System for detecting extracellular purine receptor ligands and non-human animals incorporating same

Abstract

The purpose of the present invention is to provide an evaluation system capable of systematically detecting an extracellular purine receptor ligand over time with low invasiveness, and to provide a genetically modified non-human animal expressing a first fusion protein and a second fusion protein for detecting an extracellular purine receptor ligand, and a cell thereof. The first fusion protein contains a first reporter protein and a membrane protein that binds to a purine receptor ligand, and the second fusion protein contains a second reporter protein and a protein that binds to the membrane protein that binds to the ligand.

Description

[0001] Related applications

[0002] This application is a divisional application of the patent application with international application number PCT / JP2020 / 046301, international application date December 11, 2020, Chinese application number 202080086064.1, and invention name "System for detecting extracellular purine receptor ligands and non-human animals introduced with the system". Technical Field

[0003] The present invention relates to a system for detecting extracellular purinergic receptor ligands using signal transduction via receptor proteins with purinergic receptor ligands as ligands, and to genetically modified non-human animals incorporating such systems. Furthermore, the present invention relates to methods for monitoring disease pathology and evaluating compounds using genetically modified non-human animals incorporating such systems for detecting extracellular purinergic receptor ligands. Background Art

[0004] Purinergic receptors are a group of cell surface receptors that use nucleotides such as adenosine and ATP as ligands. Purinergic receptors are reported to be involved in various diseases, including immune disorders, and the development of purinergic receptor inhibitors and purinergic receptor agonists targeting purinergic receptors is ongoing. Known purinergic receptor ligands include ATP and adenosine, as well as nucleotides including their metabolites. Signaling pathways through these purinergic receptor ligands and purinergic receptors are reported to be involved in various physiological phenomena, including nerve conduction, muscle contraction, pain perception, taste, and inflammatory responses.

[0005] Purinergic receptor ligand headed by ATP and adenosine performs function as signal transduction substance in organism, therefore, attempts to produce in organism and verification and concentration determination of behaviors such as decomposition etc. As an example thereof, a large amount of ATP in known accompanying cell death cell leaks out to extracellular, and it is reported that it plays the role of important danger signal as inflammatory process. In addition, it is reported that a large amount of ATP in cell also leaks out to extracellular during cancer cell death in cancer tissue, and ATP concentration is higher than normal tissue in cancer tissue (non-patent literature 1,3,4).

[0006] As a method for measuring the intracellular substances of the extracellular purinergic receptor ligands in the organism, microdialysis is enumerated. This method uses HPLC and other analysis methods to analyze the tissue fluid or extracellular fluid recovered from the tissue. When the assay object is ATP, in addition to the analytical methods by HPLC and other methods, the method of measuring by luciferin-luciferase assay can also be utilized. However, even when the above method is applied, it is difficult to clearly distinguish whether the purinergic receptor ligands detected and measured are from the cell or from the extracellular. In addition, the purinergic receptor ligand concentration in the organism can not be measured in real time. Further, in microdialysis, the tissue is inserted into the needle to reclaim tissue fluid. At this time, cell death or tissue necrosis at the puncture site are sometimes produced. In such a case, there is a problem that the false impression caused by the assay technique affects the measurement result. When the assay substance is ATP, it is known that its accompanying cell death and the inflammatory reaction secretion of the cell are secreted, which is not suitable for measuring by this method. Patent Document 2 reports a method for observing ATP distribution and fluctuations in vivo using non-human mammals expressing a fusion protein composed of two fluorescent proteins that function as donors and acceptors in fluorescence resonance energy transfer (FRET) bound to the amino and carboxyl termini of the ε subunit of ATP synthase, respectively. However, this method focuses on intracellular ATP and cannot assess extracellular ATP.

[0007] In addition, as an attempt to measure extracellular ATP in organism, a method (non-patent literature 1, 2, patent documentation 1) applied to genetically modified cells expressing luciferase in extracellular space has been reported. In the aforementioned method, the aforementioned genetically modified cells send signals by luciferin-luciferase reaction when exposed to an environment with a high extracellular ATP concentration, and the extracellular ATP in the organism is measured by detecting the signal. However, in this method, it is necessary to move the cells into the organism, but it is difficult to distribute the cells moved into the organism everywhere in the whole body. In the case of not knowing when ATP is released outside the cell, there is a problem of being difficult to judge when the cells are moved into for good. In addition, there is also a problem of not being able to use the cells moved into when they have been excluded by the host immune system. Further, in the above-mentioned luciferin-luciferase reaction, it is only ATP that can be measured, and other purine receptor ligands cannot be measured.

[0008] Purinergic receptor ligands, not limited to ATP, have been reported to be involved in various diseases as extracellular signaling substances. However, much remains unknown regarding which diseases, at what timescale, and how each extracellular purinergic receptor ligand functions. Therefore, to evaluate the functions of extracellular purinergic receptor ligands in living organisms and pathological conditions, an evaluation system that can detect extracellular purinergic receptor ligands systemically and in a minimally invasive manner is needed. However, such an evaluation system does not yet exist.

[0009] Prior art literature

[0010] Patent Literature

[0011] Patent Document 1: WO2006 / 126231

[0012] Patent Document 2: WO2015 / 108102

[0013] Non-patent literature

[0014] Non-patent document 1: Patrizia et al. (2008) PLoS One.3, e2599

[0015] Non-patent document 2: Francesco Di Virgilio et al. (2016) Methods Mol Biol. 1417, 115-29

[0016] Non-patent document 3: Idzko M et al. (2007) Nat Med. Aug; 13(8): 913-9

[0017] Non-patent document 4: Lommatzsch M et al. (2010) Am J Respir Crit Care Med. May 1; 181(9): 928-34 SUMMARY OF THE INVENTION

[0018] Problems to be solved by the invention

[0019] The present invention was developed in light of the aforementioned circumstances, and its purpose is to provide an evaluation system capable of low-invasive, systemic, and long-term detection of extracellular purinergic receptor ligands. More specifically, the present invention provides a genetically modified non-human animal that systemically expresses a reporter protein for detecting and evaluating extracellular purinergic receptor ligands, and also to construct an evaluation system for detecting extracellular purinergic receptor ligands. Furthermore, the present invention provides methods for detecting various diseases and monitoring the pathological conditions of various diseases, as well as screening for therapeutic agents for these diseases, using the evaluation system for detecting extracellular purinergic receptor ligands, wherein the evaluation system utilizes the aforementioned genetically modified non-human animal and the reporter protein.

[0020] Means used to solve problems

[0021] To address the above-mentioned issues, the present inventors conducted extensive research and discovered that by fusing the subunits of split luciferase to the P2Y receptor (a purinergic receptor) and the intracellular β-arrestin protein (β-Arestin), respectively, and creating genetically modified mice expressing these subunits systemically, extracellular ATP can be detected. Specifically, according to this genetic modification, when extracellular ATP binds to the P2Y receptor, the P2Y receptor and β-arrestin bind to each other within the cell, and the fused subunits appropriately converge, reconstructing and producing luciferase. Luciferase can express a luminescent signal in the presence of an appropriate substrate, and by detecting this signal, extracellular ATP can be detected. Furthermore, when ATP is measured using cells isolated from these genetically modified mice, the intensity of the detected luminescent signal can be confirmed to quantitatively determine ATP concentration in a concentration-dependent manner. Thus, the present invention can also be used for in vitro ATP concentration measurement and screening. Furthermore, when the detection of extracellular ATP in the living body of the present gene-modified mouse was studied, it was found that the luminescent signal could be detected in a concentration-dependent manner, and that extracellular ATP in the whole body could be non-invasively detected over time.

[0022] The present invention has been accomplished based on such knowledge, and relates to the following inventions in specific aspects, for example.

[0023] [1] A genetically modified non-human animal expressing a first fusion protein and a second fusion protein for detecting a purinergic receptor ligand present outside a cell, wherein:

[0024] The first fusion protein comprises a membrane protein that binds to a purinergic receptor ligand and a first reporter protein,

[0025] The second fusion protein comprises a protein that binds to the membrane protein and binds to the ligand, and a second reporter protein.

[0026] [2][1] The genetically modified non-human animal of [2], wherein the first reporter protein and the second reporter protein are subunits of the Split reporter protein.

[0027] [3][2] The genetically modified non-human animal, wherein the aforementioned Split reporter protein is Split luciferase.

[0028] [4][2] The genetically modified non-human animal of [4][2], wherein the aforementioned Split reporter protein is Split fluorescent protein.

[0029] [5][1] The genetically modified non-human animal of [5][1], wherein the first reporter protein and the second reporter protein are a combination of proteins that produce fluorescence resonance energy transfer (FRET) or bioluminescence resonance energy transfer (BRET).

[0030] [6] The genetically modified non-human animal according to any one of [1] to [5], wherein the protein binding to the membrane protein that binds to the ligand is an inhibitory protein or a portion thereof.

[0031] [7] The genetically modified non-human animal according to any one of [1] to [6], wherein the membrane protein is a G protein-coupled receptor (GPCR) or a portion thereof.

[0032] [8][7] The genetically modified non-human animal of [8][7], wherein the aforementioned GPCR is a P1 receptor.

[0033] [9][8] The genetically modified non-human animal of [9], wherein the aforementioned P1 receptor is selected from adenosine A1 receptor, adenosine A2A receptor, adenosine A2B receptor, and adenosine A3 receptor.

[0034]

[10] The genetically modified non-human animal of [8] or [9], wherein the aforementioned purinergic receptor ligand is a P1 receptor ligand.

[0035]

[11]

[10] The genetically modified non-human animal of

[11] , wherein the aforementioned P1 receptor ligand is selected from adenosine, AMP, ADP, and ATP.

[0036]

[12] [7] The genetically modified non-human animal, wherein the aforementioned GPCR is a P2 receptor.

[0037]

[13]

[12] The genetically modified non-human animal, wherein the aforementioned P2 receptor is a P2Y receptor.

[0038]

[14]

[13] The genetically modified non-human animal, wherein the aforementioned P2Y receptor is selected from P2Y1, P2Y2, P2Y4B, P2Y6, P2Y11, P2Y12, P2Y13, and P2Y14.

