WO2020013113A1 - Non-human mammalian cancer model - Google Patents
Non-human mammalian cancer model Download PDFInfo
- Publication number
- WO2020013113A1 WO2020013113A1 PCT/JP2019/026950 JP2019026950W WO2020013113A1 WO 2020013113 A1 WO2020013113 A1 WO 2020013113A1 JP 2019026950 W JP2019026950 W JP 2019026950W WO 2020013113 A1 WO2020013113 A1 WO 2020013113A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- human mammal
- gene
- cancer
- mob1a
- tamoxifen
- Prior art date
Links
- 206010028980 Neoplasm Diseases 0.000 title claims abstract description 57
- 201000011510 cancer Diseases 0.000 title claims abstract description 52
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 56
- 101150118030 MOB1A gene Proteins 0.000 claims abstract description 41
- 238000004519 manufacturing process Methods 0.000 claims abstract description 24
- 201000010536 head and neck cancer Diseases 0.000 claims abstract description 19
- 208000014829 head and neck neoplasm Diseases 0.000 claims abstract description 19
- NKANXQFJJICGDU-QPLCGJKRSA-N Tamoxifen Chemical compound C=1C=CC=CC=1C(/CC)=C(C=1C=CC(OCCN(C)C)=CC=1)/C1=CC=CC=C1 NKANXQFJJICGDU-QPLCGJKRSA-N 0.000 claims description 80
- 229960001603 tamoxifen Drugs 0.000 claims description 40
- 238000000034 method Methods 0.000 claims description 31
- 239000000126 substance Substances 0.000 claims description 21
- 206010062129 Tongue neoplasm Diseases 0.000 claims description 19
- 201000006134 tongue cancer Diseases 0.000 claims description 19
- 238000012360 testing method Methods 0.000 claims description 17
- 230000001419 dependent effect Effects 0.000 claims description 12
- 238000012216 screening Methods 0.000 claims description 11
- 239000002246 antineoplastic agent Substances 0.000 claims description 8
- 230000006872 improvement Effects 0.000 claims description 6
- 230000001629 suppression Effects 0.000 claims description 6
- 229940041181 antineoplastic drug Drugs 0.000 claims description 2
- 241001465754 Metazoa Species 0.000 abstract description 9
- 241000699666 Mus <mouse, genus> Species 0.000 description 25
- 210000002105 tongue Anatomy 0.000 description 25
- 101000775102 Homo sapiens Transcriptional coactivator YAP1 Proteins 0.000 description 22
- 102100031873 Transcriptional coactivator YAP1 Human genes 0.000 description 21
- 241000699670 Mus sp. Species 0.000 description 14
- 210000001519 tissue Anatomy 0.000 description 14
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 12
- 101150020580 yap1 gene Proteins 0.000 description 11
- 210000004027 cell Anatomy 0.000 description 10
- 208000009458 Carcinoma in Situ Diseases 0.000 description 8
- 206010058314 Dysplasia Diseases 0.000 description 8
- 231100000504 carcinogenesis Toxicity 0.000 description 8
- 230000005754 cellular signaling Effects 0.000 description 8
- 201000004933 in situ carcinoma Diseases 0.000 description 8
- ZBNZXTGUTAYRHI-UHFFFAOYSA-N Dasatinib Chemical compound C=1C(N2CCN(CCO)CC2)=NC(C)=NC=1NC(S1)=NC=C1C(=O)NC1=C(C)C=CC=C1Cl ZBNZXTGUTAYRHI-UHFFFAOYSA-N 0.000 description 7
- 239000002067 L01XE06 - Dasatinib Substances 0.000 description 7
- 238000004458 analytical method Methods 0.000 description 7
- 101150036876 cre gene Proteins 0.000 description 7
- 229960002448 dasatinib Drugs 0.000 description 7
- 239000003814 drug Substances 0.000 description 7
- 102000004169 proteins and genes Human genes 0.000 description 7
- 102100027548 WW domain-containing transcription regulator protein 1 Human genes 0.000 description 6
- 229940079593 drug Drugs 0.000 description 6
- 208000005623 Carcinogenesis Diseases 0.000 description 5
- 102100021437 MOB kinase activator 1A Human genes 0.000 description 5
- 230000036952 cancer formation Effects 0.000 description 5
- 150000001875 compounds Chemical class 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 230000006870 function Effects 0.000 description 5
- 210000003128 head Anatomy 0.000 description 5
- 230000037361 pathway Effects 0.000 description 5
- 238000010186 staining Methods 0.000 description 5
- 102100025248 C-X-C motif chemokine 10 Human genes 0.000 description 4
- 102100024997 MOB kinase activator 1B Human genes 0.000 description 4
- 101700059339 MOB1A Proteins 0.000 description 4
- 101700028414 MOB1B Proteins 0.000 description 4
- 101100222379 Rattus norvegicus Cxcl10 gene Proteins 0.000 description 4
- 238000002695 general anesthesia Methods 0.000 description 4
- 238000001727 in vivo Methods 0.000 description 4
- 238000011813 knockout mouse model Methods 0.000 description 4
- 206010041823 squamous cell carcinoma Diseases 0.000 description 4
- 230000008685 targeting Effects 0.000 description 4
- 229930186657 Lat Natural products 0.000 description 3
- 241000124008 Mammalia Species 0.000 description 3
- 229940125648 antineoplastic drug candidate Drugs 0.000 description 3
- 230000002950 deficient Effects 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 238000002513 implantation Methods 0.000 description 3
- 239000003112 inhibitor Substances 0.000 description 3
- 239000003202 long acting thyroid stimulator Substances 0.000 description 3
- 108020004999 messenger RNA Proteins 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000010172 mouse model Methods 0.000 description 3
- 230000006798 recombination Effects 0.000 description 3
- 238000005215 recombination Methods 0.000 description 3
- 238000011282 treatment Methods 0.000 description 3
- 108020004414 DNA Proteins 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- 108091000080 Phosphotransferase Proteins 0.000 description 2
- 241000700159 Rattus Species 0.000 description 2
- 108700025716 Tumor Suppressor Genes Proteins 0.000 description 2
- 102000044209 Tumor Suppressor Genes Human genes 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 230000002113 chemopreventative effect Effects 0.000 description 2
- 230000002354 daily effect Effects 0.000 description 2
- 230000002500 effect on skin Effects 0.000 description 2
- 230000001771 impaired effect Effects 0.000 description 2
- 210000000867 larynx Anatomy 0.000 description 2
- 231100000518 lethal Toxicity 0.000 description 2
- 230000001665 lethal effect Effects 0.000 description 2
- 210000000214 mouth Anatomy 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 210000003800 pharynx Anatomy 0.000 description 2
- 102000020233 phosphotransferase Human genes 0.000 description 2
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 238000007920 subcutaneous administration Methods 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- SGKRLCUYIXIAHR-AKNGSSGZSA-N (4s,4ar,5s,5ar,6r,12ar)-4-(dimethylamino)-1,5,10,11,12a-pentahydroxy-6-methyl-3,12-dioxo-4a,5,5a,6-tetrahydro-4h-tetracene-2-carboxamide Chemical compound C1=CC=C2[C@H](C)[C@@H]([C@H](O)[C@@H]3[C@](C(O)=C(C(N)=O)C(=O)[C@H]3N(C)C)(O)C3=O)C3=C(O)C2=C1O SGKRLCUYIXIAHR-AKNGSSGZSA-N 0.000 description 1
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 102000007469 Actins Human genes 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 241000283073 Equus caballus Species 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 208000001951 Fetal Death Diseases 0.000 description 1
- 206010055690 Foetal death Diseases 0.000 description 1
- 206010064571 Gene mutation Diseases 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000969581 Homo sapiens MOB kinase activator 1A Proteins 0.000 description 1
- 101100193693 Kirsten murine sarcoma virus K-RAS gene Proteins 0.000 description 1
- 101100319891 Mus musculus Yap1 gene Proteins 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 108700020796 Oncogene Proteins 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 241000009328 Perro Species 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 108010052160 Site-specific recombinase Proteins 0.000 description 1
- 241000282898 Sus scrofa Species 0.000 description 1
- 101150080074 TP53 gene Proteins 0.000 description 1
- 239000004098 Tetracycline Substances 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 102000015098 Tumor Suppressor Protein p53 Human genes 0.000 description 1
- 108010078814 Tumor Suppressor Protein p53 Proteins 0.000 description 1
- 108010040002 Tumor Suppressor Proteins Proteins 0.000 description 1
- 102000001742 Tumor Suppressor Proteins Human genes 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 238000003705 background correction Methods 0.000 description 1
- 210000002459 blastocyst Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 239000008358 core component Substances 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 108010007093 dispase Proteins 0.000 description 1
- 229960003722 doxycycline Drugs 0.000 description 1
- 238000009509 drug development Methods 0.000 description 1
- 230000005584 early death Effects 0.000 description 1
- 210000001671 embryonic stem cell Anatomy 0.000 description 1
- 210000002257 embryonic structure Anatomy 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 210000000981 epithelium Anatomy 0.000 description 1
- 229940011871 estrogen Drugs 0.000 description 1
- 239000000262 estrogen Substances 0.000 description 1
- 102000015694 estrogen receptors Human genes 0.000 description 1
- 108010038795 estrogen receptors Proteins 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 231100000479 fetal death Toxicity 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 230000005861 gene abnormality Effects 0.000 description 1
- 238000003209 gene knockout Methods 0.000 description 1
- 210000004602 germ cell Anatomy 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 230000013011 mating Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 108700025694 p53 Genes Proteins 0.000 description 1
- 229910052697 platinum Inorganic materials 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 238000010837 poor prognosis Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000007790 scraping Methods 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 238000012353 t test Methods 0.000 description 1
- 229960002180 tetracycline Drugs 0.000 description 1
- 229930101283 tetracycline Natural products 0.000 description 1
- 235000019364 tetracycline Nutrition 0.000 description 1
- 150000003522 tetracyclines Chemical class 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 230000009261 transgenic effect Effects 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
Definitions
- the present invention relates to a cancer model non-human mammal, a method for producing the animal, a method for screening an anticancer agent using the animal, and the like.
- the contents of all documents described in this specification are incorporated herein by reference.
- Head and neck cancer is a cancer with a generally poor prognosis, and is the sixth largest cancer patient in the world (600,000 affected people annually). Although treatments centered on platinum preparations have been performed in recent decades, the five-year survival rate worldwide has been around 40-50%, and improvement in treatment results has not been achieved easily. As one of the reasons, in the DNA sequence analysis of the exon region in a large-scale case of head and neck cancer published in The Cancer, Genome, Atlas (TCGA) project in 2015, the gene mutation that can be said to be universal is a tumor suppressor gene. There is only a mutation in a certain TP53 gene, no new significant cancer-promoting gene in head and neck cancers has been identified, and there is a situation where it is difficult to develop molecularly targeted therapeutics specific to head and neck cancers.
- a cancer-promoting molecule that is universally involved in the onset and progression of head and neck cancer has not been identified, and the same applies to in-vivo analysis.
- In vivo analysis of head and neck cancers has not been sufficiently advanced because the existing TP53 mutations and other signal transduction pathway abnormalities (activation of tumor promoting genes such as K-RAS and AKT) + Combination of multiple gene abnormalities such as inactivation of tumor suppressor gene), although a model for the onset of head and neck cancer has been reported, but the period leading to carcinogenesis and the observation period are several months to more than one year It may take a long time and is currently hardly used in basic research on head and neck cancer.
- the Hippo signaling system (Hippo @ Pathway) is a pathway of a tumor suppressor system having four genes of MST, SAV, MOB and LATS as a core component in mammals, and has a function of suppressing the expression of YAP1 / TAZ.
- YAP1 / TAZ is a gene (tumor promoting gene) that positively acts on tumor promotion as a transcription coupling factor (for example, see Non-Patent Document 1 and the like).
- MOB has seven homologous molecules, of which only two, MOB1A and MOB1B, can bind LATS.
- LATS alone has a weak kinase activity on YAP1 / TAZ, but acquires a very strong kinase activity upon binding of MOB1A / B and suppresses YAP1 / TAZ.
- MOB1A and MOB1B have extremely high homology and compensate for each other's functions, so that knocking out one of them does not eliminate the activity of MOB1.
- knockout of MOB1B alone showed normal development and development.
- YAP1 is enhanced to a level that is impossible in a real biological environment, or It was difficult to produce an ideal model animal, such as fetal death or early death due to cancer.
- An object of the present invention is to provide a cancer (especially head and neck cancer) model animal that can be efficiently produced in a short period of time.
- the present inventors have found that one of the Mob1a gene and the Mob1b gene may be knocked out, and the other may be able to solve the above problem by using a non-human mammal under a system that can be arbitrarily knocked out. Were found, and further improvements were made to complete the present invention.
- the invention includes, for example, the subject matter described in the following section.
- Item 1. A non-human mammal, wherein one of the Mob1a and Mob1b genes is knocked out (preferably in the whole body) and the other is under an optional knockout system.
- Item 2. Item 2. The non-human mammal according to Item 1, wherein the optionally knockout system is a Cre-LoxP system.
