be cultured is collected. Thereafter, cell dispersion treatment is performed on these tissues, and then culture is started at 33 ° C., which is the temperature at which tsA58TAg is activated, using an appropriate medium and culture dish. Cells that express tsA58TAg are immortalized and thus stably divide. In this case, non-tsA58TAg that has not undergone Cre / loxP gene recombination by performing cell culture and subculture for a certain period (a few days to a month, the period depends on the cell type / modified gene type). Expressed cells are eliminated. The immortalized cell group obtained here includes cells possessing a phenotype based on the modified gene locus X typified by a disease lesion state and the like and healthy cells that are the basis thereof.
When a part of the marker gene specific to the target cell is known, the target cell can be separated with a cell sorter using the molecule of interest as an index, or purified by a technique such as cloning.
In addition, when a disease locus contributes to carcinogenesis, the carcinogenic sensitivity promoting effect of tsA58TAg can significantly reduce the period until carcinogenesis and increase the malignancy of cancer.

(1-3) Application to human pancreatic cancer model mice (FIG. 2)
In the present invention, the “mating method using the Cre expression system” described in (1-2) above is applied to provide a pancreatic duct epithelial cell mouse that is an excellent control simultaneously with a human pancreatic cancer model mouse. .
Therefore, in the present invention, the modified locus (X) that contributes to the oncogenesis of pancreatic cancer retains <sup>`</sup> ` Kras ^G12D '' in which a mutation is introduced into the 12th glycine of the Kras gene and is immortalized by the action of Cre. “LBL-tsA58TAg / +; Kras ^{LSL-G12D / +} mouse (FIG. 2A)” having “tsA58Tag” at the position where the switch enters and “Pdx1-Cre / + mouse (FIG. 2B)” carrying the Cre gene. Mating.
Among the offspring mice, both Kras ^G12D and tsA58TAg are expressed, rapidly developing pancreatic cancer (J) and only tsA58Tag are expressed, and no significant changes in histopathology are observed (I ). Since both mice were obtained from the same parents and the other gene groups were all identical, the mouse (J) was superior in that the mouse (I) was used as a set as a control. Mouse ".

(1-4) Establishment of “human pancreatic cancer model cell line”
Subsequently, the pancreas was collected from these two types of mice (I, J), cultured, and immortalized using the ability of tsA58TAg expressed in both cells. In addition, a “pancreatic duct epithelial cell line” serving as a control is established (FIG. 5). Both are excellent biological comparison analysis tools in vitro, and are suitable for, for example, screening for pancreatic cancer-specific anticancer agents.
Here, “human pancreatic cancer model mouse” and “human pancreatic cancer cell line” hold “tsA58Tag” together with “Kras ^G12D ”, but “pancreatic duct epithelial cell mouse” and “pancreatic duct epithelial cell line” <sup>``</sup> , Because it does not retain <sup>`</sup> ` Kras <sup>G12D</sup> '' that contributes to carcinogenesis, it has only `` tsA58Tag '' related to immortalization, so it retains the properties of normal `` pancreatic duct epithelial cells '', so control Mice and culture strains can be created.

(1-5) Deposit of established cell lines
The “YamaPaCa Clone 6.2” strain among the pancreatic cancer cell clones obtained in the present invention and the “DC Clone 19.12” strain among the pancreatic duct epithelial cell clones of the control were transferred to the Patent Organism Depositary of the National Institute of Advanced Industrial Science and Technology. Deposited on March 16, 2012 and accepted as receipt numbers FERM AP-22231 and FERM AP-22230, respectively.

2. An in vivo system combining the “rapidly oncogenic pancreatic cancer model mouse” of the present invention and the “pancreatic duct epithelial cell mouse” for control
A typical "rapidly oncogenic pancreatic cancer model mouse" carrying " ^tsA58Tag " together with "Kras ^G12D " of the present invention is LBL-tsA58TAg / +; Kras ^{LSL-G12D / +} ; Pdx-1- Cre / + mouse.
A typical example of a "pancreatic duct epithelial cell mouse" that does not retain "Kras ^G12D " but retains only "tsA58Tag" is LBL-tsA58TAg / +; Pdx-1-Cre / + mouse .
“Rapid oncogenic pancreatic cancer model mouse” alone can be used for in vivo anticancer drug screening, but “pancreatic duct epithelial cell mouse” can be used as a control for screening for anticancer drug specific to pancreatic cancer. Since pancreatic cancer-specific action when a test substance is administered can be accurately analyzed, it is extremely effective as an in vivo anticancer drug screening system, particularly as a screening system for substances that are candidates for pancreatic cancer-specific anticancer drugs.