[0039]

[15] The genetically modified non-human animal of any one of

[12] to

[14] , wherein the purinergic receptor ligand is a P2 receptor ligand.

[0040]

[16]

[15] The genetically modified non-human animal of

[16] , wherein the aforementioned P2 receptor ligand is a molecule having a nucleotide backbone.

[0041]

[17] The genetically modified non-human animal of

[15] or

[16] , wherein the aforementioned P2 receptor ligand is selected from AMP, ADP, ATP, UTP, UDP, and UDP-glucose.

[0042]

[18] The genetically modified non-human animal of any one of

[15] to

[17] , wherein the P2 receptor ligand is ATP.

[0043]

[19] The genetically modified non-human animal according to any one of [1] to

[18] , wherein the first fusion protein and the second fusion protein are expressed throughout the body.

[0044]

[20] The genetically modified non-human animal of any one of [1] to

[19] , wherein the non-human animal is a non-human mammal.

[0045]

[21]

[20] A genetically modified non-human animal, wherein the non-human animal is a rodent.

[0046]

[22] The genetically modified non-human animal of

[20] or

[21] , wherein the non-human animal is a mouse.

[0047]

[23] The genetically modified non-human animal according to any one of [1] to

[22] , which is a disease model animal.

[0048]

[24]

[23] The genetically modified non-human animal, wherein the disease is selected from cancer, acute inflammation, chronic inflammation, infectious disease, fibrosis, physical or chemical organ damage, and cell damage caused by drugs such as anticancer agents.

[0049]

[25] Animal cells expressing a first fusion protein and a second fusion protein for detecting a purinergic receptor ligand present outside the cell, wherein:

[0050] The first fusion protein comprises a membrane protein that binds to a purinergic receptor ligand and a first reporter protein,

[0051] The second fusion protein comprises a protein that binds to the membrane protein and binds to the ligand, and a second reporter protein.

[0052]

[26]

[25] The animal cell, wherein the first reporter protein and the second reporter protein are subunits of the Split reporter protein.

[0053]

[27]

[26] The animal cell, wherein the aforementioned Split reporter protein is Split luciferase.

[0054]

[28]

[26] The animal cell, wherein the aforementioned Split reporter protein is Split fluorescent protein.

[0055]

[29]

[25] The animal cell of claim 1, wherein the first reporter protein and the second reporter protein are a combination of proteins that produce fluorescence resonance energy transfer (FRET) or bioluminescence resonance energy transfer (BRET).

[0056]

[30] The animal cell according to any one of

[25] to

[29] , wherein the protein bound to the membrane protein that binds to the ligand is an inhibitory protein or a portion thereof.

[0057]

[31] The animal cell of any one of

[25] to

[30] , wherein the membrane protein is a G protein-coupled receptor (GPCR) or a portion thereof.

[0058]

[32] The animal cell according to any one of

[25] to

[31] , wherein the GPCR is a P1 receptor.

[0059]

[33]

[32] The animal cell of claim 32, wherein the P1 receptor is selected from adenosine A1 receptor, adenosine A2A receptor, adenosine A2B receptor, and adenosine A3 receptor.

[0060]

[34] The animal cell of

[32] or

[33] , wherein the aforementioned purine receptor ligand is a P1 receptor ligand.

[0061]

[35]

[34] The animal cell, wherein the P1 receptor ligand is selected from adenosine, AMP, ADP, and ATP.

[0062]

[36] The animal cell of any one of

[25] to

[31] , wherein the GPCR is a P2 receptor.

[0063]

[37]

[36] The animal cell, wherein the aforementioned P2 receptor is a P2Y receptor.

[0064]

[38]

[37] The animal cell of claim 1, wherein the P2Y receptor is selected from the group consisting of P2Y1, P2Y2, P2Y4B, P2Y6, P2Y11, P2Y12, P2Y13, and P2Y14.

[0065]

[39] The animal cell of

[37] or

[38] , wherein the aforementioned purine receptor ligand is a P2 receptor ligand.

[0066]

[40]

[39] The animal cell, wherein the aforementioned P2 receptor ligand is a molecule having a nucleotide backbone.

[0067]

[41]

[39] or

[40] , wherein the P2 receptor ligand is selected from AMP, ADP, ATP, UTP, UDP, and UDP-glucose.

[0068]

[42] The animal cell of any one of

[39] to

[41] , wherein the P2 receptor ligand is ATP.

[0069]

[43] The animal cell of any one of

[25] to

[42] , wherein the animal is a mammal.

[0070]

[44] The animal cell of any one of

[25] to

[43] , wherein the animal is a rodent.

[0071]

[45] The animal cell of any one of

[25] to

[44] , wherein the animal is a mouse.

[0072]

[46] A detection kit for detecting a purinergic receptor ligand present outside a cell, comprising the animal cell according to any one of

[25] to

[45] .

[0073]

[47] A method for preparing genetically modified animal cells for detecting extracellular purine receptor ligands, comprising:

[0074] a step of introducing into the genome a gene encoding a first fusion protein comprising a membrane protein that binds to a purinergic receptor ligand present outside the cell and a first reporter protein, and

[0075] A step of introducing into the genome a gene encoding a second fusion protein comprising a protein that binds to the membrane protein and binds to the ligand and a second reporter protein.

[0076]

[48]

[47] The preparation method, wherein the first reporter protein and the second reporter protein are subunits of the Split reporter protein.

[0077]

[49]

[48] The preparation method, wherein the aforementioned Split reporter protein is Split luciferase.

[0078]

[50]

[48] The preparation method, wherein the aforementioned Split reporter protein is Split fluorescent protein.

[0079]

[51]

[47] The preparation method of

[51]

[47] , wherein the first reporter protein and the second reporter protein are a combination of proteins that produce fluorescence resonance energy transfer (FRET) or bioluminescence resonance energy transfer (BRET).

[0080]

[52] The preparation method according to any one of

[47] to

[51] , wherein the protein bound to the membrane protein that binds to the ligand is an inhibitory protein or a portion thereof.

[0081]

[53] The preparation method according to any one of

[47] to

[52] , wherein the membrane protein is a G protein-coupled receptor (GPCR) or a portion thereof.

[0082]

[54] The preparation method of

[53] , wherein the aforementioned GPCR is a P1 receptor.

[0083]

[55]

[54] The preparation method, wherein the aforementioned P1 receptor is selected from adenosine A1 receptor, adenosine A2A receptor, adenosine A2B receptor, and adenosine A3 receptor.

[0084]

[56] The preparation method of

[54] or

[55] , wherein the aforementioned purine receptor ligand is a P1 receptor ligand.

[0085]

[57]

[56] The preparation method, wherein the aforementioned P1 receptor ligand is selected from adenosine, AMP, ADP, and ATP.

[0086]

[58]

[53] The preparation method, wherein the aforementioned GPCR is a P2 receptor.

[0087]

[59]

[58] The preparation method, wherein the aforementioned P2 receptor is a P2Y receptor.

[0088]

[60]

[59] The preparation method, wherein the aforementioned P2Y receptor is selected from P2Y1, P2Y2, P2Y4B, P2Y6, P2Y11, P2Y12, P2Y13, and P2Y14.

[0089]

[61] The preparation method of any one of

[58] to

[60] , wherein the aforementioned purine receptor ligand is a P2 receptor ligand.

[0090]

[62]

[61] The preparation method, wherein the aforementioned P2 receptor ligand is a molecule having a nucleotide backbone.

[0091]

[63]

[61] or

[62] , wherein the aforementioned P2 receptor ligand is selected from AMP, ADP, ATP, UTP, UDP, and UDP glucose.

[0092]

[64] The preparation method of any one of

[61] to

[63] , wherein the aforementioned P2 receptor ligand is ATP.

[0093]

[65] The preparation method of any one of

[47] to

[64] , wherein the first fusion protein and the second fusion protein are expressed throughout the body.

[0094]

[66] The preparation method of any one of

[47] to

[65] , wherein the animal cell is a mammalian cell.

[0095]

[67] The preparation method of any one of

[47] to

[66] , wherein the animal cell is a rodent cell.

[0096]

[68] The preparation method of any one of

[47] to

[67] , wherein the aforementioned animal cell is a mouse cell.

[0097]

[69] The preparation method of any one of

[47] to

[68] , which comprises detecting the reporter protein and screening based on the detected amount thereof.

[0098]

[70] A method for preparing a genetically modified non-human animal for detecting extracellular purine receptor ligands, comprising:

[0099] a step of introducing into the genome a gene encoding a first fusion protein comprising a membrane protein that binds to a purinergic receptor ligand present outside the cell and a first reporter protein, and

[0100] A step of introducing into the genome a gene encoding a second fusion protein comprising a protein that binds to the membrane protein and binds to the ligand and a second reporter protein.

[0101]

[71]

[70] The preparation method, wherein the first reporter protein and the second reporter protein are subunits of the Split reporter protein.

[0102]

[72]

[71] The preparation method, wherein the aforementioned Split reporter protein is Split luciferase.

[0103]

[73]

[71] The preparation method, wherein the aforementioned Split reporter protein is Split fluorescent protein.

[0104]

[74]

[70] The preparation method of

[74]

[70] , wherein the first reporter protein and the second reporter protein are a combination of proteins that produce fluorescence resonance energy transfer (FRET) or bioluminescence resonance energy transfer (BRET).

[0105]

[75] The preparation method according to any one of

[70] to

[74] , wherein the protein bound to the membrane protein that binds to the ligand is an inhibitory protein or a portion thereof.

[0106]

[76] The preparation method of any one of

[70] to

[75] , wherein the membrane protein is a G protein-coupled receptor (GPCR) or a portion thereof.

[0107]

[77]

[76] The preparation method, wherein the aforementioned GPCR is a P1 receptor.

[0108]

[78]

[77] The preparation method, wherein the aforementioned P1 receptor is selected from adenosine A1 receptor, adenosine A2A receptor, adenosine A2B receptor, and adenosine A3 receptor.