- Item 3. Item 3.
- Item 4. Item 4.
- a method for producing a cancer model non-human mammal comprising the step of knocking out a Mob1a gene or a Mob1b gene using the system capable of arbitrarily knocking out the non-human mammal according to any one of Items 1 to 3.
- Item 4. A method for producing a cancer model non-human mammal, comprising a step of administering tamoxifen to the non-human mammal according to Item 3.
- a method for producing a head and neck cancer model non-human mammal comprising the step of administering tamoxifen to the head and neck of a non-human mammal according to Item 3.
- a method for producing a tongue cancer model non-human mammal comprising a step of applying tamoxifen to the tongue of the non-human mammal according to claim 3.
- Item 8. (A) a step of applying a test substance to a cancer model non-human mammal produced by the method according to any of items 4 to 7, and (B) a cancer of the cancer model non-human mammal to which the test substance is applied.
- a method for screening a candidate substance for an anticancer agent comprising a step of measuring the degree of suppression or improvement of the drug.
- a ′ one (preferably in whole body) of the Mob1a gene and the Mob1b gene, one of which is knocked out, and the other is a non-human mammal under an optionally knockout system, wherein the optionally knockout is A step of applying a tamoxifen and a test substance to a non-human mammal, which is a Cre-LoxP system that acts in a tamoxifen-dependent manner, and (B ′) a step of applying the test substance to the non-human mammal.
- a method for screening a candidate anticancer drug comprising a step of measuring the degree of suppression of cancer.
- Item 10 A head and neck cancer model non-human mammal in which the Mob1a gene and the Mob1b gene have been double knocked out in the head and neck.
- ⁇ Can provide cancer (especially head and neck cancer) model animals that can be efficiently produced in a short period of time.
- the outline of the procedure for preparing a tongue cancer model mouse using the Mob1a / b conditional double knockout mouse is shown.
- 2A shows the percentage of invasive squamous cell carcinoma (invasive @ scc), carcinoma in situ (CIS), and dysplasia (Dysplasia) observed in HE staining in FIG. 2a.
- the results of analyzing each tongue epithelial cell from a tongue cancer model mouse and control mouse and analyzing the expression of each protein in the cell by Western blot are shown.
- FIG. 3b shows the result of analyzing the band density of Fig. 3a.
- 4A shows the percentage of invasive squamous cell carcinoma (invasive @ scc), carcinoma in situ (CIS), and dysplasia (Dysplasia) observed in HE staining of FIG. 4a.
- FIG. 2 shows the results of YAP1 immunohistological staining in clinical specimens of human tongue cancer.
- the present invention preferably includes, but is not limited to, a method for producing a non-human mammal, a method for producing a cancer model non-human mammal, a method for screening a candidate anti-cancer agent using a cancer model non-human mammal, and the like.
- the present invention includes all that is disclosed herein.
- one of the Mob1a gene and the Mob1b gene is knocked out, and the other is under a system that can be arbitrarily knocked out. That is, the system may be under a system where the Mob1a gene has been knocked out and the Mob1b gene can be arbitrarily knocked out, or under a system where the Mob1b gene has been knocked out and the Mob1a gene can be arbitrarily knocked out.
- the said non-human mammal may be described as "the non-human mammal of the present invention".
- the Mob1a gene and the Mob1b gene are already known in many mammals.
- the accession number (NCBI) of the mouse Mob1 mRNA is NM_145571
- the accession number (NCBI) of the mouse Mob1 mRNA is NM_026735.
- the method of knocking out a knocked out gene is not particularly limited.
- the whole gene may be deficient, or the base sequence of the gene may be one or more, and the gene may be replaced or deleted so that gene function is lost. It may be lost or inserted.
- the knockout method is not particularly limited, and a known method can be used. For example, genetically modifying embryonic stem cells, inserting them into early embryos (blastocysts), and knocking out the germline of the transgenic cells among the born organisms Since the gene can be left in the next generation, it can be crossed to obtain a knockout organism.
- a Cre-loxP system can be mentioned as a system that can be knocked out arbitrarily.
- the Cre protein is a site-specific recombinase that causes recombination between specific sequences (loxP) of the DNA molecule.
- a mouse it can be explained as follows. Using a promoter whose expression characteristics are known, a mouse that produces Cre at a specific tissue or at a specific time is prepared. If it is desired to turn off the gene only in a specific tissue, a promoter that functions only in the specific tissue is searched for, and the Cre gene is ligated behind the promoter so that Cre is produced only in the specific tissue.
- mice having loxP inserted before and after the target gene are prepared. Of the offspring obtained by crossing these two mice, those having both genes are selected. In the selected mouse, Cre is expressed only in the specific tissue, and recombination occurs between loxPs. As a result, the target gene is knocked out only in the specific tissue.
- Cre-loxP system that allow the expression of Cre protein to be induced by a specific drug (for example, tamoxifen), and these are also well known.
- a specific drug for example, tamoxifen
- the Cre-ER protein which is a fusion protein of Cre and a mutant estrogen receptor, is usually present in the cytoplasm, but is translocated into the nucleus by binding to an estrogen derivative, tamoxifen, and becomes a loxP sequence. Recombination occurs. Using this, it is possible to adjust the working time of the Cre-loxP system in a tamoxifen-dependent manner.
- Such a drug (tamoxifen) -dependent Cre-loxP system is particularly suitable as the "arbitrarily knockout system" in the non-human mammal of the present invention.
- the non-human mammal of the present invention is a systemic conditional double knockout non-human mammal. That is, in the whole body, one of the Mob1a gene and the Mob1b gene is preferably knocked out, and the other is preferably a non-human mammal under a system capable of arbitrarily knocking out.
- a tetracycline expression control system (Tet-On @ System) is also mentioned as a system that can be arbitrarily knocked out.
- This system is a system that can turn on and off the expression of a target gene by administering and removing doxycycline, and is well known in the art.
- non-human mammal of the present invention for example, a pet or a domestic animal, or a non-human mammal used as an experimental animal is preferable.
- a pet or a domestic animal or a non-human mammal used as an experimental animal is preferable.
- a non-human mammal used as an experimental animal for example, a mouse, rat, rabbit, dog, cat, sheep, pig, goat, cow, horse And monkeys.
- rodents mouse, rat, rabbit, etc.
- the present invention also encompasses a method for producing a cancer model non-human mammal, which comprises a step of knocking out the Mob1a gene or the Mob1b gene by the system that can be arbitrarily knocked out in the non-human mammal of the present invention.
- the production method may be referred to as “the cancer model non-human mammal production method of the present invention”.
- the gene knocked out in this step is the Mob1b gene
- the Mob1b gene has been knocked out
- the Mob1b gene has been knocked out
- the Mob1a gene has an arbitrary knockout.
- the gene knocked out in this step is the Mob1a gene.
- the gene knockout performed in the step uses the system that can arbitrarily knock out.
- the method for producing a cancer model non-human mammal of the present invention preferably includes a step of administering tamoxifen to the non-human mammal.
- the administration method is not particularly limited as long as the effects of the present invention are not impaired, and examples include oral administration, transvascular administration, subcutaneous administration, and dermal administration.
- known methods such as injection and application can be appropriately selected and used.
- the method for producing a cancer model non-human mammal of the present invention is particularly suitable for producing a head and neck cancer model non-human mammal.
- tamoxifen it is particularly preferable to administer tamoxifen to the head and neck.
- the head and neck include tongue, oral cavity, pharynx, and larynx.
- tongue is particularly preferable.
- a method of applying tamoxifen to the tongue is particularly preferable.
- tamoxifen When administering tamoxifen, it is preferable to subject the non-human mammal of the present invention to general anesthesia.
- general anesthesia when tamoxifen is applied by application, since the mouse does not wake up for a long time by performing general anesthesia, the high concentration tamoxifen remains at the applied site, and the application of tamoxifen to the site is efficiently performed. Can be done.
- the present invention also includes a cancer model non-human mammal produced by the method for producing a cancer model non-human mammal of the present invention.
- a cancer model non-human mammal at the specific site where the Mob1a gene and the Mob1b gene are double-knocked out at the specific site is preferable.
- the embryo is lethal (immediately after implantation), so that a non-human mammal in which the Mob1a gene and the Mob1b gene are double-knocked out at a specific site is produced.
- a special method for example, one of the Mob1a gene and the Mob1b gene is knocked out, and the other is a non-human mammal under a system that can be arbitrarily knocked out, and In other words, it is difficult to produce a gene unless a method for knocking out a gene using a system capable of knocking out is used.
- the specific site is not particularly limited as long as the effects of the present invention are not impaired.
- a head and neck for example, tongue, oral cavity, pharynx, larynx, etc.
- tongue is particularly preferable.
- the present invention provides (A) a step of applying a test substance to the cancer model non-human mammal, and (B) a step of measuring the degree of suppression or improvement of cancer in the cancer model non-human mammal to which the test substance has been applied. And a method of screening for a candidate anticancer agent. Further, the present invention provides a non-human mammal, wherein one of the (A ′) Mob1a gene and the Mob1b gene is knocked out and the other is under a system that can be arbitrarily knocked out, wherein the knockout can be arbitrarily performed.
- a step of applying tamoxifen and a test substance to a non-human mammal wherein the system is a Cre-LoxP system that works in a tamoxifen-dependent manner, and (B ′) a cancer of the non-human mammal to which the test substance has been applied.
- a method of screening for a candidate anticancer agent which comprises the step of measuring the degree of suppression of the drug.
- the test substance is not particularly limited, and may be, for example, a compound or a composition.
- the compound may be, for example, a low molecular compound, a high molecular compound such as a nucleic acid (for example, DNA or RNA), a protein (for example, an antibody or a part thereof), or a polymer.
- the composition may be an extract or the like obtained from an organism (for example, an animal, a plant, or a microorganism), or may be a combination of two or more compounds.
- the method of applying the test substance is not particularly limited, and examples include oral administration, transvascular administration, subcutaneous administration, and dermal administration.
- the test substance When a specific drug (such as tamoxifen) is administered in the production of the cancer model non-human mammal used in the step (A), the test substance may be administered by the same administration method as the administration of the tamoxifen. .
- the method of applying tamoxifen and the test substance may be the same or different, and preferably the same.
- the administration site and administration method of tamoxifen are the same as those described in the above-mentioned method for producing a cancer model non-human mammal.
- the method for measuring the degree of suppression or improvement of cancer is not particularly limited, and a known method can be used.
- a method of directly observing a cancer tissue or a method of collecting a cancer tissue and performing staining (for example, HE staining or immunological staining) and observing the cells, and determining the degree of progress of the entire cancer from the degree of cancer of each cell.
- staining for example, HE staining or immunological staining
- Examples include a method of analysis, but are not particularly limited thereto.
- the cancer model non-human mammal preferably a head and neck cancer model non-human mammal, more preferably a tongue cancer model non-human mammal
- the Mob1a gene and the Mob1b gene are double-knocked out
- canceration is caused by the action of YAP1 instead of TAZ. Therefore, the cancer model non-human mammal can be said to be a YAP1-dependent cancer model non-human mammal.
- YAP1 is already known in many mammals. For example, the accession number (NCBI) of mouse Yap1 mRNA is NM_001171147.
- the cancer model non-human mammal can be said to be particularly useful for screening anticancer drug candidate substances targeting YAP1.
- the cancer model non-human mammal is a head and neck cancer (preferably tongue cancer) model non-human mammal, it is particularly useful for screening anticancer drug candidate substances against head and neck cancer targeting YAP1. You can say that. Similarly, it can be said that cancers at other sites are useful for screening anticancer drug candidate substances targeting YAP1.
- the non-human mammal having the Mob1a gene and the Mob1b gene double-knocked out is fatal immediately after implantation.
- it can be manufactured by manufacturing according to the manufacturing method described above.
- the term “comprising” includes “consisting essentially of” and “consisting of” (The term “comprising” “includes” “consisting” essentially “of” and “” consisting “of.”).
- the present invention encompasses any combination of the constituent features described in this specification.
- Mob1b is homozygously deficient, and the Mob1a gene is homozygous and exists under the tamoxifen-dependent Cre-LoxP system (Mob1a flox / flox, Mob1b ⁇ / ⁇ , CreERT2), double knockout mice and triple knockout mice (Mob1a flox / flox, Mob1b-/-, Yap1 flox / flox, CreERT, and Mob1a flox / flox, Mob1b-/-, Taz flox / flox, CreERT) and control mice (Mob1a flox / flox, Mob1b ⁇ / ⁇ ) were prepared by crossing the following mice as appropriate.
- CreERT2 tamoxifen-dependent Cre-LoxP system
- CreERT2 double knockout mice and triple knockout mice
- FIG. 2B is a graph showing the observed ratios of invasive squamous cell carcinoma (invasive @ scc), carcinoma in situ (CIS), and dysplasia (Dysplasia).
- canceration occurs in a very short period of time, with early mice taking one week to become cancerous and all mice becoming cancerous in only two weeks. Normally, long-term observation is required for model mice, so that individual differences are likely to occur. However, cancer model mice prepared by this method are completed in a short period of time, so that variations in various analyzes are small, which is advantageous.