3. System for in vitro analysis of pancreatic cancer combining “rapidly carcinogenic pancreatic cancer cell line” of the present invention and “pancreatic duct epithelial cell line” for control
The established "rapid carcinogenic pancreatic cancer model cell line" derived from the "rapid carcinogenic pancreatic cancer model mouse" and the "pancreatic duct epithelial cell mouse" for control is a "pancreatic duct" that can be used alone or as a control. By using “epithelial cell lines” as a pair, pancreatic cancer cell-specific intracellular actions, changes, and other kinetic analysis can be performed in vitro. In particular, both cell line pairs can be combined with pancreatic ductal adenocarcinoma (PDAC) It is effective as a biological comparative analysis tool with ductal cells (DC). A screening system using both cell line pairs is suitable for screening for anticancer agents specific to pancreatic cancer.
A typical cell line of the “rapidly oncogenic pancreatic cancer cell line” of the present invention is the FERM AP-22231 line, and a typical cell line of the “pancreatic duct epithelial cell line” for control is FERM AP-22230. Is a stock.

4). Production of rapidly carcinogenic pancreatic cancer model non-human animals
By transplanting the "rapidly oncogenic pancreatic cancer cell line" and the "pancreatic ductal epithelial cell line" for control into the back, abdominal cavity or pancreas of the non-human animal, respectively, the rapidly oncogenic pancreatic cancer model Human animals and model non-human animals for their control can be created.
These model non-human animal pairs can also be used for human pancreatic cancer cell-specific analysis as model pairs for human pancreatic cancer analysis, and are suitable for screening pancreatic cancer-specific anticancer agents that are clinically desired. is there.
Here, as the non-human animal, any animal other than a human can be used, but primates such as mice, rats, rabbits, pigs, dogs, cows, horses and monkeys, which are typical experimental animals, are included. preferable.

EXAMPLES Next, although an Example demonstrates this invention further in detail, this invention is not limited at all by these examples.
Other terms and concepts in the present invention are based on the meanings of terms that are conventionally used in the field, and various techniques used to implement the present invention include those that clearly indicate the source. Except for this, it can be easily and reliably carried out by those skilled in the art based on known documents and the like. In addition, various analyzes were performed by applying the methods described in the analytical instruments or reagents used, kit instruction manuals, catalogs, and the like.
In addition, the description content in the technical literature, the patent gazette, and the patent application specification cited in this specification shall be referred to as the description content of the present invention.

(Gene modified mouse used in this example)
LBL-tsA58TAg / + mice (such as FIG. 2A), or LSL-tsA58TAg / + mice (such as FIG. 5) which are aryl forms thereof.
^{-Kras LSL-G12D / +} mouse: The registered name of the corresponding locus on the Mouse Genome Informatics database is Kras ^tm4Tyj . A mouse that expresses Kras ^G12D , a constantly active Kras in which the 12th amino acid G is changed to D by Cre / loxP gene recombination.
Pdx1-Cre / + mice: mice that can cause Cre / loxP gene recombination throughout the pancreas.

Example 1 Preparation of Kras ^G12D- dependent pancreatic cancer model mouse
First, a Kras ^{LSL-G12D / +} mouse, which is a genetically modified mouse in which the expression of Kras ^G12D is induced by Cre / loxP gene recombination, is prepared. The mouse has a mutation in which one of the endogenous pair of Kras alleles becomes a translated G12D amino acid mutation (Kras ^G12D ), and a special LSL sequence (LSL: loxP sequence) -Transcription stop sequence-loxP sequence) is inserted, so expression of Kras ^G12D usually does not occur. Next, this Kras ^{LSL-G12D / +} mouse is ^mated with an LBL-tsA58TAg / + mouse (a mouse in which tsA58TAg is expressed by Cre / loxP gene recombination).
Then, a mouse of LBL-tsA58TAg / +; Kras ^{LSL-G12D / +} is born (FIG. 2A). When this mouse is mated with a Pdx1-Cre / + mouse (FIG. 2B) capable of causing Cre / loxP gene recombination in the pancreas, a total of eight genotype mice are produced (FIGS. 2C-J).
Among the mice, the mice of (FIGS. 2C to G) do not express a specific ^phenotype (used as a control) because ^neither tsA58TAg nor Kras ^G12D is expressed.
On the other hand, Kras ^{LSL-G12D / +} ; Pdx1-Cre / + mice (FIG. 2H) express Kras ^G12D in the pancreas, and LBL-tsA58TAg / +; Pdx1-Cre / + mice (FIG. 2I) In tsA58TAg is expressed. Furthermore, both Kras ^G12D and tsA58TAg are expressed in LBL-tsA58TAg / +; Kras ^{LSL-G12D / +} ; Pdx1-Cre / + mice (FIG. 2J). Among these, LBL-tsA58TAg / +; Kras ^{LSL-G12D / +} ; Pdx1-Cre / + mice (Fig. 2J) showed ^{rapid growth due to} the constant activity of Kras ^G12D and the tumor-promoting effect (tumor suppressor inhibitory effect) of tsA58TAg. Pancreatic cancer (de novo type) occurs. The appearance of the rapid pancreatic cancer is shown in FIG.