[0109]

[79] The preparation method of

[77] or

[78] , wherein the aforementioned purine receptor ligand is a P1 receptor ligand.

[0110]

[80] The preparation method of any one of

[77] to

[79] , wherein the P1 receptor ligand is selected from adenosine, AMP, ADP, and ATP.

[0111]

[81]

[76] The preparation method, wherein the aforementioned GPCR is a P2 receptor.

[0112]

[82] The preparation method of

[81] , wherein the aforementioned P2 receptor is a P2Y receptor.

[0113]

[83]

[82] The preparation method, wherein the aforementioned P2Y receptor is selected from P2Y1, P2Y2, P2Y4B, P2Y6, P2Y11, P2Y12, P2Y13, and P2Y14.

[0114]

[84] The preparation method of any one of

[81] to

[83] , wherein the aforementioned purine receptor ligand is a P2 receptor ligand.

[0115]

[85]

[84] The preparation method, wherein the aforementioned P2 receptor ligand is a molecule having a nucleotide backbone.

[0116] The preparation method of

[86]

[84] or

[85] , wherein the aforementioned P2 receptor ligand is selected from AMP, ADP, ATP, UTP, UDP, and UDP glucose.

[0117]

[87] The preparation method of any one of

[84] to

[86] , wherein the aforementioned P2 receptor ligand is ATP.

[0118]

[88] The preparation method of any one of

[70] to

[87] , wherein the first fusion protein and the second fusion protein are expressed throughout the body.

[0119]

[89] The preparation method of any one of

[70] to

[88] , wherein the non-human animal is a non-human mammal.

[0120]

[90] The preparation method of any one of

[70] to

[89] , wherein the non-human animal is a rodent.

[0121]

[91] The preparation method of any one of

[70] to

[90] , wherein the non-human animal is a mouse.

[0122]

[92] The preparation method of any one of

[70] to

[91] , which comprises detecting a reporter protein and screening based on the detected amount thereof.

[0123]

[93] A method for detecting the site of disease onset, comprising the step of detecting the reporter protein of

[23] or

[24] in a genetically modified non-human animal.

[0124]

[94] A method for detecting the onset stage of a disease, comprising the step of detecting a reporter protein in a genetically modified non-human animal of

[23] or

[24] .

[0125]

[95] A method for monitoring the progression of a disease, comprising the step of detecting a reporter protein in a genetically modified non-human animal of

[23] or

[24] .

[0126]

[96] A method for evaluating the efficacy of a disease preventive drug or a disease therapeutic drug, comprising the steps of administering the aforementioned preventive drug or disease therapeutic drug to a genetically modified non-human animal of

[23] or

[24] , and evaluating the efficacy of the aforementioned preventive drug or disease therapeutic drug based on the change in the detection amount of the reporter protein before and after administration.

[0127]

[97] A method for evaluating the toxicity of a disease preventive drug or a disease therapeutic drug, comprising the steps of administering the aforementioned preventive drug or disease therapeutic drug to a genetically modified non-human animal of

[23] or

[24] , and evaluating the toxicity of the aforementioned preventive drug or disease therapeutic drug based on the change in the detection amount of the reporter protein before and after administration.

[0128]

[98] A method for screening a preventive or therapeutic drug for a disease, comprising the steps of administering a test substance to a genetically modified non-human animal of

[23] or

[24] , and screening a substance effective for preventing or treating the disease based on a change in the amount of a reporter protein detected before and after administration.

[0129]

[99] A method for evaluating the effect of a drug molecule using a purine receptor ligand as an indicator, comprising the steps of administering the aforementioned drug molecule to any of the genetically modified non-human animals of [1] to

[24] or adding the aforementioned drug molecule to cells of any of the genetically modified animals of

[25] to

[45] , and evaluating the effect of the aforementioned drug molecule based on the change in the detection amount of the reporter protein before and after administration or addition.

[0130]

[100] A method for evaluating the toxicity of a drug molecule using a purine receptor ligand as an indicator, comprising the steps of administering the drug molecule to any of the genetically modified non-human animals of [1] to

[24] or adding the drug molecule to cells of any of the genetically modified animals of

[25] to

[45] , and evaluating the toxicity of the drug molecule based on the change in the detected amount of the reporter protein before and after administration or addition.

[0131]

[101] A method for evaluating the effect of a purine receptor ligand-dependent drug molecule, comprising the steps of administering the aforementioned agent to any of the genetically modified non-human animals of [1] to

[24] or adding the aforementioned drug molecule to cells of any of the genetically modified animals of

[25] to

[45] , and evaluating the effect of the aforementioned drug molecule in the presence of a purine receptor ligand represented by the detected amount of a reporter protein.

[0132]

[102] A method for screening purine receptor ligand-dependent drug molecules, comprising administering a test substance to any of the genetically modified non-human animals of [[1] to

[24] , or adding the aforementioned drug molecule to cells of any of the genetically modified animals of

[25] to

[45] , and screening the target substance based on the effect of the aforementioned drug molecule in the presence of a purine receptor ligand represented by the detected amount of a reporter protein.

[0133] Effects of the Invention

[0134] According to the present invention, an evaluation system capable of detecting extracellular purinergic receptor ligands systemically over time and with minimal invasiveness can be provided.

[0135] The present invention can provide genetically modified non-human animals that can non-invasively and temporally detect purinergic receptor ligands throughout the body. Utilizing the genetically modified non-human animals of the present invention, it is possible to determine the site of onset of diseases involving purinergic receptor ligands and study the onset period of the disease. Furthermore, these mice can also be used in drug screening, thereby effectively enabling the development of therapeutic agents for diseases involving purinergic receptor ligands. BRIEF DESCRIPTION OF THE DRAWINGS

[0136] [ Figure 1 ] Figure 1 Shows the summary of the P2Y11-split Luc (C-terminus) knock-in vector.

[0137] [ Figure 2 ] Figure 2 An overview of the Arrestin-split Luc (N-terminal) knock-in vector is shown.

[0138] [ Figure 3 ] Figure 3 This photograph shows the results of subcutaneous administration of a mixture of ATP and D-luciferin substrate solution to P2Y11-split Luc (C-terminus) (P2Y) and Arrestin-split Luc (N-terminus) (Arrb) double knock-in mice, followed by measurement of luminescence signal intensity. P2Y: P2Y11-split Luc (C-terminus) knock-in, Arrb: Arrestin-split Luc (N-terminus) knock-in, wt: wild-type.

[0139] [ Figure 4 ] Figure 4 Shown are analysis data obtained by treating fibroblasts from P2Y11-split Luc (C-terminus) and Arrestin-split Luc (N-terminus) double knock-in mice with various concentrations of ATP and D-luciferin substrate, and visualizing the detected luminescent signals.

[0140] [ Figure 5 ] Figure 5 This graph shows the intensity of luminescent signals detected when fibroblasts from P2Y11-split Luc (C-terminal) and Arrestin-split Luc (N-terminal) double knock-in mice were treated with various concentrations of ATP and D-luciferin substrate.

[0141] [ Figure 6 ] Figure 6Photographic images showing the results of subcutaneous administration of a mixture of ATP and a D-luciferin substrate solution at various concentrations to P2Y11-split Luc (C terminus) (P2Y) and Arrestin-split Luc (N terminus) (Arrb) double knock-in mice and measurement of luminescent signal intensity.

[0142] [ Figure 7 ] Figure 7 This graph shows the luminescence signal intensity after subcutaneous administration of a mixture of ATP and D-luciferin substrate solution at various concentrations to P2Y11-split Luc (C terminus) (P2Y) and Arrestin-split Luc (N terminus) (Arrb) double knock-in mice.

[0143] [ Figure 8 ] Figure 8 The photographs show the results of non-invasive visualization of ATP leakage in the liver accompanied by hepatocellular injury induced by hydrodynamic injection in P2Y11-split Luc (C terminus) (P2Y) and Arrestin-split Luc (N terminus) (Arrb) double knock-in mice by measuring the intensity of luminescent signals.

[0144] [ Figure 9 ] Figure 9 The images are photographs showing the results of (A) non-invasive visualization of ATP leakage accompanying liver canceration caused by hydrodynamic injection in P2Y11-split Luc (C terminus) (P2Y) and Arrestin-split Luc (N terminus) (Arrb) double knock-in mice, and (B) visualization of the results by measuring the luminescence signal intensity in the removed liver.

[0145] Modes for carrying out the invention

[0146] 1. Genetically modified non-human animals

[0147] The present invention relates to a genetically modified non-human animal expressing a first fusion protein and a second fusion protein for detecting a purinergic receptor ligand present outside a cell.

[0148] (1-1) First fusion protein

[0149] The "first fusion protein" in the present invention comprises a membrane protein that binds to a purinergic receptor ligand and a first reporter protein, or consists of these proteins.

[0150] In the first fusion protein, the membrane protein bound to the purinergic receptor ligand and the first reporter protein can be directly connected, or they can be connected via a linker. The membrane protein bound to the purinergic receptor ligand and the first reporter protein can be connected in any order, as long as the first fusion protein and the second fusion protein can interact as described below. In one embodiment, the membrane protein bound to the purinergic receptor ligand and the first reporter protein can be connected in this order from the N-terminus. The "linker" can utilize any conventionally known linker, for example, a peptide linker can be utilized. The number and type of amino acids in the peptide linker are not particularly limited.

[0151] (1-1-1) Membrane proteins that bind to purine receptor ligands

[0152] In the present invention, "membrane protein that binds to a purinergic receptor ligand" (hereinafter sometimes simply referred to as "membrane protein") means a protein that is bound to or exists through the cell membrane, that is, a protein that binds to a purinergic receptor ligand that exists outside the cell. The membrane protein in the present invention is not particularly limited as long as it can bind to a purinergic receptor ligand that exists outside the cell. G protein-coupled receptors (G protein-coupled receptor, hereinafter referred to as "GPCR") and ligand-dependent ion channel receptors, etc., or parts thereof, which are purinergic receptors, can be preferably used. Preferably, the membrane protein is a GPCR that is a purinergic receptor.