- the tongues of Mob1a flox / flox, Mob1b ⁇ / ⁇ , CreERT2 mice were collected, immersed in dispase II for 24 hours, and then only the tongue epithelium was separated and subjected to trypsin treatment to separate the cells to perform PrimaryPCulture.
- the cells have inherited the property that Mob1a / b can be knocked out at any time in a tamoxifen-inducible manner.
- the MOB1a / b was knocked out in a tamoxifen-inducible manner as in the mouse, and the expression of each protein was westernized. Analyzed by blot.
- the protein was specifically prepared as follows.
- the cells were collected by scraping with TNE buffer, collected by sonication for 5 minutes, and then allowed to stand on ice for 20 minutes. During that time, Voltex was performed three times. The mixture was centrifuged at 4 ° C. and 14000 RPM for 20 minutes, the supernatant was transferred to a new tube, and the protein concentration was adjusted by the BCA method. SDS was added, heated at 96 ° C. for 5 minutes, and stored at ⁇ 30 ° C.
- the antibodies used for detecting each protein are as follows. (Note that pYap1 and pTaz indicate phosphorylated Yap1 and phosphorylated Taz, respectively.) Mob1: Cell Signaling, # 4912S Lats1: Cell Signaling, # 3377S Mst1: Cell Signaling, # 3682S Actin: Cell Signaling, # 4967 Yap1: Cell Signaling, # 4912S pYap1: Cell Signaling, # 4911 Taz: Cell Signaling, # 4883S pTaz: Cell Signaling, # 75275
- TKO mice triple knockout mice in which Yap or Taz of DKO mice were knocked out were prepared, tongue cancer was induced in the same manner as described above, and tongue morphology and HE staining of tongue tissues were observed.
- the results are shown in FIG. 4a.
- the ratios of invasive squamous cell carcinoma (invasive @ scc), carcinoma in situ (CIS), and dysplasia (Dysplasia) observed by the HE staining are shown in FIG. 4B. It was found that tongue cancer induction did not occur in TKO mice in which Yap was further KO. From these results, it was found that the canceration of the DKO mouse was YAP-dependent. Therefore, the tongue cancer model mouse can be said to be a YAP-dependent carcinogenesis model.
- FIG. 5 shows the results. These results strongly suggested that YAP1 may be deeply involved in the development and progression of tongue cancer even in humans.
- Chemoprevention assay using existing YAP1 inhibitor (dasatinib)
- dasatinib YAP1 inhibitor
- dasatinib is known as a YAP1 inhibitor.
- FIG. 6 shows the results. From the results, it was found that the tongue cancer induction was not caused by the YAP1 inhibitor (dasatinib).
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Environmental Sciences (AREA)
- Pathology (AREA)
- Urology & Nephrology (AREA)
- General Physics & Mathematics (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Hematology (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Animal Behavior & Ethology (AREA)
- Zoology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Microbiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biophysics (AREA)
- Pharmacology & Pharmacy (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Provided is, inter alia, an animal cancer (particularly head and neck cancer) model that enables rapid and efficient production. More specifically, provided are, inter alia, a non-human mammal in which systemically one of the Mob1a gene and Mob1b gene is knocked out and the other resides under a system enabling optional knockout; and a method for producing a non-human mammalian cancer model comprising a step of knocking out, in the aforementioned non-human mammal, the Mob1a gene or Mob1b gene using the aforementioned system enabling optional knockout.
Description
本発明は、癌モデル非ヒト哺乳動物、当該動物の作製方法、及び当該動物を用いた抗癌剤スクリーニング方法等に関する。なお、本明細書に記載される全ての文献の内容は参照により本明細書に組み込まれる。
<< The present invention relates to a cancer model non-human mammal, a method for producing the animal, a method for screening an anticancer agent using the animal, and the like. The contents of all documents described in this specification are incorporated herein by reference.
頭頸部癌は一般的に予後不良な癌であり、世界第６位のがん患者数（年間罹患者数６０万人）である。ここ数十年プラチナ製剤を中心とした治療が行われてきたが、世界全体における５年生存率は４０－５０％程度で推移しており、治療成績の向上はなかなか図れていない。その理由の一つとして、２０１５年にＴｈｅ Ｃａｎｃｅｒ Ｇｅｎｏｍｅ Ａｔｌａｓ（ＴＣＧＡ）ｐｒｏｊｅｃｔより発表された頭頸部癌の大規模症例によるエキソン領域のＤＮＡ塩基配列解析では、普遍的と言える遺伝子変異は腫瘍抑制遺伝子であるＴＰ５３遺伝子の変異のみであり、頭頸部癌における新規の有意な癌促進遺伝子は同定されず、頭頸部癌特異的な分子標的治療薬の開発が困難な状況がある。
Head and neck cancer is a cancer with a generally poor prognosis, and is the sixth largest cancer patient in the world (600,000 affected people annually). Although treatments centered on platinum preparations have been performed in recent decades, the five-year survival rate worldwide has been around 40-50%, and improvement in treatment results has not been achieved easily. As one of the reasons, in the DNA sequence analysis of the exon region in a large-scale case of head and neck cancer published in The Cancer, Genome, Atlas (TCGA) project in 2015, the gene mutation that can be said to be universal is a tumor suppressor gene. There is only a mutation in a certain TP53 gene, no new significant cancer-promoting gene in head and neck cancers has been identified, and there is a situation where it is difficult to develop molecularly targeted therapeutics specific to head and neck cancers.
このように、頭頸部癌の発症や進展に普遍的に関わる癌促進分子は同定されておらず、それはｉｎ ｖｉｖｏ解析でも同様である。頭頸部癌でのｉｎ ｖｉｖｏ解析は未だ十分には進んでいない状況であり、その理由として、既存のものとしてＴＰ５３変異と他のシグナル伝達経路異常（Ｋ－ＲＡＳやＡＫＴなど腫瘍促進遺伝子の活性化＋腫瘍抑制遺伝子の不活化を合わせる等、複数の遺伝子異常を組み合わせたもの）による頭頸部癌発症モデルは報告されているものの、その発癌に至る期間および観察期間は数ヶ月から１年以上もの長期間を要することもあり、現在、頭頸部癌基礎研究において殆ど活用されていない。
Thus, a cancer-promoting molecule that is universally involved in the onset and progression of head and neck cancer has not been identified, and the same applies to in-vivo analysis. In vivo analysis of head and neck cancers has not been sufficiently advanced because the existing TP53 mutations and other signal transduction pathway abnormalities (activation of tumor promoting genes such as K-RAS and AKT) + Combination of multiple gene abnormalities such as inactivation of tumor suppressor gene), although a model for the onset of head and neck cancer has been reported, but the period leading to carcinogenesis and the observation period are several months to more than one year It may take a long time and is currently hardly used in basic research on head and neck cancer.
ところで、ヒッポシグナル伝達系（Ｈｉｐｐｏ Ｐａｔｈｗａｙ）は、哺乳動物においては、ＭＳＴ、ＳＡＶ、ＭＯＢ、ＬＡＴＳの４遺伝子をコアコンポーネントとする腫瘍抑制系のＰａｔｈｗａｙであり、ＹＡＰ１／ＴＡＺの発現を抑制する働きを有する。ＹＡＰ１／ＴＡＺは転写共役因子として、腫瘍促進に対し正に働く遺伝子（腫瘍促進遺伝子）である（例えば非特許文献１等を参照）。
By the way, the Hippo signaling system (Hippo @ Pathway) is a pathway of a tumor suppressor system having four genes of MST, SAV, MOB and LATS as a core component in mammals, and has a function of suppressing the expression of YAP1 / TAZ. Have. YAP1 / TAZ is a gene (tumor promoting gene) that positively acts on tumor promotion as a transcription coupling factor (for example, see Non-Patent Document 1 and the like).
ＭＯＢには７つの相同分子があるが、このうちＭＯＢ１ＡとＭＯＢ１Ｂの２つのみがＬＡＴＳと結合できる。ＬＡＴＳは単独ではＹＡＰ１／ＴＡＺに対するそのキナーゼ活性は弱いものの、ＭＯＢ１Ａ／Ｂが結合することによって非常に強いキナーゼ活性を獲得し、ＹＡＰ１／ＴＡＺを抑制する。また、ＭＯＢ１ＡとＭＯＢ１Ｂとは相同性が極めて高く、互いにその機能を代償するため、いずれか一方をノックアウトするのみではＭＯＢの活性は消失しない。なお、ＭＯＢ１Ａ／ＭＯＢ１Ｂを二重に完全欠損（ホモ欠損）するマウスを作製したところ着床直後に致死（胎生致死）となったが、ＭＯＢ１Ｂのみをノックアウトしたところ正常な発達・発育を認めた。
MOB has seven homologous molecules, of which only two, MOB1A and MOB1B, can bind LATS. LATS alone has a weak kinase activity on YAP1 / TAZ, but acquires a very strong kinase activity upon binding of MOB1A / B and suppresses YAP1 / TAZ. In addition, MOB1A and MOB1B have extremely high homology and compensate for each other's functions, so that knocking out one of them does not eliminate the activity of MOB1. In addition, when a mouse in which MOB1A / MOB1B was completely completely deficient (homodeficient) was produced, the mouse became lethal (embryo-killed) immediately after implantation. However, knockout of MOB1B alone showed normal development and development.
Ｈｉｐｐｏ Ｐａｔｈｗａｙは近年非常に注目されており、特に腫瘍促進遺伝子ＹＡＰ１をターゲットとした薬剤開発が進められているが、そのｉｎ ｖｉｖｏの薬効解析には最短でも複数ヶ月～１年程度の長い観察期間を要するため、実験期間および使用薬剤量の面から、より短期間での解析が可能なモデル動物が切望されている。
Hippo @ Pathway has attracted much attention in recent years, and drug development targeting the tumor-promoting gene YAP1 has been particularly advanced. However, in vivo analysis of the efficacy of in vivo requires a long observation period of at least several months to one year. Therefore, in view of the experimental period and the amount of drug used, a model animal that can be analyzed in a shorter period of time has been desired.
ただ、ＹＡＰ１そのものを発現亢進させる、あるいはＨｉｐｐｏ Ｐａｔｈｗａｙに関連する遺伝子の発現を抑制するなどして、モデル動物を作製しようとしても、現実の生体環境内ではありえないほどＹＡＰ１発現が亢進したり、あるいは、胎生致死若しくは癌により早期に死亡するなど、理想的なモデル動物を作製することは難しかった。
However, even if the expression of YAP1 itself is enhanced or the expression of a gene related to Hippo @ Pathway is suppressed, the expression of YAP1 is enhanced to a level that is impossible in a real biological environment, or It was difficult to produce an ideal model animal, such as fetal death or early death due to cancer.
本発明は、短期間で効率よく作製可能な癌（特に頭頸部癌）モデル動物の提供を課題とする。
An object of the present invention is to provide a cancer (especially head and neck cancer) model animal that can be efficiently produced in a short period of time.
本発明者らは、Ｍｏｂ１ａ遺伝子及びＭｏｂ１ｂ遺伝子のうち、一方はノックアウトされており、もう一方は任意にノックアウトできるシステム下にある、非ヒト哺乳動物を利用することで、上記課題を解決できる可能性を見出し、さらに改良を重ねて本発明を完成させるに至った。
The present inventors have found that one of the Mob1a gene and the Mob1b gene may be knocked out, and the other may be able to solve the above problem by using a non-human mammal under a system that can be arbitrarily knocked out. Were found, and further improvements were made to complete the present invention.
本発明は例えば以下の項に記載の主題を包含する。
項１．
（好ましくは、全身において）Ｍｏｂ１ａ遺伝子及びＭｏｂ１ｂ遺伝子のうち、一方はノックアウトされており、もう一方は任意にノックアウトできるシステム下にある、非ヒト哺乳動物。
項２．
前記任意にノックアウトできるシステムが、Ｃｒｅ－ＬｏｘＰシステムである、項１に記載の非ヒト哺乳動物。
項３．
前記Ｃｒｅ－ＬｏｘＰシステムが、タモキシフェン依存的にはたらくＣｒｅ－ＬｏｘＰシステムである、項２に記載の非ヒト哺乳動物。
項４．
項１～３のいずれかに記載の非ヒト哺乳動物において、前記任意にノックアウトできるシステムにより、Ｍｏｂ１ａ遺伝子又はＭｏｂ１ｂ遺伝子をノックアウトする工程を含む、癌モデル非ヒト哺乳動物の製造方法。
項５．
項３に記載の非ヒト哺乳動物に、タモキシフェンを投与する工程を含む、癌モデル非ヒト哺乳動物の製造方法。
項６．
項３に記載の非ヒト哺乳動物の頭頸部に、タモキシフェンを投与する工程を含む、頭頸部癌モデル非ヒト哺乳動物の製造方法。
項７．
請求項３に記載の非ヒト哺乳動物の舌に、タモキシフェンを塗布する工程を含む、舌癌モデル非ヒト哺乳動物の製造方法。
項８．
（Ａ）項４～７のいずれかに記載の方法で製造された癌モデル非ヒト哺乳動物に被験物質を適用する工程、及び
（Ｂ）被験物質が適用された癌モデル非ヒト哺乳動物の癌の抑制若しくは改善の程度を測定する工程を含む、抗癌剤候補物質をスクリーニングする方法。
項９．
（Ａ’）（好ましくは、全身において）Ｍｏｂ１ａ遺伝子及びＭｏｂ１ｂ遺伝子のうち、一方はノックアウトされており、もう一方は任意にノックアウトできるシステム下にある、非ヒト哺乳動物であって、前記任意にノックアウトできるシステムが、タモキシフェン依存的にはたらくＣｒｅ－ＬｏｘＰシステムである、非ヒト哺乳動物に対して、タモキシフェン及び被験物質を適用する工程、及び
（Ｂ’）被験物質が適用された当該非ヒト哺乳動物の癌の抑制の程度を測定する工程
を含む、抗癌剤候補物質をスクリーニングする方法。
項１０．
頭頸部においてＭｏｂ１ａ遺伝子及びＭｏｂ１ｂ遺伝子がダブルノックアウトされた、頭頸部癌モデル非ヒト哺乳動物。 The invention includes, for example, the subject matter described in the following section.