FIG. 3 is an anatomical photograph of various types of genotype mice (16 days old), of which (A) to (D) are detachment photographs, (E) to (H) are laparotomy photographs, and (I) to (L). ) Is a photograph after removing the stomach, duodenum, pancreas and spleen and fixing them in formalin. In LBL-tsA58TAg / +; Kras ^{LSL-G12D / +} ; Pdx1-Cre / + (Fig. 3D, H, L), rapid pancreatic cancer develops and bloody ascites (Fig. 3D) and PDAC significantly increase The pancreas (Fig. 3H, L) is confirmed. Other genotype mice show little difference in appearance at 16 days of age.

(Example 2) Comparison of survival time of rapid pancreatic cancer model mouse of the present invention and conventional pancreatic cancer model mouse
FIG. 4A shows the survival curves of the rapid pancreatic cancer model mouse obtained in Example 1 and mice obtained by various genotypes obtained simultaneously.
Rapid pancreatic cancer model mice, LBL-tsA58TAg / +; Kras ^{LSL-G12D / +} ; Pdx1-Cre / + mice were found to generally die before weaning (within 3 weeks of age).
4B and 4C are survival curves of pancreatic cancer model mice shown in previous papers. Compared with these, the shortening of the survival time of LBL-tsA58TAg / +; Kras ^{LSL-G12D / +} ; Pdx1-Cre / + mice in the present invention is remarkable.

(Example 3) Immortal culture of pancreatic cancer cells and comparative cells
Of the group of mice obtained in Example 1, LBL-tsA58TAg / +, a rapid pancreatic cancer model; Kras ^{LSL-G12D / +} ; Pdx1-Cre / + mice (FIG. 5 right) and LBL-tsA58TAg for comparison and control / +; In Pdx1-Cre / + mice (FIG. 5 left), since tsA58TAg is expressed in the pancreas, immortalized culture is possible.
After collecting the pancreas from the mouse, the cells were dispersed by combining physical treatment such as cutting with scissors and scalpel and grinding with a slide glass, and enzymatic treatment such as collagenase, and then coated with type I collagen Start culture in the dish. At this time, a special medium (DMEM high glucose version, manufactured by Wako Pure Chemical Industries, 10% FCS, 100 unit / ml penicillin G • 100 μg / ml streptomycin (sulfate) Life Technologies, manufactured by 1 × MITO Becton Dickinson) is used. The culture temperature is set to 33 ° C. (CO ₂ is 5%), which is a temperature suitable for activation of SV40 tsA58 T antigen. When the culture is performed under such conditions, the cells fixed on the bottom surface of the culture dish are in an infinitely proliferating state (immortalized state) because the T antigen is expressed.
Thereafter, as with normal cell culture work, peeling from the bottom of the dish, passage, and cryopreservation are performed by enzyme treatment such as trypsin. Cells that have not undergone Cre / loxP gene recombination are negative for T antigen and are therefore excluded during the culture process. Perform cell cloning, etc. if necessary.