[0153] GPCRs are primarily seven-transmembrane receptors, with seven α-helices extending through the plasma membrane, an N-terminal region located outside the cell, and a C-terminal region located inside the cell. Ligand binding to the extracellular region activates the GPCR, causing structural changes. The intracellular region is then phosphorylated by G protein-coupled receptor kinases.

[0154] Examples of GPCRs that are purinergic receptors include, but are not limited to, P1 receptors and P2 receptors. Preferably, the membrane protein is a P1 receptor or a P2 receptor.

[0155] Purinergic receptors, P1 receptors, are GPCRs that use adenosine, AMP, ADP, and ATP as ligands and can be classified into A1 receptors, A2A receptors, A2B receptors, and A3 receptors. In the present invention, one or more receptors selected from these can be used as membrane proteins.

[0156] Purinergic receptors are GPCRs that use AMP, ADP, ATP, UTP, UDP, or UDP-glucose as ligands, preferably P2Y receptors. P2Y receptors can be classified into P2Y1 receptors, P2Y2 receptors, P2Y4B receptors, P2Y6 receptors, and P2Y 11 receptors, P2Y 12 receptors, P2Y 13 receptors, and P2Y 14In the present invention, one or more receptors selected from these can be used as membrane proteins. More preferably, P2Y2 receptors with high ATP specificity, P2Y 11 receptors, particularly preferably P2Y 11 receptor.

[0157] In the present invention, the membrane protein that binds to the purinergic receptor ligand can be a known one, and a known one registered in a public database such as NCBI or GenBank can be used. For example, for the A1 receptor belonging to the P1 receptor family, the one from human is registered as NP_000665.1, and the one from mouse is registered as NP_001008533.1. For the P2Y2 receptor, the one from human is registered as NP_002555.3, and the one from mouse is registered as NP_001289275.1. 11 The receptor from human is registered as NP_002557.2. The membrane protein that binds to the purinergic receptor ligand is preferably derived from an animal of the same species as the host. In the present invention, for example, P2Y represented by the amino acid sequence of SEQ ID NO: 9 can be 11 The receptors are utilized as membrane proteins that bind to purinergic receptor ligands.

[0158] In the present invention, as a "membrane protein that binds to a purinergic receptor ligand," analogs and mutants thereof can also be used as long as they can bind to a purinergic receptor ligand present outside the cell. Analogs and mutants of "membrane protein that binds to a purinergic receptor ligand" mean (i) a protein consisting of an amino acid sequence in which 1 to 50, for example, 1 to 20, or 1 to 10 amino acids are deleted, substituted, added, or inserted in the amino acid sequence of the "membrane protein that binds to a purinergic receptor ligand," and can bind to a purinergic receptor ligand present outside the cell, or (ii) a protein consisting of an amino acid sequence having a sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 99% or more to the amino acid sequence of the "membrane protein that binds to a purinergic receptor ligand," and can bind to a purinergic receptor ligand present outside the cell. The sequence identity of the amino acid sequence can be calculated based on a known method, for example, by applying BLAST (Basic Local Alignment Search Tool at the National Center for Biological Information), for example, with default settings. The sequence identity of the amino acid sequence can be calculated based on all proteins that can bind to purinergic receptor ligands, or it can be calculated based on binding domains that have binding activity to purinergic receptor ligands. For example, when the sequence homology of the binding domain in a receptor that binds to a specific purinergic receptor ligand is high, since the sequence homology of other than the binding domain, such as the transmembrane domain, is low, even when the homology as a whole membrane protein is not high, the sequence homology of the specific binding domain is understood to be high.

[0159] In addition, in the present invention, the terms "a portion thereof" and "a portion thereof" used in connection with membrane proteins mean a protein consisting of a portion of the amino acid sequence of a membrane protein and capable of binding to at least a purinergic receptor ligand present outside the cell. Examples of such proteins include proteins consisting of a portion of a domain of the amino acid sequence of a membrane protein that is essential for binding to at least a purinergic receptor ligand present outside the cell (e.g., a binding domain in a receptor that binds to a specific purinergic receptor ligand) and an essential domain for binding to the cell membrane (e.g., a transmembrane domain). The various domains of a membrane protein can be determined based on amino acid sequence information and gene information registered in public databases such as NCBI and GenBank.

[0160] (1-1-2) First reporter protein

[0161] In the present invention, the "first reporter protein" refers to a protein that functions as a reporter protein together with the second reporter protein in the second fusion protein described later. "Functioning together as a reporter protein" means that the first reporter protein and the second reporter protein only function as reporter proteins after they come into proximity or bind to each other, becoming an indicator showing the expression and localization of a specific molecule. Examples of proteins that "function together as reporter proteins" include split reporter proteins and combinations of proteins that produce fluorescence resonance energy transfer (FRET) or bioluminescence resonance energy transfer (BRET).

[0162] A "split reporter protein" is a reporter protein that is split into two or more subunits. When each subunit exists separately, it does not function as a reporter protein. However, when the two or more split subunits are brought into proximity or combined, the reporter protein can be reconstructed and function can be displayed. A "split-subunit reporter protein" can be split into each subunit if the protein originally consists of two or more subunits. Even a reporter protein originally composed of a single protein can be artificially split into multiple subunits for use. Examples of reporter proteins include, but are not limited to, green, red, blue, or yellow fluorescent proteins and enzymes such as luciferase, β-galactosidase, alkaline phosphatase, horseradish peroxidase, β-glucuronidase, chloramphenicol acetyltransferase, invertase, dihydrofolate reductase, and β-lactamase. A specific example of a split reporter protein is split luciferase. Another specific example includes, but are not limited to, split fluorescent proteins such as split GFP, split YFP, and split CYP. Furthermore, while already reported split reporter proteins can be used, artificial splitting and application of specific reporter proteins is also possible.

[0163] "FRET" refers to the phenomenon in which, when two fluorescent proteins (a first fluorescent protein (donor) and a second fluorescent protein (acceptor) have overlapping wavelengths of fluorescence and are brought into close proximity, the energy absorbed by the excitation of the first fluorescent protein serves as energy for the excitation of the second fluorescent protein, causing the second fluorescent protein to fluoresce. Furthermore, "BRET" refers to the phenomenon in which, when a bioluminescent protein is used in place of the first fluorescent protein (donor) in FRET, the energy absorbed by the bioluminescent protein when the second fluorescent protein is close to the bioluminescent protein serves as energy for the excitation of the second fluorescent protein, causing the second fluorescent protein to fluoresce. Luciferase is an example of a bioluminescent protein.

[0164] Therefore, as the "first reporter protein" in the present invention, one subunit of a split reporter protein, one fluorescent protein in a combination of fluorescent proteins capable of FRET, or any one of a combination of a bioluminescent protein and a fluorescent protein capable of BRET can be used.

[0165] (1-2) Second fusion protein

[0166] The "second fusion protein" in the present invention comprises a protein that can bind to the aforementioned membrane protein that binds to the purine receptor ligand present outside the cell (hereinafter sometimes simply described as "protein that can bind to the membrane protein that binds to the ligand") and a second reporter protein, or is composed of these proteins.

[0167] In the second fusion protein, the protein that can bind to the membrane protein that binds to the ligand and the second reporter protein can be directly connected or connected via a linker. The protein that can bind to the membrane protein that binds to the ligand and the second reporter protein can be connected in any order, as long as the first fusion protein and the second fusion protein can interact as described below. In one embodiment, the second reporter protein and the protein that can bind to the membrane protein that binds to the ligand can be connected in this order from the N-terminus. The "linker" can utilize any conventionally known linker, for example, a peptide linker can be utilized. The number and type of amino acids in the peptide linker are not particularly limited.

[0168] (1-2-1) Proteins that can bind to ligand-bound membrane proteins

[0169] In the present invention, "a protein capable of binding to the aforementioned membrane protein that binds to a purinergic receptor ligand present extracellularly" means a protein that specifically or selectively binds to the aforementioned membrane protein, i.e., a protein that binds to a purinergic receptor ligand present extracellularly. Such a protein in the present invention is not particularly limited, and preferably, membrane protein activation regulators, antibodies, receptor kinases, transcription factors, or portions thereof can be used. When the membrane protein in the present invention is a GPCR, inhibitory proteins and G proteins, or GPCR kinases, which are GPCR activation regulators, can be preferably used.

[0170] "Inhibitor" is an activation regulatory factor that is phosphorylated in the intracellular region of a GPCR activated by the action of a G protein-coupled receptor kinase in a ligand-activated extracellular region. It produces desensitization of the GPCR. Inhibitors are known in mammals as 4 subclasses, and there are inhibitory proteins -1, inhibitory proteins -2 (also referred to as "β-inhibitors -1"), inhibitory proteins -3 (also referred to as "β-inhibitors -2"), and inhibitory proteins -4 (also referred to as "X-inhibitors"). In the present invention, inhibitory proteins -2 (β-inhibitors -1) and inhibitory proteins -3 (β-inhibitors -2) can be preferably applied, and inhibitory proteins -2 (β-inhibitors -1) and inhibitory proteins -3 (β-inhibitors -2) are particularly preferably used in combination. By combining inhibitory proteins -2 (β-inhibitors -1) and inhibitory proteins -3 (β-inhibitors -2), both of their mediated signal transductions can be detected.

[0171] In the present invention, proteins capable of binding to ligand-binding membrane proteins can be known proteins, and known proteins whose amino acid sequence information and gene information are registered in public databases such as NCBI and GenBank can be used. For example, β-arrestin-1 and β-arrestin-2 are registered as NP_004032.2 and NP_004304.1 for human, and as NP_796205.1 and NP_001258287.1 for mouse, respectively. These proteins can be used in the present invention. For example, β-arrestin-1 and β-arrestin-2 represented by the amino acid sequences of SEQ ID NOs. 10 and 11 can be used as proteins capable of binding to ligand-binding membrane proteins in the present invention.