Item 1.
A non-human mammal, wherein one of the Mob1a and Mob1b genes is knocked out (preferably in the whole body) and the other is under an optional knockout system.
Item 2.
Item 2. The non-human mammal according to Item 1, wherein the optionally knockout system is a Cre-LoxP system.
Item 3.
Item 3. The non-human mammal according toitem 2, wherein the Cre-LoxP system is a Cre-LoxP system that works in a tamoxifen-dependent manner.
Item 4.
Item 4. A method for producing a cancer model non-human mammal, comprising the step of knocking out a Mob1a gene or a Mob1b gene using the system capable of arbitrarily knocking out the non-human mammal according to any one ofItems 1 to 3.
Item 5.
Item 4. A method for producing a cancer model non-human mammal, comprising a step of administering tamoxifen to the non-human mammal according to Item 3.
Item 6.
Item 4. A method for producing a head and neck cancer model non-human mammal, comprising the step of administering tamoxifen to the head and neck of a non-human mammal according to Item 3.
Item 7.
A method for producing a tongue cancer model non-human mammal, comprising a step of applying tamoxifen to the tongue of the non-human mammal according to claim 3.
Item 8.
(A) a step of applying a test substance to a cancer model non-human mammal produced by the method according to any of items 4 to 7, and (B) a cancer of the cancer model non-human mammal to which the test substance is applied. A method for screening a candidate substance for an anticancer agent, comprising a step of measuring the degree of suppression or improvement of the drug.
Item 9.
(A ′) one (preferably in whole body) of the Mob1a gene and the Mob1b gene, one of which is knocked out, and the other is a non-human mammal under an optionally knockout system, wherein the optionally knockout is A step of applying a tamoxifen and a test substance to a non-human mammal, which is a Cre-LoxP system that acts in a tamoxifen-dependent manner, and (B ′) a step of applying the test substance to the non-human mammal. A method for screening a candidate anticancer drug, comprising a step of measuring the degree of suppression of cancer.
Item 10.
A head and neck cancer model non-human mammal in which the Mob1a gene and the Mob1b gene have been double knocked out in the head and neck.
項１．
（好ましくは、全身において）Ｍｏｂ１ａ遺伝子及びＭｏｂ１ｂ遺伝子のうち、一方はノックアウトされており、もう一方は任意にノックアウトできるシステム下にある、非ヒト哺乳動物。
項２．
前記任意にノックアウトできるシステムが、Ｃｒｅ－ＬｏｘＰシステムである、項１に記載の非ヒト哺乳動物。
項３．
前記Ｃｒｅ－ＬｏｘＰシステムが、タモキシフェン依存的にはたらくＣｒｅ－ＬｏｘＰシステムである、項２に記載の非ヒト哺乳動物。
項４．
項１～３のいずれかに記載の非ヒト哺乳動物において、前記任意にノックアウトできるシステムにより、Ｍｏｂ１ａ遺伝子又はＭｏｂ１ｂ遺伝子をノックアウトする工程を含む、癌モデル非ヒト哺乳動物の製造方法。
項５．
項３に記載の非ヒト哺乳動物に、タモキシフェンを投与する工程を含む、癌モデル非ヒト哺乳動物の製造方法。
項６．
項３に記載の非ヒト哺乳動物の頭頸部に、タモキシフェンを投与する工程を含む、頭頸部癌モデル非ヒト哺乳動物の製造方法。
項７．
請求項３に記載の非ヒト哺乳動物の舌に、タモキシフェンを塗布する工程を含む、舌癌モデル非ヒト哺乳動物の製造方法。
項８．
（Ａ）項４～７のいずれかに記載の方法で製造された癌モデル非ヒト哺乳動物に被験物質を適用する工程、及び
（Ｂ）被験物質が適用された癌モデル非ヒト哺乳動物の癌の抑制若しくは改善の程度を測定する工程を含む、抗癌剤候補物質をスクリーニングする方法。
項９．
（Ａ’）（好ましくは、全身において）Ｍｏｂ１ａ遺伝子及びＭｏｂ１ｂ遺伝子のうち、一方はノックアウトされており、もう一方は任意にノックアウトできるシステム下にある、非ヒト哺乳動物であって、前記任意にノックアウトできるシステムが、タモキシフェン依存的にはたらくＣｒｅ－ＬｏｘＰシステムである、非ヒト哺乳動物に対して、タモキシフェン及び被験物質を適用する工程、及び
（Ｂ’）被験物質が適用された当該非ヒト哺乳動物の癌の抑制の程度を測定する工程
を含む、抗癌剤候補物質をスクリーニングする方法。
項１０．
頭頸部においてＭｏｂ１ａ遺伝子及びＭｏｂ１ｂ遺伝子がダブルノックアウトされた、頭頸部癌モデル非ヒト哺乳動物。 The invention includes, for example, the subject matter described in the following section.
A non-human mammal, wherein one of the Mob1a and Mob1b genes is knocked out (preferably in the whole body) and the other is under an optional knockout system.
Item 3.
Item 3. The non-human mammal according to
Item 4.
Item 4. A method for producing a cancer model non-human mammal, comprising the step of knocking out a Mob1a gene or a Mob1b gene using the system capable of arbitrarily knocking out the non-human mammal according to any one of
Item 5.
Item 4. A method for producing a cancer model non-human mammal, comprising a step of administering tamoxifen to the non-human mammal according to Item 3.
Item 6.
Item 4. A method for producing a head and neck cancer model non-human mammal, comprising the step of administering tamoxifen to the head and neck of a non-human mammal according to Item 3.
Item 7.
A method for producing a tongue cancer model non-human mammal, comprising a step of applying tamoxifen to the tongue of the non-human mammal according to claim 3.
Item 8.
(A) a step of applying a test substance to a cancer model non-human mammal produced by the method according to any of items 4 to 7, and (B) a cancer of the cancer model non-human mammal to which the test substance is applied. A method for screening a candidate substance for an anticancer agent, comprising a step of measuring the degree of suppression or improvement of the drug.
Item 9.
(A ′) one (preferably in whole body) of the Mob1a gene and the Mob1b gene, one of which is knocked out, and the other is a non-human mammal under an optionally knockout system, wherein the optionally knockout is A step of applying a tamoxifen and a test substance to a non-human mammal, which is a Cre-LoxP system that acts in a tamoxifen-dependent manner, and (B ′) a step of applying the test substance to the non-human mammal. A method for screening a candidate anticancer drug, comprising a step of measuring the degree of suppression of cancer.
A head and neck cancer model non-human mammal in which the Mob1a gene and the Mob1b gene have been double knocked out in the head and neck.
短期間で効率よく作製可能な癌（特に頭頸部癌）モデル動物の提供が可能となる。
癌 Can provide cancer (especially head and neck cancer) model animals that can be efficiently produced in a short period of time.
以下、本発明に包含される各実施形態について、さらに詳細に説明する。本発明は、非ヒト哺乳動物、癌モデル非ヒト哺乳動物の製造方法、癌モデル非ヒト哺乳動物を用いた抗癌剤候補物質のスクリーニング方法等を好ましく包含するが、これらに限定されるわけではなく、本発明は本明細書に開示される全てを包含する。
Hereinafter, each embodiment included in the present invention will be described in more detail. The present invention preferably includes, but is not limited to, a method for producing a non-human mammal, a method for producing a cancer model non-human mammal, a method for screening a candidate anti-cancer agent using a cancer model non-human mammal, and the like. The present invention includes all that is disclosed herein.
本発明に包含される非ヒト哺乳動物は、Ｍｏｂ１ａ遺伝子及びＭｏｂ１ｂ遺伝子のうち、一方はノックアウトされており、もう一方は任意にノックアウトできるシステム下にある。すなわち、Ｍｏｂ１ａ遺伝子がノックアウトされており、Ｍｏｂ１ｂ遺伝子が任意にノックアウトできるシステム下にあってもよいし、Ｍｏｂ１ｂ遺伝子がノックアウトされており、Ｍｏｂ１ａ遺伝子が任意にノックアウトできるシステム下にあってもよい。なお、当該非ヒト哺乳動物を、「本発明の非ヒト哺乳動物」と表記することがある。また、Ｍｏｂ１ａ遺伝子及びＭｏｂ１ｂ遺伝子は多くの哺乳類で既に知られており、例えばマウスのＭｏｂ１ａ ｍＲＮＡのアクセッション番号（ＮＣＢＩ）はNM_145571であり、マウスのＭｏｂ１ｂ ｍＲＮＡのアクセッション番号（ＮＣＢＩ）はNM_026735である。
非 Among the non-human mammals included in the present invention, one of the Mob1a gene and the Mob1b gene is knocked out, and the other is under a system that can be arbitrarily knocked out. That is, the system may be under a system where the Mob1a gene has been knocked out and the Mob1b gene can be arbitrarily knocked out, or under a system where the Mob1b gene has been knocked out and the Mob1a gene can be arbitrarily knocked out. In addition, the said non-human mammal may be described as "the non-human mammal of the present invention". In addition, the Mob1a gene and the Mob1b gene are already known in many mammals. For example, the accession number (NCBI) of the mouse Mob1 mRNA is NM_145571, and the accession number (NCBI) of the mouse Mob1 mRNA is NM_026735. .
ノックアウトされている遺伝子におけるノックアウトの方法は特に制限されず、例えば、遺伝子全体が欠損した状態であってもよいし、遺伝子の塩基配列が１又は２以上、遺伝子機能が失われるように置換、欠失、又は挿入された状態であってもよい。ノックアウトの手法も特に制限されず、公知の方法を用いることができる。例えば、胚性幹細胞に対して遺伝子組み替えを行い、初期胚（胚盤胞）の中に挿入し、そして生まれてくる生物のうちで、遺伝子組換え細胞が生殖系列に分化したものが、ノックアウトされた遺伝子を次世代に残すことができるので、これを交配してノックアウト生物を得ることができる。
The method of knocking out a knocked out gene is not particularly limited. For example, the whole gene may be deficient, or the base sequence of the gene may be one or more, and the gene may be replaced or deleted so that gene function is lost. It may be lost or inserted. The knockout method is not particularly limited, and a known method can be used. For example, genetically modifying embryonic stem cells, inserting them into early embryos (blastocysts), and knocking out the germline of the transgenic cells among the born organisms Since the gene can be left in the next generation, it can be crossed to obtain a knockout organism.
また、任意にノックアウトできるシステムとしては、例えばＣｒｅ－ｌｏｘＰシステムが挙げられる。当該システムは当該技術分野において周知である。Ｃｒｅタンパク質は部位特異的組換え酵素であり、ＤＮＡ分子の特定の配列（ｌｏｘＰ）同士の間で組換えを起こす。マウスを例に取ると、次のように説明することができる。発現特性が既知のプロモーターを利用して、Ｃｒｅを特定の組織あるいは特定の時期に生産するマウスを用意する。特的の組織でだけ遺伝子をオフにしたいなら、当該特定の組織だけで機能するプロモーターをさがし、その後ろにＣｒｅの遺伝子を繋げて、当該特定の組織でだけＣｒｅが生産されるようにする。次に、目的遺伝子の前後にｌｏｘＰを挿入したマウスを用意する。この２つのマウスを交配させて得られた子孫のうち、双方の遺伝子を持つものを選択する。当該選択されたマウスは、当該特定の組織でのみＣｒｅが発現し、よってｌｏｘＰ同士の間で組み換えが起こり、結果として当該特定の組織でのみ目的遺伝子がノックアウトされる。
シ ス テ ム Also, as a system that can be knocked out arbitrarily, for example, a Cre-loxP system can be mentioned. Such systems are well-known in the art. The Cre protein is a site-specific recombinase that causes recombination between specific sequences (loxP) of the DNA molecule. Taking a mouse as an example, it can be explained as follows. Using a promoter whose expression characteristics are known, a mouse that produces Cre at a specific tissue or at a specific time is prepared. If it is desired to turn off the gene only in a specific tissue, a promoter that functions only in the specific tissue is searched for, and the Cre gene is ligated behind the promoter so that Cre is produced only in the specific tissue. Next, a mouse having loxP inserted before and after the target gene is prepared. Of the offspring obtained by crossing these two mice, those having both genes are selected. In the selected mouse, Cre is expressed only in the specific tissue, and recombination occurs between loxPs. As a result, the target gene is knocked out only in the specific tissue.