Among the LBL-tsA58TAg / +; Pdx1-Cre / + mouse-derived cell clone group and the LBL-tsA58TAg / +; Kras ^{LSL-G12D / +} ; Pdx1-Cre / + mouse-derived cell clone group thus established Also includes pancreatic-derived cells that are not pancreatic ductal epithelial. Therefore, it is possible to preselect with pancreatic duct epithelial cell markers such as cytokeratins 8 and 19 which are indicators of pancreatic duct epitheliality (FIG. 6A).
“YamaPaCa Clone 6” strain (FERM AP-22231) of the pancreatic cancer cell clone (LBL-tsA58TAg / +; Kras ^{LSL-G12D / +} ; derived from Pdx1-Cre / + mouse) selected in this way and a control A micrograph of “DC Clone 19” strain (FERM AP-22230) of a pancreatic duct epithelial cell clone (LBL-tsA58TAg / +; derived from Pdx1-Cre / + mouse) is shown in FIG. 5B. In addition, immunostaining of both pancreatic duct epithelial markers Ck8 and Ck19 was confirmed by images, and it was confirmed that there was no contamination of pancreatic cells that were not pancreatic epithelial (FIG. 6A). In the following examples, a set of these two strains was used as the system of the present invention.

(Example 4) Expression level analysis result of specific marker gene group
As an example that the system of the present invention can be used for important experiments such as identification of cancer marker genes, the results of real-time PCR analysis of the expression of known pancreatic cancer marker genes in the above normal pancreatic duct epithelial clones and pancreatic cancer clones are shown. Shown (FIG. 6B).
In this analysis, three LBL-tsA58TAg / +; Pdx1-Cre / + mouse-derived control normal pancreatic duct epithelial cell clones (DC Clone1, 15, 19) and LBL-tsA58TAg / +; Kras ^{LSL-G12D / +} ; Pdx1-Cre / + mouse-derived pancreatic cancer cell clones (YamaPaCa Clone6, 8, 25) were analyzed. The expression levels of the following three genes (Tacstd2, SerpinB2, Itgb6), known as pancreatic cancer markers, are all higher in LBL-tsA58TAg / +; Kras ^{G12D / +} ; Pdx1-Cre / + pancreatic cancer cell clones It shows a tendency and can be determined to be a clone having properties as pancreatic cancer (FIGS. 6Ba to c). IL-24 (FIG. 6Bd), whose expression is known to be induced by Kras activation, is also expressed in LBL-tsA58TAg / +; Kras ^{LSL-G12D / +} ; Pdx1-Cre / + pancreatic cancer cells. Since the clone group shows a high value, it can be confirmed that Kras is activated as planned in the same cell group.

(Example 5) Confirmation of whether transplantation experiment is possible
Next, LBL-tsA58TAg / +; Kras ^{LSL-G12D / +} ; Pdx-1-Cre / + mouse-derived pancreatic cancer cell clone group and LBL-tsA58TAg / +; Pdx-1-Cre / + mouse-derived control Using normal pancreatic duct epithelial cell clone groups, 1 × 10 ⁶ cells were transplanted subcutaneously into the back of nude mice, and the state of tumor growth was measured every week for 5 weeks (FIG. 7A). As shown in this figure, it can be seen that only pancreatic cancer cell clones are transplantable strains established in nude mice. It can also be confirmed that clones YamaPaCa Clone 6 and 25, in which the expression level of the pancreatic cancer marker gene group is larger, grow larger than YamaPaCa Clone 8 in which the expression level is smaller. On the other hand, the normal pancreatic duct epithelial cell clone for comparison does not settle (only DC Clone 19 is displayed in the graph, and DC Clone 1 and 15 do not settle as well). Thus, it can be judged that a mouse pancreatic cancer clone expressing both Kras ^G12D and tsA58TAg is a transplantable and reliable pancreatic cancer cell.

  The rapidly oncogenic pancreatic cancer cell line “YamaPaCa Clone 6.2” established in the present invention and the pancreatic duct epithelial cell line “DC Clone 19.12”, which are the controls thereof, were transferred to the National Institute of Advanced Industrial Science and Technology (AIST). On March 16, 2012, and were accepted as receipt numbers FERM AP-22231 and FERM AP-22230, respectively.