[0172] In the present invention, as a "protein capable of binding to a ligand-binding membrane protein," analogs and mutants thereof can also be used as long as they can bind to the ligand-binding membrane protein. Analogs and mutants of the "protein capable of binding to a ligand-binding membrane protein" mean (a) a protein consisting of an amino acid sequence in which 1 to 50, for example, 1 to 20, or 1 to 10 amino acids are deleted, substituted, added, or inserted in the amino acid sequence of the "protein capable of binding to a ligand-binding membrane protein," and which can bind to the ligand-binding membrane protein; or (b) a protein consisting of an amino acid sequence having a sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 99% or more with the amino acid sequence of the "protein capable of binding to a ligand-binding membrane protein," and which can bind to the ligand-binding membrane protein. The sequence identity of the amino acid sequence can be calculated as described above, based on all proteins that can bind to the ligand-binding membrane protein, or based on the binding domain that can bind to the ligand-binding membrane protein.

[0173] Furthermore, in the present invention, the terms "a portion thereof" and "a portion thereof" used in connection with a protein capable of binding to a ligand-binding membrane protein mean a protein consisting of a portion of the amino acid sequence of the protein and capable of binding to at least the ligand-binding membrane protein. Examples of such proteins include those consisting of a portion of a domain, such as a domain required for binding to the ligand-binding membrane protein, within the amino acid sequence of the protein capable of binding to the ligand-binding membrane protein. For example, when the protein capable of binding to the ligand-binding membrane protein is an antibody, examples of such a portion include partial fragments of the antibody having binding activity to the ligand-binding membrane protein, such as Fab, F(ab')2, and scFV.

[0174] (1-2-2) Second reporter protein

[0175] The "second reporter protein" in the present invention refers to a protein that functions as a reporter protein together with the first reporter protein in the first fusion protein. The "second reporter protein" in the present invention may include a subunit of a split reporter protein that differs from the first reporter protein, a fluorescent protein that differs from the first reporter protein in a combination of fluorescent proteins capable of FRET, or a protein that differs from the first reporter protein in a combination of a bioluminescent protein and a fluorescent protein capable of BRET.

[0176] (1-3) Genetically modified non-human animals

[0177] "Genetic modification" in this invention is used in its broadest sense to refer to all operations that alter a host organism's nucleic acid sequence, including the introduction of genes or partial sequences from a foreign organism, or nucleic acid sequences that function as promoters, into the host organism. Specifically, genetic modification encompasses the deletion of a full-length or partial sequence of a host organism's gene, or the replacement of a foreign gene with the same function as the host organism's gene. Furthermore, genetic modification encompasses the application of genetic modification techniques to amino acid substitutions in a base sequence encoding a specific protein, and the creation of a base sequence encoding a fusion protein by ligating it to a base sequence encoding another protein.

[0178] The "non-human animals" in the present invention are not particularly limited, but are preferably non-human mammals. For example, rodents such as mice, rats, and hamsters, non-human primates such as monkeys and chimpanzees, other mammals such as rabbits, sheep, cattle, and pigs, and birds, amphibians, reptiles, and fish can be used as non-human animals in the present invention. Rodents are particularly preferred, and mice are most preferred.

[0179] The genetically modified non-human animal of the present invention has cells expressing the first fusion protein and the second fusion protein, and preferably has cells expressing the first fusion protein and the second fusion protein throughout the body.

[0180] In the genetically modified non-human animals of the present invention, if a purinergic receptor ligand present outside of cells expressing the first and second fusion proteins binds to the membrane protein in the first fusion protein, the protein in the second fusion protein that can bind to the membrane protein bound to the ligand also binds to it. As a result, the first reporter protein in the first fusion protein and the second reporter protein in the second fusion protein come into proximity or bind, functioning together as reporter proteins. By detecting this reporter protein, the location and amount of the purinergic receptor ligand present outside of cells in the genetically modified non-human animals of the present invention can be assessed.

[0181] The method for detecting purinergic receptor ligands present outside cells of the genetically modified non-human animal of the present invention will be described later.

[0182] (1-3-1) Method for preparing genetically modified non-human animals

[0183] The genetically modified non-human animals of the present invention can be prepared using conventionally known genetic modification methods, such as by introducing genes encoding the first and second fusion proteins into the non-human animal genome. As used herein, "gene" refers to DNA, RNA, or a DNA / RNA hybrid, and is not particularly limited in form as long as it encodes a specified protein.

[0184] The gene encoding the first fusion protein can be obtained by ligating a gene encoding a membrane protein that binds to a purinergic receptor ligand and a gene encoding a first reporter protein directly or via a base sequence encoding a linker.

[0185] The gene encoding the membrane protein binding to the purinergic receptor ligand can be a gene obtained by cloning from any gene library using the gene information registered in the above-mentioned public databases, or a gene obtained by chemically preparing by gene synthesis technology.

[0186] The gene encoding the first reporter protein may be a commercially available product, or may be chemically prepared by gene synthesis together with a gene encoding a membrane protein that binds to a purine receptor ligand.

[0187] Alternatively, the gene encoding the second fusion protein can be obtained by ligating genes encoding a protein capable of binding to a membrane protein that binds to a ligand and a second reporter protein directly or via a base sequence encoding a linker.

[0188] The gene encoding the protein that can bind to the membrane protein that binds to the ligand can be obtained by using gene information registered in the above-mentioned public databases, cloning from any gene library, or chemically preparing by gene synthesis technology.

[0189] The gene encoding the second reporter protein may be a commercially available product, or may be chemically prepared by gene synthesis together with a gene encoding a protein capable of binding to the ligand-binding membrane protein.

[0190] The gene encoding the first fusion protein and the gene encoding the second fusion protein, respectively, in addition to the above-mentioned genes, may also contain regulatory sequences for transcription and translation (for example, promoter sequences, enhancer sequences, splice acceptor sequences, terminator sequences, poly A sequences, etc.), selection marker genes (for example, neomycin resistance gene, hygromycin resistance gene, puromycin resistance gene, diphtheria toxin A gene, herpes simplex virus thymidine kinase gene, etc.), tag sequences for separation and purification, etc., as needed.

[0191] The method for introducing the gene encoding the first fusion protein and the gene encoding the second fusion protein into non-human animals is not particularly limited. For example, a DNA vector (plasmid vector, cosmid vector, bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC) and other non-plasmid vectors, etc.) or a viral vector (e.g., a retroviral vector, etc.) can be appropriately applied to the microinjection method of the fertilized egg pronucleus, and the electroporation (electroporation) method and lipofection method of cells such as embryonic stem cells, sperm stem cells, and artificial pluripotent stem cells (iPS cells). The gene encoding the first fusion protein and the gene encoding the second fusion protein can be integrated into the same vector or into different vectors.

[0192] In addition, the gene encoding the first fusion protein and the gene encoding the second fusion protein can also be inserted into the host chromosome separately. As a non-limiting embodiment, the insertion of each gene into the host chromosome can be achieved by random integration at any position, or by using zinc finger nucleases (U.S. Patent Nos. 6,534,261, 6,607,882, 6,746,838, 6,794,136, 6,824,978, 6,866,997, 6,933,113, 6,979,539, 7,013,219, 7,030,215, 7,220,719 , 7,241,573, 7,241,574, 7,585,849, 7,595,376, 6,903,185, 6,479,626), TALEN (U.S. Patent Nos. 8,420,782, 8,440,431, 8,440,432, 8,450,471) or CRISPR-Cas9 technology to change the target position (U.S. Patent Nos. 8697359, 8795965, 8771945). In addition, as a non-limiting embodiment of the present invention, the insertion position of the gene encoding the first fusion protein and the gene encoding the second fusion protein on the chromosome is not particularly limited. For example, the Rosa26 gene, Hippo gene, TIGRE gene, etc., which are known to be stable expression regions of foreign genes, can be operably inserted into a specified position using homologous recombination or the like. "Operable" in this specification means that the inserted gene encoding the first fusion protein and the gene encoding the second fusion protein express the first fusion protein and the second fusion protein respectively under the control of the transcriptional regulatory sequence on the host chromosome or under the control of the transcriptional regulatory sequence possessed by each gene.

[0193] The genetically modified non-human animals of the present invention can be prepared by introducing both the gene encoding the first fusion protein and the gene encoding the second fusion protein into one individual (cell), or by mating animals into which the gene encoding the first fusion protein and the gene encoding the second fusion protein have been introduced, thereby obtaining offspring and selecting offspring having both genes.

[0194] The genetically modified non-human animal of the present invention is heterozygous or homozygous for each of the gene encoding the first fusion protein and the gene encoding the second fusion protein, and preferably is homozygous for both genes.

[0195] The genetically modified non-human animals of the present invention can be screened based on the detection level of the reporter protein, thereby obtaining genetically modified non-human animals having a desired detection level of the reporter protein. Genetically modified non-human animals thus obtained having a desired detection level of the reporter protein can be used in the methods for evaluating and screening purinergic receptor ligand-dependent drug molecules described below.

[0196] (1-3-2) Disease model animals

[0197] The genetically modified non-human animals of the present invention can be disease model animals. "Disease" is preferably a disease characterized by the presence of extracellular purinergic receptor ligands. Examples of such diseases include cancer, acute inflammation, chronic inflammation, infectious diseases, fibrosis, physical or chemical organ damage, and cell damage caused by anticancer agents.

[0198] In the present invention, "cancer" means a malignant neoplasm, which may be metastatic or non-metastatic. For example, non-limiting examples of cancers arising from epithelial tissues such as the digestive tract and skin include brain tumors, skin cancer, head and neck cancer, esophageal cancer, lung cancer, stomach cancer, duodenal cancer, breast cancer, prostate cancer, cervical cancer, uterine cancer, pancreatic cancer, liver cancer, large intestine cancer, colon cancer, bladder cancer, and ovarian cancer. Furthermore, non-limiting examples of sarcomas arising from non-epithelial tissues (stroma) such as muscle include osteosarcoma, chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma, liposarcoma, and angiosarcoma. Furthermore, as non-limiting examples of blood cancers from hematopoietic organs, examples include malignant lymphomas such as Hodgkin's lymphoma and non-Hodgkin's lymphoma, leukemias including acute myeloid leukemia or chronic myeloid leukemia, and acute lymphatic leukemia or chronic lymphatic leukemia, and multiple myeloma. "Cancer" in the present invention also includes "neoplasm". Neoplasms are tumors that are characterized by partial angiogenesis. Neoplasms also mean all newly formed tissue tumors, for example, benign such as hemangiomas, gliomas, teratomas, or malignant such as cancers, sarcomas, gliomas, astrocytomas, neuroblastomas, retinoblastomas, etc.