また、Ｃｒｅ－ｌｏｘＰシステムには、特定の薬剤（例えばタモキシフェン）によりＣｒｅタンパク質の発現を誘導できるようにしたバリエーションも存在しており、これも周知である。より具体的には、例えば、Ｃｒｅと変異エストロゲン受容体の融合タンパク質であるＣｒｅ－ＥＲタンパク質は通常細胞質に存在するが、エストロゲン誘導体であるタモキシフェンと結合することにより核内に移行し、ｌｏｘＰ配列に対して組換えを起こす。これを利用してＣｒｅ－ｌｏｘＰシステムの働く時期をタモキシフェン依存的に調節することが可能である。このような薬剤（タモキシフェン）依存的にはたらくＣｒｅ－ｌｏｘＰシステムは、本発明の非ヒト哺乳動物における「任意にノックアウトできるシステム」として特に好適である。
なお、本発明の非ヒト哺乳動物は、全身性のコンディショナルダブルノックアウト非ヒト哺乳動物であることが好ましい。すなわち、全身において、Ｍｏｂ１ａ遺伝子及びＭｏｂ１ｂ遺伝子のうち、一方はノックアウトされており、もう一方は任意にノックアウトできるシステム下にある非ヒト哺乳動物であることが好ましい。 There are also variations in the Cre-loxP system that allow the expression of Cre protein to be induced by a specific drug (for example, tamoxifen), and these are also well known. More specifically, for example, the Cre-ER protein, which is a fusion protein of Cre and a mutant estrogen receptor, is usually present in the cytoplasm, but is translocated into the nucleus by binding to an estrogen derivative, tamoxifen, and becomes a loxP sequence. Recombination occurs. Using this, it is possible to adjust the working time of the Cre-loxP system in a tamoxifen-dependent manner. Such a drug (tamoxifen) -dependent Cre-loxP system is particularly suitable as the "arbitrarily knockout system" in the non-human mammal of the present invention.
Preferably, the non-human mammal of the present invention is a systemic conditional double knockout non-human mammal. That is, in the whole body, one of the Mob1a gene and the Mob1b gene is preferably knocked out, and the other is preferably a non-human mammal under a system capable of arbitrarily knocking out.
なお、本発明の非ヒト哺乳動物は、全身性のコンディショナルダブルノックアウト非ヒト哺乳動物であることが好ましい。すなわち、全身において、Ｍｏｂ１ａ遺伝子及びＭｏｂ１ｂ遺伝子のうち、一方はノックアウトされており、もう一方は任意にノックアウトできるシステム下にある非ヒト哺乳動物であることが好ましい。 There are also variations in the Cre-loxP system that allow the expression of Cre protein to be induced by a specific drug (for example, tamoxifen), and these are also well known. More specifically, for example, the Cre-ER protein, which is a fusion protein of Cre and a mutant estrogen receptor, is usually present in the cytoplasm, but is translocated into the nucleus by binding to an estrogen derivative, tamoxifen, and becomes a loxP sequence. Recombination occurs. Using this, it is possible to adjust the working time of the Cre-loxP system in a tamoxifen-dependent manner. Such a drug (tamoxifen) -dependent Cre-loxP system is particularly suitable as the "arbitrarily knockout system" in the non-human mammal of the present invention.
Preferably, the non-human mammal of the present invention is a systemic conditional double knockout non-human mammal. That is, in the whole body, one of the Mob1a gene and the Mob1b gene is preferably knocked out, and the other is preferably a non-human mammal under a system capable of arbitrarily knocking out.
また、Ｃｒｅ－ｌｏｘＰシステムの他、例えばテトラサイクリン発現調整システム（Ｔｅｔ－Ｏｎ Ｓｙｓｔｅｍ）も前記任意にノックアウトできるシステムとして挙げられる。当該システムは、ドキシサイクリンの投与及び除去により目的遺伝子の発現をＯＮ及びＯＦＦとすることができるシステムであり、当該技術分野において周知である。
の 他 In addition to the Cre-loxP system, for example, a tetracycline expression control system (Tet-On @ System) is also mentioned as a system that can be arbitrarily knocked out. This system is a system that can turn on and off the expression of a target gene by administering and removing doxycycline, and is well known in the art.
本発明の非ヒト哺乳動物の種としては、例えばペット又は家畜、あるいは実験動物として用いられる非ヒト哺乳動物が好ましく、例えばマウス、ラット、ウサギ、イヌ、ネコ、ヒツジ、ブタ、ヤギ、ウシ、ウマ、サル等が挙げられる。中でも、げっ歯類（マウス、ラット、ウサギなど）が好ましい。
As the species of the non-human mammal of the present invention, for example, a pet or a domestic animal, or a non-human mammal used as an experimental animal is preferable. For example, a mouse, rat, rabbit, dog, cat, sheep, pig, goat, cow, horse And monkeys. Among them, rodents (mouse, rat, rabbit, etc.) are preferred.
本発明は、前記本発明の非ヒト哺乳動物において、前記任意にノックアウトできるシステムにより、Ｍｏｂ１ａ遺伝子又はＭｏｂ１ｂ遺伝子をノックアウトする工程を含む、癌モデル非ヒト哺乳動物の製造方法も包含する。当該製造方法を、「本発明の癌モデル非ヒト哺乳動物製造方法」と表記することがある。なお、Ｍｏｂ１ａ遺伝子がノックアウトされており、Ｍｏｂ１ｂ遺伝子が任意にノックアウトできるシステム下にある場合には、当該工程でノックアウトされる遺伝子はＭｏｂ１ｂ遺伝子であり、Ｍｏｂ１ｂ遺伝子がノックアウトされており、Ｍｏｂ１ａ遺伝子が任意にノックアウトできるシステム下にある場合には、当該工程でノックアウトされる遺伝子はＭｏｂ１ａ遺伝子である。いずれの場合であっても、当該工程により行われる遺伝子ノックアウトには、前記任意にノックアウトできるシステムが利用される。
The present invention also encompasses a method for producing a cancer model non-human mammal, which comprises a step of knocking out the Mob1a gene or the Mob1b gene by the system that can be arbitrarily knocked out in the non-human mammal of the present invention. The production method may be referred to as “the cancer model non-human mammal production method of the present invention”. When the Mob1a gene has been knocked out and the Mob1b gene is under a system that can be arbitrarily knocked out, the gene knocked out in this step is the Mob1b gene, the Mob1b gene has been knocked out, and the Mob1a gene has an arbitrary knockout. When the system is under a system that allows knockout, the gene knocked out in this step is the Mob1a gene. In any case, the gene knockout performed in the step uses the system that can arbitrarily knock out.
上述の通り、前記任意にノックアウトできるシステムとしては、特にタモキシフェン依存的にはたらくＣｒｅ－ｌｏｘＰシステムが好ましい。よって、本発明の癌モデル非ヒト哺乳動物製造方法としては、前記非ヒト哺乳動物にタモキシフェンを投与する工程を含むことが好ましい。本発明の効果が損なわれない限り投与方法は特に制限されず、例えば経口投与、経血管投与、皮下投与、皮膚投与等が挙げられる。具体的な投与手段についても、注射や塗布など、公知の方法を適宜選択して用いることができる。本発明の癌モデル非ヒト哺乳動物製造方法は、特に頭頸部癌モデル非ヒト哺乳動物の製造に好適であり、この場合特に頭頸部にタモキシフェンを投与することが好ましい。頭頸部としては、例えば舌、口腔、咽頭、喉頭等が挙げられるが、中でも特に舌が好ましく、この場合、舌にタモキシフェンを塗布する方法が特に好ましい。
As described above, as the system that can be knocked out arbitrarily, a Cre-loxP system that works in a tamoxifen-dependent manner is particularly preferable. Therefore, the method for producing a cancer model non-human mammal of the present invention preferably includes a step of administering tamoxifen to the non-human mammal. The administration method is not particularly limited as long as the effects of the present invention are not impaired, and examples include oral administration, transvascular administration, subcutaneous administration, and dermal administration. As for the specific administration means, known methods such as injection and application can be appropriately selected and used. The method for producing a cancer model non-human mammal of the present invention is particularly suitable for producing a head and neck cancer model non-human mammal. In this case, it is particularly preferable to administer tamoxifen to the head and neck. Examples of the head and neck include tongue, oral cavity, pharynx, and larynx. Among them, tongue is particularly preferable. In this case, a method of applying tamoxifen to the tongue is particularly preferable.
なお、タモキシフェンを投与するにあたっては、本発明の非ヒト哺乳動物に全身麻酔を施すことが好ましい。特に、塗布によりタモキシフェンを投与する場合には、全身麻酔を施すことによりマウスが長時間覚醒しないため、高濃度タモキシフェンが塗布した部位に残った状態となり、タモキシフェンの当該部位への適用が効率的に行われ得る。
When administering tamoxifen, it is preferable to subject the non-human mammal of the present invention to general anesthesia. In particular, when tamoxifen is applied by application, since the mouse does not wake up for a long time by performing general anesthesia, the high concentration tamoxifen remains at the applied site, and the application of tamoxifen to the site is efficiently performed. Can be done.
また、本発明は、前記本発明の癌モデル非ヒト哺乳動物製造方法により製造された癌モデル非ヒト哺乳動物も包含する。当該癌モデル非ヒト哺乳動物の中でも、特定部位においてＭｏｂ１ａ遺伝子及びＭｏｂ１ｂ遺伝子がダブルノックアウトされた、当該特定部位での癌モデル非ヒト哺乳動物が好ましい。そもそも、前述の通り、Ｍｏｂ１ａ遺伝子及びＭｏｂ１ｂ遺伝子をダブルノックアウトした場合には胎生（着床直後）致死となるため、特定の部位においてＭｏｂ１ａ遺伝子及びＭｏｂ１ｂ遺伝子がダブルノックアウトされた非ヒト哺乳動物を製造するためには、特殊な方法（例えば、Ｍｏｂ１ａ遺伝子及びＭｏｂ１ｂ遺伝子のうち、一方はノックアウトされており、もう一方は任意にノックアウトできるシステム下にある、非ヒト哺乳動物を用いて、特定部位において前記任意にノックアウトできるシステムを利用した遺伝子ノックアウトを行う方法）でなければ製造することが困難である。当該特定部位としては、本発明の効果を損なわない限り特に限定されないが、たとえば頭頸部（例えば舌、口腔、咽頭、喉頭等）が好ましく、中でも舌が好ましい。
The present invention also includes a cancer model non-human mammal produced by the method for producing a cancer model non-human mammal of the present invention. Among the cancer model non-human mammals, a cancer model non-human mammal at the specific site where the Mob1a gene and the Mob1b gene are double-knocked out at the specific site is preferable. In the first place, as described above, when the Mob1a gene and the Mob1b gene are double-knocked out, the embryo is lethal (immediately after implantation), so that a non-human mammal in which the Mob1a gene and the Mob1b gene are double-knocked out at a specific site is produced. To do so, a special method (for example, one of the Mob1a gene and the Mob1b gene is knocked out, and the other is a non-human mammal under a system that can be arbitrarily knocked out, and In other words, it is difficult to produce a gene unless a method for knocking out a gene using a system capable of knocking out is used. The specific site is not particularly limited as long as the effects of the present invention are not impaired. For example, a head and neck (for example, tongue, oral cavity, pharynx, larynx, etc.) is preferable, and tongue is particularly preferable.
本発明は、（Ａ）前記癌モデル非ヒト哺乳動物に被験物質を適用する工程、及び（Ｂ）被験物質が適用された癌モデル非ヒト哺乳動物の癌の抑制若しくは改善の程度を測定する工程を含む、抗癌剤候補物質をスクリーニングする方法をも包含する。また、本発明は、（Ａ’）Ｍｏｂ１ａ遺伝子及びＭｏｂ１ｂ遺伝子のうち、一方はノックアウトされており、もう一方は任意にノックアウトできるシステム下にある、非ヒト哺乳動物であって、前記任意にノックアウトできるシステムが、タモキシフェン依存的にはたらくＣｒｅ－ＬｏｘＰシステムである、非ヒト哺乳動物に対して、タモキシフェン及び被験物質を適用する工程、及び（Ｂ’）被験物質が適用された当該非ヒト哺乳動物の癌の抑制の程度を測定する工程を含む、抗癌剤候補物質をスクリーニングする方法をも包含する。
The present invention provides (A) a step of applying a test substance to the cancer model non-human mammal, and (B) a step of measuring the degree of suppression or improvement of cancer in the cancer model non-human mammal to which the test substance has been applied. And a method of screening for a candidate anticancer agent. Further, the present invention provides a non-human mammal, wherein one of the (A ′) Mob1a gene and the Mob1b gene is knocked out and the other is under a system that can be arbitrarily knocked out, wherein the knockout can be arbitrarily performed. A step of applying tamoxifen and a test substance to a non-human mammal, wherein the system is a Cre-LoxP system that works in a tamoxifen-dependent manner, and (B ′) a cancer of the non-human mammal to which the test substance has been applied. And a method of screening for a candidate anticancer agent, which comprises the step of measuring the degree of suppression of the drug.