Claims (13)

It was selected from a group of offspring mice produced by mating a mouse with “ ^tsA58Tag ” that holds “Kras ^G12D ” and has an immortalization switch by the action of Cre and a mouse that has the Cre gene. , characterized by comprising the transgenic mice following (a) and (b), a mouse model pairs for human pancreatic ductal adenocarcinoma analysis;
(A) "Kras ^G12D" and "tsA58Tag" human pancreatic ductal adenocarcinoma model mice with rapid carcinogenicity holding a,
(B) The control model mouse that holds only “tsA58Tag” without holding “Kras ^G12D ”. Human pancreatic ductal adenocarcinoma mouse model of (a) is, LBL-tsA58TAg / +; Kras LSL-G12D / +; Pdx-1-Cre / + is a mouse, the control mouse model of (b), LBL-tsA58TAg / +; it is Pdx-1-Cre / + mice, human pancreatic ductal adenocarcinoma analysis model mice pair according to claim 1. Derived from the pancreas of control model mouse according to claim 1 or 2 (a) human pancreatic ductal adenocarcinoma mouse model and (b) has been established, to consist of cell lines below (c) and (d) wherein the cultured cell line pairs for human pancreatic ductal adenocarcinoma in vitro analysis;
(C) "Kras ^G12D" and "tsA58Tag" human pancreatic ductal adenocarcinoma model cell lines with quick carcinogenic holding a,
(D) A pancreatic duct epithelial cell line that retains only “tsA58Tag” without retaining “Kras ^G12D ”. Human pancreatic ductal adenocarcinoma lines (c) is, LBL-tsA58TAg / +; Kras LSL-G12D / +; a Pdx-1-Cre / + mice of FERM AP-22231 strain, pancreatic ductal epithelial cells (d) strain, LBL-tsA58TAg / +; is Pdx-1-Cre / + mice of FERM AP-22230 strain, constructed from human pancreatic ductal adenocarcinoma model cell lines and pancreatic duct epithelial cell line according to claim 3 human pancreatic ductal adenocarcinoma in vitro analysis for the cultured cell line pair. Is a human pancreatic ductal adenocarcinoma model cell lines with quick oncogenic FERM AP-22231 strain. Is a pancreatic ductal epithelial cell line as a control of human pancreatic ductal adenocarcinoma model cell lines with quick oncogenic FERM AP-22230 strain. It becomes claim 3 or 5 rapidly oncogenic human pancreatic ductal adenocarcinoma model cell lines and having the (c) according to the pancreatic duct epithelial cell line (d) from the non-human animal planting each, the following (e) and (f) model, comprising the non-human animal, a human pancreatic ductal adenocarcinoma in vivo analysis model non-human animal pairs;
(E) "Kras ^G12D" and "tsA58Tag" rapid carcinogenicity human pancreatic ductal adenocarcinoma model non-human animal having a holding the,
(F) ^Pancreatic duct epithelial cell model non-human animal that does not retain “Kras ^G12D ” but retains only “tsA58Tag”. Characterized by using the claim 1 or 2 human pancreatic ductal adenocarcinoma analysis model mice pairs described, or model for human pancreatic ductal adenocarcinoma in vivo analysis according to claim 7 of a non-human animal pair, how the specific action analyzed or evaluated in vivo against pancreatic ductal adenocarcinoma of the test substance. Characterized by using the claim 1 or 2 human pancreatic ductal adenocarcinoma analysis model mice pairs described, or model for human pancreatic ductal adenocarcinoma in vivo analysis according to claim 7 of a non-human animal pair, human pancreatic ductal adenocarcinoma-specific anticancer agents screening methods. For the model mouse pairs (a) and (b) below born from the same parents consisting of a mouse having “Kras ^G12D ” and “tsA58Tag” and a mouse carrying the Cre gene, the step of administering a test substance as an essential method for analyzing or assessing the specific effect on pancreatic ductal adenocarcinoma of the test substance;
(A) "Kras ^G12D" and "tsA58Tag" human pancreatic ductal adenocarcinoma model mice with rapid carcinogenicity holding a,
(B) The control model mouse that holds only “tsA58Tag” without holding “Kras ^G12D ”. Human pancreatic ductal adenocarcinoma mouse model of (a) is, LBL-tsA58TAg / +; Kras LSL-G12D / +; Pdx-1-Cre / + is a mouse, the control mouse model of (b), LBL-tsA58TAg / The method for analysis or evaluation according to claim 10, which is +; Pdx-1-Cre / + mouse. Claim 3 or characterized by using the 4 human pancreatic ductal adenocarcinoma in vitro analysis cell line pair, human pancreatic ductal adenocarcinoma-specific anticancer agents screening methods. Wherein the plant is a human pancreatic ductal adenocarcinoma model cell lines with rapid carcinogenicity of FERM AP-22231 strain to the non-human animal, the method producing pancreatic ductal adenocarcinoma model non-human animal.