[0199] The genetically modified non-human animals of the present invention can be prepared as cancer model animals having cancerous tissue. "Cancer tissue" means tissue containing at least one cancer cell. Thus, for example, if cancerous tissue contains cancer cells and blood vessels, it refers to all cell types that contribute to the formation of a tumor (tumor mass) containing cancer cells and endothelial cells. Tumor means a lesion of tumor tissue (afoci of tumor tissue), and "tumor" generally means a benign or malignant neoplasm.

[0200] In the genetically modified non-human animals of the present invention, cancer model animals can be prepared by administering carcinogenic substances, expressing oncogenes by genetic modification, transplanting cancer cells, and the like based on conventionally known methods.

[0201] The disease model animals of the present invention can be used in the detection of disease onset sites and onset periods, monitoring of disease progression, evaluation of drug molecules effective in disease prevention or treatment, evaluation of drug toxicity and side effects, and screening methods described below.

[0202] 2. Genetically modified animal cells

[0203] The present invention further relates to genetically modified animal cells expressing the first fusion protein and the second fusion protein for detecting extracellular purinergic receptor ligands.

[0204] The "first fusion protein" and "second fusion protein" in the present invention are as described above.

[0205] The "animal cell" in the present invention means a cell from an animal belonging to the phylum Vertebrata, or a cell from an invertebrate (an animal other than an animal belonging to the phylum Vertebrata), without particular limitation. Preferably, the "animal cell" in the present invention means a cell from an animal belonging to the phylum Vertebrata. The phylum Vertebrata includes jawless animals and gnathostomes, and the gnathostomes include the classes Mammalia, Aves, Amphibia, Reptilia, and the like. More preferably, the "animal cell" in the present invention is a cell from an animal belonging to the class Mammalia, which is called a mammal, without particular limitation, and is particularly preferably a cell from a mouse, rat, human, monkey, pig, dog, sheep, goat, and the like.

[0206] In the genetically modified animal cells of the present invention, when a purinergic receptor ligand present extracellularly binds to the membrane protein in the first fusion protein, the protein in the second fusion protein that is capable of binding to the membrane protein bound to the ligand binds to it. This brings the first reporter protein in the first fusion protein into proximity with or binds to the second reporter protein in the second fusion protein, functioning together as a reporter protein. By detecting this reporter protein, the presence and amount of the purinergic receptor ligand present extracellularly in the genetically modified animal cells of the present invention can be assessed.

[0207] The method for detecting the purine receptor ligand present outside the cells of the genetically modified animal cells of the present invention will be described later.

[0208] (2-1) Method for preparing genetically modified animal cells

[0209] The genetically modified animal cells of the present invention can be prepared by applying conventionally known genetic modification methods, and can be prepared by genetically introducing the gene encoding the first fusion protein and the gene encoding the second fusion protein into animal cells.

[0210] The gene encoding the first fusion protein and the gene encoding the second fusion protein can be introduced into animal cells by appropriately applying known methods, for example, DNA vectors (plasmid vectors, cosmid vectors, and non-plasmid vectors such as bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs), etc.) or viral vectors (e.g., retroviral vectors, etc.), electroporation (electroporation), lipofection, etc. The gene encoding the first fusion protein and the gene encoding the second fusion protein can be integrated into the same vector or into different vectors.

[0211] In addition, the gene encoding the first fusion protein and the gene encoding the second fusion protein can also be inserted into the host chromosome separately. As a non-limiting way, the insertion of each gene into the host chromosome can be carried out by changing the arbitrary position through random integration, or by changing the target position by applying zinc finger nuclease, TALEN or CRISPR-Cas9 technology. As a non-limiting way in the present invention, the insertion position of the gene encoding the first fusion protein and the gene encoding the second fusion protein on the chromosome is not particularly limited. For example, Rosa26 gene, Hippo gene, TIGRE gene, etc. are listed, and they can be operably inserted by homologous recombination to a specified position.

[0212] The genetically modified animal cell of the present invention is heterozygous or homozygous for each of the gene encoding the first fusion protein and the gene encoding the second fusion protein, and preferably is homozygous for both genes.

[0213] Alternatively, the genetically modified animal cells of the present invention can also be prepared by isolating from the genetically modified non-human animals of the present invention described above.

[0214] The genetically modified animal cells of the present invention include not only cells used in vitro, such as isolated cells and cells established as cell lines, but also cells in vivo. Specifically, the genetically modified animal cells of the present invention include cells in vivo in the genetically modified non-human animals described above, and the genetically modified animal cells of the present invention transplanted into animals.

[0215] In the genetically modified animal cells of the present invention, a purinergic receptor ligand present outside the cell binds to the membrane protein bound to the purinergic receptor ligand in the first fusion protein, thereby activating the membrane protein. The protein bound to the activated membrane protein in the second fusion protein then binds to the membrane protein. As a result, the first reporter protein in the first fusion protein and the second reporter protein in the second fusion protein come into proximity or bind, functioning together as reporter proteins. By detecting and measuring these reporter proteins, the amount of purinergic receptor ligand present outside the cell and the presence or absence of its influence can be assessed.

[0216] The genetically modified animal cells of the present invention can be provided as part of a test kit for detecting extracellular purinergic receptor ligands. In addition to the cells, the kit may also include instructions for cell culture methods, methods for detecting the reporter protein, and a calibration curve showing the range between the amount of reporter protein detected and the amount of extracellular purinergic receptor ligand. Furthermore, if the reporter protein is an enzyme, the kit may include an appropriate substrate.

[0217] The method for detecting and evaluating the purinergic receptor ligand present outside the cells of the genetically modified animal cells of the present invention will be described later.

[0218] 3. Various evaluation methods using genetically modified animals and genetically modified animal cells

[0219] (3-1) Detection of extracellular purine receptor ligands

[0220] In one embodiment, the genetically modified animals and genetically modified animal cells of the present invention can be used in methods for detecting extracellular purinergic receptor ligands. In the present invention, "detection" means qualitative determination and / or quantitative measurement, and "detection of a purinergic receptor ligand" means either qualitative determination of the presence of a purinergic receptor ligand or quantitative measurement of the concentration of an extracellular purinergic receptor ligand, or both.

[0221] Purinergic receptor ligands in the present invention can be detected by detecting reporter proteins. As described above, in the genetically modified animals and genetically modified animal cells of the present invention, when a purinergic receptor ligand present outside the cell binds to the aforementioned membrane protein in the first fusion protein, a protein in the second fusion protein that can bind to the membrane protein bound to the aforementioned ligand binds to it. As a result, the first reporter protein in the first fusion protein and the second reporter protein in the second fusion protein come into proximity or bind, functioning together as reporter proteins. By detecting this reporter protein, purinergic receptor ligands present outside the cell can be detected.

[0222] The method for detecting the reporter protein can be appropriately selected according to the reporter protein used, and conventional methods can be applied. For example, when a fluorescent protein is used as the reporter protein, it can be performed by irradiating the fluorescent protein with light of the excitation wavelength and detecting the emitted fluorescence. Specific examples of such fluorescent proteins include GFP, CYP, and YFP. In addition, when an enzyme is used as the reporter protein, it can be performed by administering or adding a substrate for the enzyme as needed, allowing the enzyme to react with its substrate, and detecting the reactant. In the case where the reactant is a fluorescent substance, it can be performed by irradiating the fluorescent substance with light of the excitation wavelength and detecting the emitted fluorescence. In addition, when the reactant emits light, it can be performed by detecting the luminescence. Alternatively, when the reactant is a dye, it can be detected by measuring the absorbance of the dye. More specifically, when luciferase is used as the reporter protein, it can be performed by administering or adding a cell membrane-permeable luciferin as a substrate, generating a luciferin-luciferase reaction, and detecting the luminescence of the generated protein.

[0223] For example, according to the present invention, ATP can be cited as a purinergic receptor ligand, and the genetically modified animals and genetically modified cells of the present invention can be used to detect ATP present outside cells.

[0224] (3-2) Evaluation and screening of drug molecules

[0225] In another embodiment, the genetically modified animals and genetically modified animal cells of the present invention can be used in a method for evaluating the effects of drug molecules using extracellular purine receptor ligands as indicators.

[0226] In this method, the test substance is administered to the genetically modified animal of the present invention, or added to the culture medium for culturing cells of the genetically modified animal of the present invention. When the reporter protein is an enzyme, the substrate of the enzyme is further administered or added as needed, and the reporter protein is detected. The amount is compared with the amount detected before administration or addition, and the efficacy of the test substance can be evaluated.

[0227] The "test substance" is not particularly limited, and includes low molecular weight compounds, amino acids, nucleic acids, lipids, sugars, extracts of natural products, etc. Natural compound libraries, synthetic compound libraries, metabolite libraries, existing drug libraries, etc. can be used.

[0228] Administration of the test substance and substrate (if necessary) to the genetically modified animal of the present invention can be carried out by any means, for example, by injection such as intravenous injection, intradermal injection, subcutaneous injection, intramuscular injection, and intraperitoneal injection (but not limited to these). Furthermore, the test substance and substrate may be administered to the genetically modified animal of the present invention simultaneously or separately, and the administration methods may be the same or different.

[0229] The test substance and the substrate (if necessary) may be added to the culture medium for culturing the genetically modified animal cells of the present invention simultaneously or separately.

[0230] When a test substance is administered or added, if the detected amount of the reporter protein decreases compared to before administration or addition, the test substance can be evaluated as having an effect of inhibiting the production of a purine receptor ligand. On the other hand, if the detected amount of the reporter protein increases compared to before administration or addition, the test substance can be evaluated as having an effect of enhancing the production of a purine receptor ligand. Furthermore, based on this evaluation, drug molecules having each effect can be screened.