被験物質は特に制限されず、例えば、化合物及び組成物であり得る。化合物としては、例えば低分子化合物や、核酸（例えばＤＮＡ、ＲＮＡ等）やタンパク質（例えば抗体又はその一部等）、ポリマー等の高分子化合物であってよい。組成物としても、生物（例えば動物、植物、微生物等）から得た抽出物等であってもよく、化合物を２種以上組み合わせたものであってもよい。被験物質の適用方法についても特に制限されるものではなく、例えば、例えば経口投与、経血管投与、皮下投与、皮膚投与等が挙げられる。なお、（Ａ）工程で用いる癌モデル非ヒト哺乳動物の製造において、特定薬剤（タモキシフェン等）の投与を行った場合には、当該タモキシフェンの投与と同じ投与方法により被験物質を投与してもよい。また、（Ａ’）工程においては、タモキシフェンと被験物質の適用方法は同じであっても異なっていてもよく、同じであることが好ましい。タモキシフェンの投与部位や投与方法等については、上記の癌モデル非ヒト哺乳動物製造方法で説明したのと同様である。
The test substance is not particularly limited, and may be, for example, a compound or a composition. The compound may be, for example, a low molecular compound, a high molecular compound such as a nucleic acid (for example, DNA or RNA), a protein (for example, an antibody or a part thereof), or a polymer. The composition may be an extract or the like obtained from an organism (for example, an animal, a plant, or a microorganism), or may be a combination of two or more compounds. The method of applying the test substance is not particularly limited, and examples include oral administration, transvascular administration, subcutaneous administration, and dermal administration. When a specific drug (such as tamoxifen) is administered in the production of the cancer model non-human mammal used in the step (A), the test substance may be administered by the same administration method as the administration of the tamoxifen. . In step (A '), the method of applying tamoxifen and the test substance may be the same or different, and preferably the same. The administration site and administration method of tamoxifen are the same as those described in the above-mentioned method for producing a cancer model non-human mammal.
また、（Ｂ）工程や（Ｂ’）工程において、癌の抑制若しくは改善の程度を測定する方法としては、特に制限されず、公知の方法を用いることができる。例えば癌組織を直接観察する方法や、癌組織を採取して染色（例えばＨＥ染色や免疫学的染色）を行ったうえで細胞観察し、各細胞の癌の程度から全体の癌の進行度を解析する方法等が挙げられるが、特にこれらに制限されるものではない。
方法 In the steps (B) and (B ′), the method for measuring the degree of suppression or improvement of cancer is not particularly limited, and a known method can be used. For example, a method of directly observing a cancer tissue, or a method of collecting a cancer tissue and performing staining (for example, HE staining or immunological staining) and observing the cells, and determining the degree of progress of the entire cancer from the degree of cancer of each cell. Examples include a method of analysis, but are not particularly limited thereto.
Ｍｏｂ１ａ遺伝子及びＭｏｂ１ｂ遺伝子がダブルノックアウトされた本発明の癌モデル非ヒト哺乳動物（好ましくは頭頸部癌モデル非ヒト哺乳動物、より好ましくは舌癌モデル非ヒト哺乳動物）においては、Ｈｉｐｐｏ ＰａｔｈｗａｙにおけるＹＡＰ１及びＴＡＺのうち、ＴＡＺではなくＹＡＰ１のはたらきにより癌化が起こっている。このため、当該癌モデル非ヒト哺乳動物は、ＹＡＰ１依存性の癌モデル非ヒト哺乳動物ということもできる。ＹＡＰ１は多くの哺乳類で既に知られており、例えばマウスのＹａｐ１のｍＲＮＡのアクセッション番号（ＮＣＢＩ）はNM_001171147である。
In the cancer model non-human mammal (preferably a head and neck cancer model non-human mammal, more preferably a tongue cancer model non-human mammal) of the present invention in which the Mob1a gene and the Mob1b gene are double-knocked out, YAP1 in Hippo @ Pathway and Among TAZs, canceration is caused by the action of YAP1 instead of TAZ. Therefore, the cancer model non-human mammal can be said to be a YAP1-dependent cancer model non-human mammal. YAP1 is already known in many mammals. For example, the accession number (NCBI) of mouse Yap1 mRNA is NM_001171147.
当該癌モデル非ヒト哺乳動物は、発癌がＹＡＰ１に依存していることから、ＹＡＰ１をターゲットとした抗癌剤候補物質をスクリーニングするために、特に有用であるということができる。例えば、当該癌モデル非ヒト哺乳動物が頭頸部癌（好ましくは舌癌）モデル非ヒト哺乳動物である場合には、ＹＡＰ１をターゲットとした頭頸部癌に対する抗癌剤候補物質をスクリーニングするために、特に有用であるといえる。また、その他の部位の癌についても同様に、ＹＡＰ１をターゲットとした抗癌剤候補物質をスクリーニングするために有用であるといえる。
Since the cancer model depends on YAP1 for carcinogenesis, the cancer model non-human mammal can be said to be particularly useful for screening anticancer drug candidate substances targeting YAP1. For example, when the cancer model non-human mammal is a head and neck cancer (preferably tongue cancer) model non-human mammal, it is particularly useful for screening anticancer drug candidate substances against head and neck cancer targeting YAP1. You can say that. Similarly, it can be said that cancers at other sites are useful for screening anticancer drug candidate substances targeting YAP1.
なお、上述の通り、Ｍｏｂ１ａ遺伝子及びＭｏｂ１ｂ遺伝子をダブルノックアウトした非ヒト哺乳動物を交配により作製しようとすると、着床直後に致死となるところ、Ｍｏｂ１ａ遺伝子及びＭｏｂ１ｂ遺伝子をダブルノックアウトした非ヒト哺乳動物は、例えば上述した製造方法により製造することによって製造が可能となったものである。
As described above, when trying to produce a non-human mammal having the Mob1a gene and the Mob1b gene double-knocked out by mating, the non-human mammal having the Mob1a gene and the Mob1b gene double-knocked out is fatal immediately after implantation. For example, it can be manufactured by manufacturing according to the manufacturing method described above.
なお、本明細書において「含む」とは、「本質的にからなる」と、「からなる」をも包含する（The term "comprising" includes "consisting essentially of” and "consisting of."）。また、本発明は、本明細書に説明した構成要件を任意の組み合わせを全て包含する。
Note that in this specification, the term "comprising" includes "consisting essentially of" and "consisting of" (The term "comprising" "includes" "consisting" essentially "of" and "" consisting "of."). In addition, the present invention encompasses any combination of the constituent features described in this specification.
また、上述した本発明の各実施形態について説明した各種特性（性質、構造、機能等）は、本発明に包含される主題を特定するにあたり、どのように組み合わせられてもよい。すなわち、本発明には、本明細書に記載される組み合わせ可能な各特性のあらゆる組み合わせからなる主題が全て包含される。
The various characteristics (properties, structures, functions, and the like) described in the embodiments of the present invention described above may be combined in any way when specifying the subject matter included in the present invention. That is, the present invention encompasses all subjects including all combinations of the characteristics that can be combined as described in this specification.
以下、本発明をより具体的に説明するが、本発明は下記の例に限定されるものではない。
Hereinafter, the present invention will be described more specifically, but the present invention is not limited to the following examples.
Ｍｏｂ１ａ／ｂコンディショナルダブルノックアウト（ＤＫＯ）マウスの製造
Ｍｏｂ１ｂをホモで欠損しており、Ｍｏｂ１ａ遺伝子がホモでタモキシフェン依存的Ｃｒｅ－ＬｏｘＰシステム下に存在するマウス（Mob1a flox/flox, Mob1b -/-, CreERT2）をはじめ、以下の検討に用いたダブルノックアウトマウス及びトリプルノックアウトマウス（Mob1a flox/flox, Mob1b -/-, Yap1 flox/flox, CreERT、及びMob1a flox/flox, Mob1b -/-, Taz flox/flox, CreERT）、並びにコントロールマウス（Mob1a flox/flox, Mob1b -/-）は、次のマウスを適宜交配することによって作製した。 Production of Mob1a / b conditional double knockout (DKO) mice Mob1b is homozygously deficient, and the Mob1a gene is homozygous and exists under the tamoxifen-dependent Cre-LoxP system (Mob1a flox / flox, Mob1b − / −, CreERT2), double knockout mice and triple knockout mice (Mob1a flox / flox, Mob1b-/-, Yap1 flox / flox, CreERT, and Mob1a flox / flox, Mob1b-/-, Taz flox / flox, CreERT) and control mice (Mob1a flox / flox, Mob1b − / −) were prepared by crossing the following mice as appropriate.
Ｍｏｂ１ｂをホモで欠損しており、Ｍｏｂ１ａ遺伝子がホモでタモキシフェン依存的Ｃｒｅ－ＬｏｘＰシステム下に存在するマウス（Mob1a flox/flox, Mob1b -/-, CreERT2）をはじめ、以下の検討に用いたダブルノックアウトマウス及びトリプルノックアウトマウス（Mob1a flox/flox, Mob1b -/-, Yap1 flox/flox, CreERT、及びMob1a flox/flox, Mob1b -/-, Taz flox/flox, CreERT）、並びにコントロールマウス（Mob1a flox/flox, Mob1b -/-）は、次のマウスを適宜交配することによって作製した。 Production of Mob1a / b conditional double knockout (DKO) mice Mob1b is homozygously deficient, and the Mob1a gene is homozygous and exists under the tamoxifen-dependent Cre-LoxP system (Mob1a flox / flox, Mob1b − / −, CreERT2), double knockout mice and triple knockout mice (Mob1a flox / flox, Mob1b-/-, Yap1 flox / flox, CreERT, and Mob1a flox / flox, Mob1b-/-, Taz flox / flox, CreERT) and control mice (Mob1a flox / flox, Mob1b − / −) were prepared by crossing the following mice as appropriate.
（１）Mob1a flox/flox; Mob1b -/-
（２）Rosa26CreER-Tg
（３）Taz flox/flox
（４）Yap1 flox/flox (1) Mob1a flox / flox; Mob1b-/-
(2) Rosa26CreER-Tg
(3) Taz flox / flox
(4) Yap1 flox / flox
（２）Rosa26CreER-Tg
（３）Taz flox/flox
（４）Yap1 flox/flox (1) Mob1a flox / flox; Mob1b-/-
(2) Rosa26CreER-Tg
(3) Taz flox / flox
(4) Yap1 flox / flox
なお、（１）は上記非特許文献２（Nishio M et al, J Clin Invest. 2012 Dec;122(12):4505-18.）に記載の方法に従って作製した。（２）はThe Jackson Laboratoryから入手した（上記非特許文献５：Srinivas S et al, BMC Dev Biol. 2001;1:4.参照）。（３）はDr J. Wrana（トロント大学）から頂いた。（４）はYap1 flox/flox ＥＳ細胞（Knockout Mouse Project Repository, UC Davis, CA, USA.）を発生させて作製した。
（Incidentally, (1) was prepared according to the method described in Non-patent Document 2 (Nishio Met et al, JClin Invest. 2012 Dec; 122 (12): 4505-18.). (2) was obtained from The Jackson Laboratory (see Non-Patent Document 5: Srinivas Set et al, BMC Dev Biol. 2001; 1: 4.). (3) was provided by Dr J. Wrana (University of Toronto). (4) was prepared by generating Yap1 flox / flox ES cells (Knockout Mouse Project Repository, UC Davis, CA, USA.).
舌癌モデルマウスの調製
３週齢のＭｏｂ１ａ／ｂコンディショナルＤＫＯマウスを全身麻酔し、その舌にタモキシフェン（１００％エタノールに１０ｍｇ／ｍｌの濃度で溶解させたもの）を５回塗布した。当該操作を３日間連続で行った。当該手順の概要を図１に示す。なお、全身麻酔のため約１２時間はマウスは覚醒せず、このために高濃度タモキシフェンが舌に残った状態となり、タモキシフェンの舌への適用が効率的に行われることになる。 Preparation of Tongue Cancer Model Mice A 3-week-old Mob1a / b conditional DKO mouse was anesthetized under general anesthesia, and tamoxifen (dissolved in 100% ethanol at a concentration of 10 mg / ml) was applied to the tongue five times. This operation was performed for three consecutive days. An outline of the procedure is shown in FIG. In addition, the mouse does not awake for about 12 hours due to general anesthesia, so that a high-concentration tamoxifen remains in the tongue, and the tamoxifen can be efficiently applied to the tongue.
３週齢のＭｏｂ１ａ／ｂコンディショナルＤＫＯマウスを全身麻酔し、その舌にタモキシフェン（１００％エタノールに１０ｍｇ／ｍｌの濃度で溶解させたもの）を５回塗布した。当該操作を３日間連続で行った。当該手順の概要を図１に示す。なお、全身麻酔のため約１２時間はマウスは覚醒せず、このために高濃度タモキシフェンが舌に残った状態となり、タモキシフェンの舌への適用が効率的に行われることになる。 Preparation of Tongue Cancer Model Mice A 3-week-old Mob1a / b conditional DKO mouse was anesthetized under general anesthesia, and tamoxifen (dissolved in 100% ethanol at a concentration of 10 mg / ml) was applied to the tongue five times. This operation was performed for three consecutive days. An outline of the procedure is shown in FIG. In addition, the mouse does not awake for about 12 hours due to general anesthesia, so that a high-concentration tamoxifen remains in the tongue, and the tamoxifen can be efficiently applied to the tongue.
タモキシフェン塗布開始から１週間後、２週間後、及び４週間後に舌組織を採取し、ヘマトキシリン・エオジン染色（ＨＥ染色）を行って観察した。観察結果を図２ａに示す。また、観察された、浸潤扁平上皮癌（ｉｎｖａｓｉｖｅ ｓｃｃ）、上皮内癌（ＣＩＳ）、及び形成異常（Ｄｙｓｐｌａｓｉａ）の割合をグラフして図２ｂに示す。
One week, two weeks, and four weeks after the start of the application of tamoxifen, the tongue tissue was collected and observed by hematoxylin and eosin staining (HE staining). The observation results are shown in FIG. 2a. FIG. 2B is a graph showing the observed ratios of invasive squamous cell carcinoma (invasive @ scc), carcinoma in situ (CIS), and dysplasia (Dysplasia).
当該結果から、早いマウスなら癌化まで１週間、全マウスが癌化するのもわずか２週間という、非常に短期間で癌化が起こることがわかった。通常モデルマウスは長期間の観察が必要なため個体差が出やすいが、当該手法により調製した癌モデルマウスは短期間で観察が終了するため、各種解析のばらつきが小さくてすみ、有利である。
From the results, it was found that canceration occurs in a very short period of time, with early mice taking one week to become cancerous and all mice becoming cancerous in only two weeks. Normally, long-term observation is required for model mice, so that individual differences are likely to occur. However, cancer model mice prepared by this method are completed in a short period of time, so that variations in various analyzes are small, which is advantageous.
舌癌モデルマウスの癌化メカニズムの検討
上記の舌癌モデルマウスの癌化がＹａｐ１主体で起こったものかＴａｚ主体で起こったものかを検討した。 Examination of the mechanism of carcinogenesis of tongue cancer model mouse It was examined whether the carcinogenesis of the tongue cancer model mouse was mainly caused by Yap1 or mainly by Taz.
上記の舌癌モデルマウスの癌化がＹａｐ１主体で起こったものかＴａｚ主体で起こったものかを検討した。 Examination of the mechanism of carcinogenesis of tongue cancer model mouse It was examined whether the carcinogenesis of the tongue cancer model mouse was mainly caused by Yap1 or mainly by Taz.
Mob1a flox/flox, Mob1b-/-, CreERT2マウスの舌を採取し、２４時間ディスパーゼＩＩに浸した後に舌上皮のみを分離してトリプシン処理を行うことにより細胞をバラバラにし、Ｐｒｉｍａｒｙ Ｃｕｌｔｕｒｅを行った。当該細胞はタモキシフェン誘導性にMob1a/bを任意の時期でノックアウト出来るような性質を引き継いでおり、当該細胞において、マウスと同様にタモキシフェン誘導性にMOB1a/bをノックアウトして各タンパク質の発現をウエスタンブロットにより解析した。タンパク質の調製は、具体的には、次のようにして行った。ＰＢＳ（－）でｗａｓｈ後にＴＮＥバッファーで細胞をＳｃｒａｐｅして回収し、超音波破砕５分、その後氷上で２０ｍｉｎ静置した。その間、３回Ｖｏｌｔｅｘした。４℃、１４０００ＲＰＭで２０ｍｉｎ遠心し、上清を新たなｔｕｂｅへ移し、ＢＣＡ法でタンパク濃度を揃えた。ＳＤＳを添加し、９６℃で５ｍｉｎ加温し、－３０℃で保存した。
The tongues of Mob1a flox / flox, Mob1b − / −, CreERT2 mice were collected, immersed in dispase II for 24 hours, and then only the tongue epithelium was separated and subjected to trypsin treatment to separate the cells to perform PrimaryＰCulture. The cells have inherited the property that Mob1a / b can be knocked out at any time in a tamoxifen-inducible manner.In the cells, the MOB1a / b was knocked out in a tamoxifen-inducible manner as in the mouse, and the expression of each protein was westernized. Analyzed by blot. The protein was specifically prepared as follows. After washing with PBS (-), the cells were collected by scraping with TNE buffer, collected by sonication for 5 minutes, and then allowed to stand on ice for 20 minutes. During that time, Voltex was performed three times. The mixture was centrifuged at 4 ° C. and 14000 RPM for 20 minutes, the supernatant was transferred to a new tube, and the protein concentration was adjusted by the BCA method. SDS was added, heated at 96 ° C. for 5 minutes, and stored at −30 ° C.
また、各タンパク質検出のために用いた抗体は次の通りである。（なお、ｐＹａｐ１及びｐＴａｚは、それぞれリン酸化Ｙａｐ１及びリン酸化Ｔａｚを示す。）
Ｍｏｂ１：Ｃｅｌｌ Ｓｉｇｎａｌｉｎｇ， ＃４９１２Ｓ
Ｌａｔｓ１：Ｃｅｌｌ Ｓｉｇｎａｌｉｎｇ， ＃３４７７Ｓ
Ｍｓｔ１：Ｃｅｌｌ Ｓｉｇｎａｌｉｎｇ， ＃３６８２Ｓ
Ａｃｔｉｎ：Ｃｅｌｌ Ｓｉｇｎａｌｉｎｇ， ＃４９６７
Ｙａｐ１：Ｃｅｌｌ Ｓｉｇｎａｌｉｎｇ， ＃４９１２Ｓ
ｐＹａｐ１：Ｃｅｌｌ Ｓｉｇｎａｌｉｎｇ， ＃４９１１
Ｔａｚ：Ｃｅｌｌ Ｓｉｇｎａｌｉｎｇ， ＃４８８３Ｓ
ｐＴａｚ：Ｃｅｌｌ Ｓｉｇｎａｌｉｎｇ， ＃７５２７５ The antibodies used for detecting each protein are as follows. (Note that pYap1 and pTaz indicate phosphorylated Yap1 and phosphorylated Taz, respectively.)
Mob1: Cell Signaling, # 4912S
Lats1: Cell Signaling, # 3377S
Mst1: Cell Signaling, # 3682S
Actin: Cell Signaling, # 4967
Yap1: Cell Signaling, # 4912S
pYap1: Cell Signaling, # 4911
Taz: Cell Signaling, # 4883S
pTaz: Cell Signaling, # 75275
Ｍｏｂ１：Ｃｅｌｌ Ｓｉｇｎａｌｉｎｇ， ＃４９１２Ｓ
Ｌａｔｓ１：Ｃｅｌｌ Ｓｉｇｎａｌｉｎｇ， ＃３４７７Ｓ
Ｍｓｔ１：Ｃｅｌｌ Ｓｉｇｎａｌｉｎｇ， ＃３６８２Ｓ
Ａｃｔｉｎ：Ｃｅｌｌ Ｓｉｇｎａｌｉｎｇ， ＃４９６７
Ｙａｐ１：Ｃｅｌｌ Ｓｉｇｎａｌｉｎｇ， ＃４９１２Ｓ
ｐＹａｐ１：Ｃｅｌｌ Ｓｉｇｎａｌｉｎｇ， ＃４９１１
Ｔａｚ：Ｃｅｌｌ Ｓｉｇｎａｌｉｎｇ， ＃４８８３Ｓ
ｐＴａｚ：Ｃｅｌｌ Ｓｉｇｎａｌｉｎｇ， ＃７５２７５ The antibodies used for detecting each protein are as follows. (Note that pYap1 and pTaz indicate phosphorylated Yap1 and phosphorylated Taz, respectively.)
Mob1: Cell Signaling, # 4912S
Lats1: Cell Signaling, # 3377S
Mst1: Cell Signaling, # 3682S
Actin: Cell Signaling, # 4967
Yap1: Cell Signaling, # 4912S
pYap1: Cell Signaling, # 4911
Taz: Cell Signaling, # 4883S
pTaz: Cell Signaling, # 75275
ウエスタンブロットにより解析結果を図３ａに示す。ＤＫＯマウスでは、Ｍｏｂ１はノックアウトされていることが確認できた。また、ＤＫＯマウスではＹａｐ１の発現は亢進している一方、Ｔａｚの発現はほとんど変わらないことが確認できた。さらに、当該解析結果のバンドの濃度をSoftware(Fujifilm Multi Gauge)を用いて定量（バックグラウンド補正あり）した。それぞれn=3で測定し、t検定でp値を算出し、有意差を判定した。結果を図３ｂに示す。図３ｂにおいて、＊は有意差有り（ｐ＜０．０５）を示す。当該解析結果からも、Ｙａｐ１は有意差を持って発現が亢進すること、及びＴａｚの発現は変化が無いこと、が裏付けられた。
解析 The results of analysis by Western blot are shown in FIG. In the DKO mouse, it was confirmed that Mob1 was knocked out. In addition, it was confirmed that the expression of Yap1 was enhanced in the DKO mouse, but the expression of Taz was hardly changed. Further, the concentration of the band as a result of the analysis was quantified (with background correction) using Software (Fujifilm Multi Multi Gauge). Each was measured at n = 3, the p value was calculated by the t test, and a significant difference was determined. The results are shown in FIG. 3b. In FIG. 3b, * indicates a significant difference (p <0.05). The analysis results also confirmed that the expression of Yap1 was enhanced with a significant difference, and that the expression of Taz did not change.
そこで、ＤＫＯマウスのＹａｐ又はＴａｚをノックアウトしたトリプルノックアウト（ＴＫＯ）マウスを調製し、上記と同様にして舌癌誘導を行い、舌の形態観察及び舌組織のＨＥ染色観察を行った。結果を図４ａに示す。さらに、当該ＨＥ染色で観察された、浸潤扁平上皮癌（ｉｎｖａｓｉｖｅ ｓｃｃ）、上皮内癌（ＣＩＳ）、及び形成異常（Ｄｙｓｐｌａｓｉａ）の割合をグラフして図４ｂに示す。Ｙａｐを更にＫＯしたＴＫＯマウスであれば舌癌誘導が起こらないことがわかった。この結果から、前記ＤＫＯマウスの癌化はＹＡＰ依存性であることがわかった。よって、前記舌癌モデルマウスは、ＹＡＰ依存性の発癌モデルということができる。
Thus, triple knockout (TKO) mice in which Yap or Taz of DKO mice were knocked out were prepared, tongue cancer was induced in the same manner as described above, and tongue morphology and HE staining of tongue tissues were observed. The results are shown in FIG. 4a. Furthermore, the ratios of invasive squamous cell carcinoma (invasive @ scc), carcinoma in situ (CIS), and dysplasia (Dysplasia) observed by the HE staining are shown in FIG. 4B. It was found that tongue cancer induction did not occur in TKO mice in which Yap was further KO. From these results, it was found that the canceration of the DKO mouse was YAP-dependent. Therefore, the tongue cancer model mouse can be said to be a YAP-dependent carcinogenesis model.
ヒト舌癌の臨床標本におけるＹＡＰ１染色
九州がんセンターで外科的治療した舌癌標本（ホルマリン固定後にパラフィン包埋したもの）に対して、ＹＡＰ１抗体（SIGMA, SAB4500094）を用いて、免疫組織学的染色を行った。結果を図５に示す。当該結果から、ヒトにおいても、舌癌の発症および進展にＹＡＰ１が深く関与している可能性が強く示唆された。 YAP1 staining in human tongue cancer clinical specimens Tongue cancer specimens surgically treated at Kyushu Cancer Center (formalin-fixed and paraffin-embedded) were immunohistochemically analyzed using YAP1 antibody (SIGMA, SAB4500094). Staining was performed. FIG. 5 shows the results. These results strongly suggested that YAP1 may be deeply involved in the development and progression of tongue cancer even in humans.
九州がんセンターで外科的治療した舌癌標本（ホルマリン固定後にパラフィン包埋したもの）に対して、ＹＡＰ１抗体（SIGMA, SAB4500094）を用いて、免疫組織学的染色を行った。結果を図５に示す。当該結果から、ヒトにおいても、舌癌の発症および進展にＹＡＰ１が深く関与している可能性が強く示唆された。 YAP1 staining in human tongue cancer clinical specimens Tongue cancer specimens surgically treated at Kyushu Cancer Center (formalin-fixed and paraffin-embedded) were immunohistochemically analyzed using YAP1 antibody (SIGMA, SAB4500094). Staining was performed. FIG. 5 shows the results. These results strongly suggested that YAP1 may be deeply involved in the development and progression of tongue cancer even in humans.