[0231] When a test substance exhibits cell-damaging or organ-damaging properties as either its primary or side effect, the detection of purinergic receptor ligands leaking from damaged tissues or secreted by immune cells present in damaged tissues can be applied to the toxicity assessment of the test substance. In particular, when administered to genetically modified animals, organs exhibiting toxicity can be non-invasively identified, and changes over time can be captured.

[0232] More specifically, ATP is an example of a purinergic receptor ligand. According to this method, using ATP as an indicator, the drug effect of enhancing or inhibiting ATP production can be evaluated, and drug molecules having this effect can be screened.

[0233] (3-3) Evaluation and screening of purine receptor ligand-dependent drug molecules

[0234] In another embodiment, the genetically modified animals and genetically modified animal cells of the present invention can be used in a method for evaluating the effects of purine receptor ligand-dependent drug molecules.

[0235] The "purinergic receptor ligand-dependent drug molecule" in the present invention means a drug molecule that is activated or inactivated depending on the presence or amount of a purinergic receptor ligand present outside the cell.

[0236] In this method, the test substance is administered to the genetically modified animal of the present invention, or added to a culture medium for culturing cells of the genetically modified animal of the present invention, and the efficacy of the test substance can be evaluated in the presence of a predetermined amount of extracellular purinergic receptor ligand.

[0237] In this method, the amount of purinergic receptor ligand present outside the cells of the genetically modified animals or genetically modified animal cells of the present invention can be confirmed by detecting a reporter protein. Detection of the reporter protein is sufficient as long as the amount of extracellular purinergic receptor ligand is clear at least when the test substance is acting. Detection of the reporter protein can be performed before, after, or both of the administration or addition of the test substance. Preferably, detection of the reporter protein can be performed before administration or addition of the test substance.

[0238] In this method, the genetically modified animals of the present invention that have been pre-screened based on the detection level of a predetermined reporter protein can be used. Alternatively, the amount of purinergic receptor ligand present outside the cells of the genetically modified animals of the present invention or the genetically modified animal cells of the present invention can be adjusted by administering the purinergic receptor ligand to the genetically modified animals of the present invention or adding the purinergic receptor ligand to the culture medium in which the genetically modified animal cells of the present invention are cultured.

[0239] The "test substance" is as described above, and the test substance and substrate (if necessary) can be administered or added by any means as described above.

[0240] The detection of the reporter protein can be performed as described above depending on the reporter protein used.

[0241] When changes in drug efficacy are observed depending on the presence or amount of extracellular purine receptor ligands, the test substance can be evaluated as a purine receptor ligand-dependent drug molecule, and purine receptor ligand-dependent drug molecules can be screened based on this evaluation.

[0242] More specifically, ATP can be cited as a purinergic receptor ligand. According to the present method, the effects of ATP-dependent drug molecules can be evaluated and ATP-dependent drug molecules can be screened.

[0243] (3-4) Screening of drug molecules for monitoring disease pathology, prevention or treatment of diseases

[0244] In another embodiment, the genetically modified animals of the present invention can be used in methods for detecting the site and time of disease onset and monitoring the pathological state of the disease. In this method, by detecting a reporter protein in the above-described disease model animals, the site, extent, and time of disease onset characterized by the presence of extracellular purinergic receptor ligands can be identified. By identifying these over time, the progression of the disease pathological state can be monitored.

[0245] In another embodiment, the genetically modified animals of the present invention can be used in methods for evaluating preventive or therapeutic drugs for diseases and for screening effective preventive or therapeutic drugs.

[0246] In this method, the test substance is administered to the above-mentioned disease model animal, and when the reporter protein is an enzyme, the substrate of the enzyme is further administered, the reporter protein is detected, and its amount is compared with the detected amount before administration to evaluate the efficacy of the test substance.

[0247] When the test substance is administered and the amount of the reporter protein detected decreases compared to before administration, the test substance can be evaluated as being effective in preventing or treating a disease. Furthermore, based on this evaluation, drug molecules having the respective effects can be screened.

[0248] The "test substance" is as described above, and the test substance and substrate (if necessary) can be administered by any means as described above.

[0249] The detection of the reporter protein can be performed as described above depending on the reporter protein used.

[0250] More specifically, ATP is an example of a purinergic receptor ligand. Using ATP as an indicator, the present method can evaluate the efficacy of drug molecules effective in the prevention or treatment of cancer, thereby enabling screening for drug molecules effective in the prevention or treatment of cancer. Furthermore, the present method can screen for drug molecules effective in the prevention or treatment of diseases other than cancer and side effects caused by other agents. Non-limiting examples of diseases and side effects other than cancer include acute inflammation, chronic inflammation, infectious diseases, fibrosis, physical or chemical organ damage, and cell damage caused by anticancer agents. Using ATP as an indicator, the efficacy of drug molecules effective in the prevention or treatment of these diseases and side effects can be evaluated.

[0251] In addition, in this specification, unless a term is described to mean a limitation of quantity of "1" or "plurality", the term described in this specification is not to be interpreted as a term having the meaning of "1 or more" and is not to be interpreted as a term having a specific limitation of quantity.

[0252] It is obvious to those skilled in the art that any combination of one or more aspects described in this specification is also encompassed by the present invention, as long as there is no technical contradiction based on their common technical knowledge.

[0253] All prior art documents cited in this specification are incorporated herein by reference.

[0254] This specification includes the contents described in the specification and / or drawings of Japanese Patent Application No. 2019-225404, which is the basis of the priority of this application. Example

[0255] Next, the present invention will be further specifically described with reference to examples, but the present invention is not limited to the following examples.

[0256] [Example 1] Preparation of P2Y11-split Luc (C-terminus) and Arrestin-split Luc (N-terminus) double knock-in mice

[0257] 1.1. Construction of P2Y11-split Luc (C-terminal) knock-in vector

[0258] A splice acceptor, poly A addition signal, and FLAG tag were added to the sequence combining the C-terminal region of the human P2Y11 gene and the luciferase gene to create a "P2Y11-split Luc (C-terminal)" expression cassette (SEQ ID NO: 1). This sequence was cloned into the homologous recombination vector pZDonor-mRosa26 vector (SIGMA-Aldrich Inc. #D9196) for knocking in the mouse Rosa gene region using methods known to those skilled in the art to construct a P2Y11-split Luc (C-terminal) knock-in vector (SEQ ID NO: 2). Figure 1 ).

[0259] 1.2. Construction of Arrestin-split Luc (N-terminal) knock-in vector

[0260] To the mouse arrestin-2 (β-arrestin-1) gene ( Figure 2 The sequence of the N-terminal region of the luciferase gene and the mouse arrestin-3 (β-arrestin-2) gene ( Figure 2The sequence of the N-terminal region of the luciferase gene was added with a Myc tag or a His tag, respectively, and connected with a 2A peptide. The expression cassette was combined with the mouse β-actin promoter to form an "Arrestin-split Luc (N-terminal)" (SEQ ID NO: 2). Using methods known to those skilled in the art, this sequence was cloned into the homologous recombination vector pZDonor-mRosa26 vector (SIGMA-Aldrich Inc. #D9196) for knocking in the mouse Rosa gene region to construct the Arrestin-split Luc (N-terminal) knock-in vector ( Figure 2 ).

[0261] 1.3. DNA Microinjection of P2Y11-split Luc (C-terminus) and Arrestin-split Luc (N-terminus) Knock-in Vectors into Mouse Fertilized Eggs

[0262] A solution of a P2Y11-split Luc (C-terminal) knock-in vector or an Arrestin-split Luc (N-terminal) knock-in vector mixed with ZFN mRNA targeting the mouse Rosa gene (SIGMA-Aldrich Inc. #M4574) was injected into pronuclear-stage mouse fertilized eggs. Embryos were cultured overnight at 37°C and then transferred to the uterus of a 0.5-day pseudopregnant ICR recipient female to produce offspring. The knock-in allele was detected by PCR and founder mice were selected. Respectively, primers mR1387F (SEQ ID NO: 3) and h11-480R (SEQ ID NO: 4) and FLucC-1321F (SEQ ID NO: 5) and mR3334R (SEQ ID NO: 6) were used in the knock-in allele detection of P2Y11-split Luc (C terminus) knock-in mice, and primers mR1247Fq (SEQ ID NO: 3) and cmv-R (SEQ ID NO: 7) and mArb2a-F2 (SEQ ID NO: 8) and mR3334R (SEQ ID NO: 6) were used in the knock-in allele detection of Arrestin-split Luc (N terminus) knock-in mice.

[0263] 1.4. Preparation of P2Y11-split Luc (C-terminus) and Arrestin-split Luc (N-terminus) Double Knock-in Mice

[0264] The resulting founder mice were mated with C57BL / 6N mice after sexual maturity, and transmission of the knock-in allele to the next generation was confirmed by PCR using genomic DNA extracted from tissues of the next generation of mice as a template. P2Y11-split Luc (C-terminus) knock-in mice were then crossed with Arrestin-split Luc (N-terminus) knock-in mice to generate P2Y11-split Luc (C-terminus) and Arrestin-split Luc (N-terminus) double knock-in mice.

[0265] [Example 2] In vivo ATP signal detection in P2Y11-split Luc (C-terminus) and Arrestin-split Luc (N-terminus) double knock-in mice

[0266] A 50 μL mixture of 1 mM ATP and 15 mg / mL VivoGlo luciferin (Promega #P1043) was subcutaneously administered to mice double-knocked in P2Y11-split Luc (C-terminus) and Arrestin-split Luc (N-terminus). Luminescence signal intensity was measured using an IVIS Spectrum CT (PerkinElmer) 10-20 minutes after ATP administration. Measurement conditions were: Exposure time = 180 sec, Binning = Medium, F / Stop = 1, and luminescence signal was analyzed as a physical quantity (photons / second).

[0267] As a result, luminescence signals were detected by ATP administration in P2Y11-split Luc (C-terminal) and Arrestin-split Luc (N-terminal) double knock-in mice. However, no luminescence signals were detected in mice with only one allele each and wild-type mice ( Figure 3 These results confirmed that Arrestin is recruited by ATP binding to P2Y11 and functions as a reaction-specific reporter mouse capable of generating a luminescent signal by reconstructing split-luciferase.