既存のＹＡＰ１阻害剤（ダサチニブ）を用いたｃｈｅｍｏｐｒｅｖｅｎｔｉｏｎ ａｓｓａｙ（化学予防アッセイ）
Ｍｏｂ１ａ／ｂコンディショナルＤＫＯマウスの舌にタモキシフェンを塗布開始するより３日前に、ダサチニブを５mg/kgの濃度で腹腔内連日投与を開始した。その後、生後３週齢となった時からタモキシフェンを３日間舌に塗布し、更に当該塗布開始から２週間ダサチニブを毎日腹腔内投与した。ダサチニブはマウス１匹あたり５mg/kg/回の濃度で合計１７日間連続投与されたこととなる。 Chemoprevention assay (chemoprevention assay) using existing YAP1 inhibitor (dasatinib)
Three days before starting to apply tamoxifen to the tongue of Mob1a / b conditional DKO mice, daily intraperitoneal administration of dasatinib at a concentration of 5 mg / kg was started. Thereafter, from the age of 3 weeks after birth, tamoxifen was applied to the tongue for 3 days, and dasatinib was intraperitoneally administered daily for 2 weeks from the start of the application. Dasatinib was administered at a concentration of 5 mg / kg / mouse / mouse for a total of 17 consecutive days.
Ｍｏｂ１ａ／ｂコンディショナルＤＫＯマウスの舌にタモキシフェンを塗布開始するより３日前に、ダサチニブを５mg/kgの濃度で腹腔内連日投与を開始した。その後、生後３週齢となった時からタモキシフェンを３日間舌に塗布し、更に当該塗布開始から２週間ダサチニブを毎日腹腔内投与した。ダサチニブはマウス１匹あたり５mg/kg/回の濃度で合計１７日間連続投与されたこととなる。 Chemoprevention assay (chemoprevention assay) using existing YAP1 inhibitor (dasatinib)
Three days before starting to apply tamoxifen to the tongue of Mob1a / b conditional DKO mice, daily intraperitoneal administration of dasatinib at a concentration of 5 mg / kg was started. Thereafter, from the age of 3 weeks after birth, tamoxifen was applied to the tongue for 3 days, and dasatinib was intraperitoneally administered daily for 2 weeks from the start of the application. Dasatinib was administered at a concentration of 5 mg / kg / mouse / mouse for a total of 17 consecutive days.
なお、ダサチニブはＹＡＰ１阻害剤として知られている。
ダ In addition, dasatinib is known as a YAP1 inhibitor.
タモキシフェン投与開始から２週間後に舌の形態観察及び舌組織のＨＥ染色観察を行った。結果を図６に示す。当該結果からＹＡＰ１阻害剤（ダサチニブ）により、舌癌誘導は起こらないことがわかった。
Two weeks after the start of the tamoxifen administration, the morphology of the tongue and the HE staining of the tongue tissue were observed. FIG. 6 shows the results. From the results, it was found that the tongue cancer induction was not caused by the YAP1 inhibitor (dasatinib).
Claims (10)
- 全身において、Ｍｏｂ１ａ遺伝子及びＭｏｂ１ｂ遺伝子のうち、一方はノックアウトされており、もう一方は任意にノックアウトできるシステム下にある、非ヒト哺乳動物。 A non-human mammal in which one of the Mob1a gene and the Mob1b gene is knocked out and the other is under a system that can be arbitrarily knocked out.
- 前記任意にノックアウトできるシステムが、Ｃｒｅ－ＬｏｘＰシステムである、請求項１に記載の非ヒト哺乳動物。 The non-human mammal according to claim 1, wherein the optionally knockout system is the Cre-LoxP system.
- 前記Ｃｒｅ－ＬｏｘＰシステムが、タモキシフェン依存的にはたらくＣｒｅ－ＬｏｘＰシステムである、請求項２に記載の非ヒト哺乳動物。 3. The non-human mammal according to claim 2, wherein the Cre-LoxP system is a tamoxifen-dependent Cre-LoxP system.
- 請求項１～３のいずれかに記載の非ヒト哺乳動物において、前記任意にノックアウトできるシステムにより、Ｍｏｂ１ａ遺伝子又はＭｏｂ１ｂ遺伝子をノックアウトする工程を含む、癌モデル非ヒト哺乳動物の製造方法。 4. A method for producing a cancer model non-human mammal, comprising a step of knocking out a Mob1a gene or a Mob1b gene using the system capable of arbitrarily knocking out the non-human mammal according to any one of claims 1 to 3.
- 請求項３に記載の非ヒト哺乳動物に、タモキシフェンを投与する工程を含む、癌モデル非ヒト哺乳動物の製造方法。 A method for producing a cancer model non-human mammal, comprising a step of administering tamoxifen to the non-human mammal according to claim 3.
- 請求項３に記載の非ヒト哺乳動物の頭頸部に、タモキシフェンを投与する工程を含む、頭頸部癌モデル非ヒト哺乳動物の製造方法。 A method for producing a head and neck cancer model non-human mammal, comprising a step of administering tamoxifen to the head and neck of the non-human mammal according to claim 3.
- 請求項３に記載の非ヒト哺乳動物の舌に、タモキシフェンを塗布する工程を含む、舌癌モデル非ヒト哺乳動物の製造方法。 A method for producing a tongue cancer model non-human mammal, comprising a step of applying tamoxifen to the tongue of the non-human mammal according to claim 3.
- （Ａ）請求項４～７のいずれかに記載の方法で製造された癌モデル非ヒト哺乳動物に被験物質を適用する工程、及び
（Ｂ）被験物質が適用された癌モデル非ヒト哺乳動物の癌の抑制若しくは改善の程度を測定する工程を含む、抗癌剤候補物質をスクリーニングする方法。 (A) a step of applying a test substance to the cancer model non-human mammal produced by the method according to any one of claims 4 to 7, and (B) a step of applying the test substance to the cancer model non-human mammal to which the test substance is applied. A method for screening a candidate anticancer agent, comprising a step of measuring the degree of suppression or improvement of cancer. - （Ａ’）全身において、Ｍｏｂ１ａ遺伝子及びＭｏｂ１ｂ遺伝子のうち、一方はノックアウトされており、もう一方は任意にノックアウトできるシステム下にある、非ヒト哺乳動物であって、前記任意にノックアウトできるシステムが、タモキシフェン依存的にはたらくＣｒｅ－ＬｏｘＰシステムである、非ヒト哺乳動物に対して、タモキシフェン及び被験物質を適用する工程、及び
（Ｂ’）被験物質が適用された当該非ヒト哺乳動物の癌の抑制の程度を測定する工程
を含む、抗癌剤候補物質をスクリーニングする方法。 (A ′) In the whole body, one of the Mob1a gene and the Mob1b gene is knocked out, and the other is a non-human mammal under a system that can be arbitrarily knocked out. A step of applying tamoxifen and a test substance to a non-human mammal, which is a Cre-LoxP system that acts in a tamoxifen-dependent manner, and (B ′) suppressing cancer in the non-human mammal to which the test substance has been applied. A method for screening a candidate anticancer drug, comprising a step of measuring the degree. - 頭頸部においてＭｏｂ１ａ遺伝子及びＭｏｂ１ｂ遺伝子がダブルノックアウトされた、頭頸部癌モデル非ヒト哺乳動物。 A head and neck cancer model non-human mammal in which the Mob1a gene and the Mob1b gene have been double knocked out in the head and neck.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2018130573 | 2018-07-10 | ||
| JP2018-130573 | 2018-07-10 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2020013113A1 true WO2020013113A1 (en) | 2020-01-16 |
Family
ID=69141490
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/JP2019/026950 WO2020013113A1 (en) | 2018-07-10 | 2019-07-08 | Non-human mammalian cancer model |
Country Status (1)
| Country | Link |
|---|---|
| WO (1) | WO2020013113A1 (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2016088919A (en) * | 2014-11-11 | 2016-05-23 | 国立研究開発法人産業技術総合研究所 | Anticancer agent comprising ivermectin or milbemycin d as active ingredient |
| JP2017214304A (en) * | 2016-05-30 | 2017-12-07 | 一般社団法人バイオ産業情報化コンソーシアム | Novel compound and method for producing the same, and pharmaceutical composition containing novel compound |
-
2019
- 2019-07-08 WO PCT/JP2019/026950 patent/WO2020013113A1/en active Application Filing
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2016088919A (en) * | 2014-11-11 | 2016-05-23 | 国立研究開発法人産業技術総合研究所 | Anticancer agent comprising ivermectin or milbemycin d as active ingredient |
| JP2017214304A (en) * | 2016-05-30 | 2017-12-07 | 一般社団法人バイオ産業情報化コンソーシアム | Novel compound and method for producing the same, and pharmaceutical composition containing novel compound |
Non-Patent Citations (4)
| Title |
|---|
| GOTO, HIRAKI ET AL.: "Loss of Mobla/b in mice results in chondrodysplasia due to YAP1/TAZ-TEAD- dependent repression of SOX9", DEVELOPMENT, vol. 145, no. 6, 16 March 2018 (2018-03-16), pages 1 - 11, XP055674419 * |
| NISHIO, MIKI ET AL.: "Cancer susceptibility and embryonic lethality in Mob la/1b double-mutant mice", THE JOURNAL OF CLINCAL INVESTIGATION, vol. 122, no. 12, 3 December 2012 (2012-12-03), pages 4505 - 4518, XP055674421 * |
| SUZUKI, SATOSHI: "Keratinocytes homeostasis by contact inhibition signaling and its disorders", ANNUAL REPORT OF COSMETOLOGY, vol. 22, 2014, pages 112 - 117 * |
| WEI, ZICHENG ET AL.: "Overexpression of Hippo pathway effector TAZ in tongue squamous cell carcinoma: correlation with clinicopathological features and patients' prognosis", JOURNAL OF ORAL PATHOLOGY & MEDICINE, vol. 42, no. 10, November 2013 (2013-11-01), pages 747 - 754 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Giesige et al. | AAV-mediated follistatin gene therapy improves functional outcomes in the TIC-DUX4 mouse model of FSHD | |
| Hall et al. | Conditional overexpression of TGF-β1 disrupts mouse salivary gland development and function | |
| Zhang et al. | Haploinsufficiency of Klippel-Trenaunay syndrome gene Aggf1 inhibits developmental and pathological angiogenesis by inactivating PI3K and AKT and disrupts vascular integrity by activating VE-cadherin | |
| Tsai et al. | CPEB4 knockout mice exhibit normal hippocampus-related synaptic plasticity and memory | |
| Zanotti et al. | Sustained Notch2 signaling in osteoblasts, but not in osteoclasts, is linked to osteopenia in a mouse model of Hajdu-Cheney syndrome | |
| Miwa et al. | Generation and characterization of PDGFR α‐GFPC re ER t2 knock‐In mouse line | |
| EP2914101B1 (en) | Animal model for type 2 diabetes and obesity | |
| Fujino et al. | c-MAF deletion in adult C57BL/6J mice induces cataract formation and abnormal differentiation of lens fiber cells | |
| WO2012176860A1 (en) | Method for screening substances having body-weight-regulating effect | |
| WO2016063202A1 (en) | The role of a cluster of long noncoding rna transcripts in protecting the heart from pathological hypertrophy | |
| JP5834297B2 (en) | Collagen production enhancer | |
| KR101894670B1 (en) | Method of screening agents for treating disorder related to dysregulation of hepatic lipid metabolism through inhibition of PPARγ-mediated transcription | |
| JP4613824B2 (en) | Transgenic non-human mammal | |
| WO2020013113A1 (en) | Non-human mammalian cancer model | |
| Takikawa et al. | Deletion of SIRT1 in myeloid cells impairs glucose metabolism with enhancing inflammatory response to adipose tissue hypoxia | |
| CN110691512B (en) | Genetically engineered non-human mammals, methods of construction and uses thereof | |
| JP5250810B2 (en) | Screening for substances that enhance utrophin gene expression | |
| KR102433456B1 (en) | Tph1 knockout mouse | |
| JP2020089332A (en) | Non-human genetically modified animal and its making method | |
| Gong et al. | Activating the expression of human K-rasG12D stimulates oncogenic transformation in transgenic goat fetal fibroblast cells | |
| JP2024002576A (en) | Basal cell-like breast cancer model non-human mammal | |
| JP2019170294A (en) | Parkinson's disease model non-human animal | |
| Gu et al. | Generation of p27icreER transgenic mice: A tool for inducible gene expression in supporting cells in the cochlea | |
| WO2022176917A1 (en) | Mesenchymal stem cells and adipocytes for preparing mitokine mixture, and therapeutic or prophylactic drug | |
| JP5669055B2 (en) | Double knockout non-human animals |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 19833580 Country of ref document: EP Kind code of ref document: A1 |
|
| NENP | Non-entry into the national phase |
Ref country code: DE |
|
| 122 | Ep: pct application non-entry in european phase |
Ref document number: 19833580 Country of ref document: EP Kind code of ref document: A1 |
|
| NENP | Non-entry into the national phase |
Ref country code: JP |