[0268] [Example 3] Establishment of fibroblasts from P2Y11-split Luc (C-terminus) and Arrestin-split Luc (N-terminus) double knock-in mice and in vitro ATP signal detection

[0269] 3.1. Establishment of fibroblasts from P2Y11-split Luc (C-terminus) and Arrestin-split Luc (N-terminus) double knock-in mice

[0270] Skin tissue was collected from P2Y11-split Luc (C-terminus) and Arrestin-split Luc (N-terminus) double knock-in mice and finely dissected in culture medium using surgical scissors. The tissue was then placed on a coverslip with the dermis side touching the bottom of a culture dish and cultured in D-MEM supplemented with 10% FBS and 1x NEAA. Fibroblasts, whose migration and proliferation were confirmed, were recovered by trypsinization and repeatedly passaged to establish a fibroblast cell line.

[0271] 3.2. In vitro ATP concentration detection

[0272] Fibroblasts from mice double-knocked in with P2Y11-split Luc (C-terminus) and Arrestin-split Luc (N-terminus) were seeded in 24-well culture plates. The next day, ATP and VivoGlo luciferin (Promega #P1043) were added to a final concentration of 0-1000 μM, and luminescence signals were detected using an IVIS Spectrum CT (PerkinElmer). The assay conditions were exposure time = 180 sec, Binning = Medium, and F / Stop = 1, with luminescence signals analyzed as physical quantities (photons / second).

[0273] As a result, concentration-dependent signal intensities were detected at 0 μM, 10 μM, 50 μM, 100 μM, 200 μM, 400 μM, 600 μM, 800 μM, and 1000 μM ATP, and the obtained regression equation was y=437.91x+10840(R 2 =0.9727)( Figure 4 、 Figure 5 ).

[0274] [Example 4] In vivo imaging of ATP with known concentrations

[0275] A mixture of 50uL of ATP at final concentrations of 0mM, 1mM, 2mM, 4mM, and 8mM and 15mg / mL VivoGlo luciferin (Promega #P1043) was administered subcutaneously to P2Y11-split Luc (C-terminus) and Arrestin-split Luc (N-terminus) double knock-in mice. 10 to 20 minutes after ATP administration, the luminescence signal intensity was measured using IVIS Spectrum CT (PerkinElmer). The measurement conditions were exposure time = 60sec, Binning = Medium, F / Stop = 1, and the luminescence signal was analyzed as a physical quantity (photons / second). As a result, the ATP concentration-dependent signal intensity was detected, and the regression equation obtained was y = 1E+06x + 3E+06(R 2= 0.9964). This confirmed that it functions as a concentration-dependent reporter mouse ( Figure 6 、 Figure 7 ).

[0276] [Example 5] Visualization of ATP by inflammatory stimulation

[0277] P2Y11-split Luc (C-terminal) and Arrestin-split Luc (N-terminal) double knock-in mice were injected hydrodynamically to visualize ATP leakage from the liver associated with hepatocellular damage. The day after hydrodynamic injection, 150 mg / kg of VivoGlo luciferin (Promega #P1043) was administered intraperitoneally. Luminescence signal intensity was measured using an IVIS Spectrum CT (PerkinElmer). Measurement conditions were exposure time = 60 sec, Binning = Medium, F / Stop = 1, and luminescence signal was analyzed as a physical quantity (photons / second).

[0278] The results confirmed that a high luminescence signal was detected in the liver of the hydrodynamic injection group compared to the group without hydrodynamic injection (-), which became a non-invasive visual reporter of ATP leakage during inflammatory stimulation ( Figure 8 ).

[0279] [Example 6] Visualization of ATP in tissues induced by carcinogenesis

[0280] In mice with P2Y11-split Luc (C-terminus) and Arrestin-split Luc (N-terminus) double knock-in, ATP concentrations in normal tissue and tumor formation sites were compared and visualized when carcinogenesis was induced in the liver using hydrodynamic DNA delivery. Specifically, a mutant Kras expression vector and a gRNA-Cas9 expression vector targeting p53, p16, and Smad4 were mixed and administered intravenously via the caudal vein at a dose of 3-5 μg / mouse of each vector, equivalent to approximately 10% of the mouse's body weight. Mutant Kras expression was achieved using the expression vector pMacII, described in The Journal of Biological Chemistry (2011) 286, 20109-20116. The sequence encoding the mutant Kras (G12D) shown in SEQ ID NO: 12 was cloned into the expression vector pMacII and used. This construct expresses the mutant Kras (G12D) under the control of the CMV enhancer and the mouse β-actin promoter. The gRNA-Cas9 expression vectors targeting each target gene used partial sequences of the p53, p16, and Smad4 genes as gRNA sequences and were cloned into pGENA22 (Horizon Discovery). Three gRNA-Cas9 expression vectors were prepared for each target gene, mixed, and administered. For the gRNA sequence of p53, the sequences shown in sequence numbers 13, 14, and 15 were applied. For the gRNA sequence of p16, the sequences shown in sequence numbers 16, 17, and 18 were applied. For the gRNA sequence of Smad4, the sequences shown in sequence numbers 19, 20, and 21 were applied. For analysis of changes in ATP concentration in tissues associated with canceration in the liver, 150 mg / kg of VivoGlo luciferin (Promega#P1043) was administered intraperitoneally, and the luminescent signal intensity was measured using IVIS Spectrum CT (PerkinElmer). The measurement conditions were exposure time = 120 sec (in vivo), 1 sec (excised liver), Binning = Medium, F / Stop = 1, and the luminescence signal was analyzed as a physical quantity (photons / second).

[0281] As a result, by administering mutant Kras expression vectors and gRNA-Cas9 expression vectors targeting p53, p16, and Smad4 genes, strong luminescence signals were detected by non-invasive visualization in individuals with tumor formation and growth in the liver ( Figure 9 (A)). Furthermore, after the liver was removed, in vitro analysis was performed and compared with normal tissue or normal areas, high luminescence signals were detected in the cancerous areas and tumor formation sites, and high ATP concentrations were confirmed in the proliferation areas of cancer cells ( Figure 9(B))。

Claims

1. A method for detecting extracellular ATP in a low-invasive manner over time and systemically, characterized in that: The method utilizes genetically modified animal cells, The genetically modified animal cell is a genetically modified animal cell expressing a first fusion protein and a second fusion protein for detecting ATP present outside the cell. The first fusion protein comprises P2Y11 that binds to ATP and a first reporter protein, The second fusion protein comprises the P2Y11-binding protein that binds to the ATP and a second reporter protein, The first and second reporter proteins are subunits of Split luciferase. The method includes the step of quantitatively measuring the ATP concentration based on the intensity of the luminescent signal of luciferin generated by the catalytic activity of the luciferase reconstructed and generated by protein assembly of each subunit.

2. The method according to claim 1, wherein The genetically modified animal cell comprises in its genome: a gene encoding a first fusion protein comprising P2Y11 that binds to extracellular ATP and a first reporter protein, and A gene encoding a second fusion protein comprising the P2Y11 binding protein that binds to the ATP and a second reporter protein.

3. A method for detecting extracellular ATP in a low-invasive manner over time and systemically, characterized in that: The method utilizes a genetically modified non-human animal, The genetically modified non-human animal is a genetically modified non-human animal expressing a first fusion protein and a second fusion protein for detecting ATP present outside cells. The first fusion protein comprises P2Y11 that binds to ATP and a first reporter protein, The second fusion protein comprises the P2Y11-binding protein that binds to the ATP and a second reporter protein, The first and second reporter proteins are subunits of Split luciferase. The method includes the step of quantitatively measuring the ATP concentration based on the intensity of the luminescent signal of luciferin based on the catalytic activity of the luciferase reconstructed and generated by protein assembly of each subunit.

4. The method according to claim 3, wherein The genetically modified non-human animal comprises in its genome: a gene encoding a first fusion protein comprising P2Y11 that binds to extracellular ATP and a first reporter protein, and A gene encoding a second fusion protein comprising the P2Y11 binding protein that binds to the ATP and a second reporter protein.

5. A method for evaluating the efficacy of a preventive or therapeutic drug for a disease, characterized in that: The method utilizes a genetically modified non-human animal, The disease is a disease involving purine receptor ligand (ATP), The genetically modified non-human animal is a genetically modified non-human animal expressing a first fusion protein and a second fusion protein for detecting ATP present outside cells. The first fusion protein comprises P2Y11 that binds to ATP and a first reporter protein, The second fusion protein comprises the P2Y11-binding protein that binds to the ATP and a second reporter protein, The first and second reporter proteins are subunits of Split luciferase. The method includes administering the preventive drug or disease treatment drug to the genetically modified non-human animal, quantitatively measuring the ATP concentration by measuring the intensity of the luminescent signal of the luciferin based on the catalytic activity of the luciferase reconstructed and generated by the protein assembly of each subunit, thereby evaluating the change in the detection amount of the reporter protein before and after administration, and evaluating the efficacy of the preventive drug or disease treatment drug based on the change.

6. A method for screening a preventive drug or a therapeutic drug for a disease, characterized in that: The method utilizes a genetically modified non-human animal, The disease is a disease involving purine receptor ligand (ATP), The genetically modified non-human animal is a genetically modified non-human animal expressing a first fusion protein and a second fusion protein for detecting ATP present outside cells. The first fusion protein comprises P2Y11 that binds to ATP and a first reporter protein, The second fusion protein comprises the P2Y11-binding protein that binds to the ATP and a second reporter protein, The first and second reporter proteins are subunits of Split luciferase. The method includes administering a test substance to the genetically modified non-human animal, quantitatively measuring the ATP concentration by measuring the intensity of the luminescent signal of the luciferin based on the catalytic activity of the luciferase reconstructed and generated by the protein assembly of each subunit, thereby evaluating the change in the detection amount of the reporter protein before and after administration, and screening for substances effective in preventing or treating the disease based on the change.

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