System for detecting extracellular purinergic receptor ligands and non-human animals incorporating said system

Abstract

The purpose of the present invention is to provide an evaluation system that enables less invasive, over-time and systemic detection of an extracellular purinergic receptor ligand. Provided is a transgenic nonhuman animal that expresses a first fused protein and a second fused protein for detecting a purinergic receptor ligand present outside cells, wherein the first fused protein contains a membrane protein capable of binding to the purinergic receptor ligand and a first reporter protein and the second fused protein contains a protein capable of binding to the membrane protein to which the ligand is bound and a second reporter protein. Also provided is a cell of the transgenic nonhuman animal.

Description

[Technical Field]

[0001] The present invention relates to a system for detecting extracellular purinergic receptor ligands utilizing signal transduction of receptor proteins that have purinergic receptor ligands as ligands, and to genetically modified non-human animals into which the system has been introduced, as well as to methods for monitoring disease conditions and evaluating compounds using genetically modified non-human animals into which the system for detecting extracellular purinergic receptor ligands has been introduced. [Background technology]

[0002] Purinergic receptors are a group of cell surface receptors whose ligands are nucleotides such as adenosine and ATP. Purinergic receptors have been reported to be involved in various diseases, including immune disorders, and the development of purinergic receptor inhibitors and agonists that target purinergic receptors is also underway. Known purinergic receptor ligands include ATP, adenosine, and nucleotides including their metabolites. Signal transduction pathways mediated by these purinergic receptor ligands and purinergic receptors have been reported to be involved in a variety of physiological phenomena, including neurotransmission, muscle contraction, pain sensation, taste, and inflammatory responses.

[0003] Purinergic receptor ligands, including ATP and adenosine, function as signal transducers in the body, and studies have been conducted to verify their in vivo production, degradation, and concentration measurements. For example, it is known that large amounts of intracellular ATP leak extracellularly upon cell death, and it has been reported that this plays an important role as a danger signal in the inflammatory process. Furthermore, it has been reported that ATP concentrations in cancer tissues are higher than in normal tissues because large amounts of intracellular ATP leak extracellularly upon cancer cell death (Non-Patent Documents 1, 3, 4).

[0004] Microdialysis is a method for measuring biological substances, including extracellular purinergic receptor ligands, in vivo. This method involves analyzing tissue fluid or extracellular fluid collected from tissue using HPLC or other methods. When measuring ATP, in addition to analytical methods such as HPLC, luciferin-luciferase assays can also be used. However, even with these methods, it is difficult to clearly distinguish whether the detected and measured purinergic receptor ligands are intracellular or extracellular, and it is not possible to measure the concentration of purinergic receptor ligands in vivo in real time. Furthermore, microdialysis involves inserting a needle into tissue to collect tissue fluid, which can cause cell death or tissue necrosis at the insertion site. In such cases, there is a problem of artifacts caused by the measurement technique affecting the measurement results. ATP is known to be secreted in response to cell death and inflammatory responses, making it less suitable for measurement by this method. Patent Document 2 reports a method for observing the distribution and fluctuation of ATP in vivo using a non-human mammal that expresses a fusion protein consisting of two types of fluorescent proteins that can serve as a donor and acceptor in fluorescence resonance energy transfer (FER) attached to the amino- and carboxy-terminal ends of the ε subunit of ATP synthase. However, this method focuses on intracellular ATP and cannot evaluate extracellular ATP.

[0005] In addition, a method using genetically modified cells that express luciferase extracellularly has been reported as an attempt to measure extracellular ATP in vivo (Non-Patent Documents 1 and 2, Patent Document 1). In this method, when the genetically modified cells are exposed to an environment with a high extracellular ATP concentration, they emit a signal via the luciferin-luciferase reaction, and this signal is detected to measure extracellular ATP in vivo. However, this method requires the cells to be transferred into the body, but it is difficult to distribute the transferred cells throughout the body. Furthermore, when the time of extracellular release of ATP is unknown, it is difficult to determine the appropriate time to transfer the cells. Another problem remains: the method cannot be used if the transferred cells are eliminated by the host's immune system. Furthermore, the luciferin-luciferase reaction can only measure ATP, and cannot measure other purinergic receptor ligands.

[0006] Although purinergic receptor ligands, including ATP, have been reported to be associated with various diseases as extracellular signaling substances, there are many unknowns regarding what extracellular purinergic receptor ligands function in what diseases, at what timing, and how they function. Therefore, to evaluate the function of extracellular purinergic receptor ligands in vivo and in pathological conditions, an evaluation system capable of detecting extracellular purinergic receptor ligands systemically, minimally invasively, over time, and systematically is needed, but such an evaluation system has not yet existed. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] WO2006 / 126231 [Patent Document 2] WO2015 / 108102 [Non-patent literature]

[0008] [Non-Patent Document 1] Patrizia et.al.(2008)PLoS One.3,e2599 [Non-patent document 2] Francesco Di Virgilio et.al.(2016)Methods Mol Biol.1417,115-29 [Non-patent document 3] Idzko M et.al.(2007)Nat Med.Aug;13(8):913-9 [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 [Problem to be solved by the invention]

[0009] The present invention has been made in light of the above-described circumstances, and aims to provide an evaluation system capable of detecting extracellular purinergic receptor ligands systemically, minimally invasively, over time, and in a time-dependent manner. More specifically, the present invention aims to provide a genetically modified non-human animal that systemically expresses a reporter protein for detecting and evaluating extracellular purinergic receptor ligands, and to construct an evaluation system for detecting extracellular purinergic receptor ligands. A further aim of the present invention is to provide methods for detecting various diseases, monitoring the pathological conditions of various diseases, and screening for therapeutic agents for these diseases, using the genetically modified non-human animal and the evaluation system that uses the reporter protein to detect extracellular purinergic receptor ligands. [Means for solving the problem]

[0010] The present inventors conducted extensive research to solve the above-mentioned problems. They fused each subunit of split luciferase to the P2Y purinergic receptor (P2Y ) and its intracellular binding β-arrestin, respectively, and generated genetically engineered mice expressing these fused subunits throughout the body. They found that this method enabled detection of extracellular ATP. Specifically, with this genetic modification, when extracellular ATP binds to the P2Y receptor, the P2Y receptor and β-arrestin bind intracellularly, resulting in the appropriate association of the fused subunit proteins, reconstituting and producing luciferase. Luciferase expresses a luminescent signal in the presence of an appropriate substrate, and extracellular ATP can be detected by detecting this signal. Furthermore, when ATP was measured using cells isolated from the genetically engineered mice, the intensity of the detected luminescent signal was concentration-dependent, confirming that ATP concentration could be quantitatively measured. This allows for applications such as in vitro ATP concentration measurement and screening. Furthermore, when we examined the detection of extracellular ATP in the living body of this genetically modified mouse, we found that the luminescent signal could be detected in a concentration-dependent manner, making it possible to non-invasively detect extracellular ATP throughout the body over time.

[0011] The present invention has been completed based on these findings, and in specific embodiments, relates to the following inventions, for example. [1] A genetically modified non-human animal that expresses a first fusion protein and a second fusion protein for detecting a purinergic receptor ligand present extracellularly, the first fusion protein comprises a membrane protein that binds to a purinergic receptor ligand and a first reporter protein; A genetically modified non-human animal, wherein the second fusion protein comprises a protein that binds to the membrane protein to which the ligand is bound and a second reporter protein. [2] The genetically modified non-human animal of [1], wherein the first reporter protein and the second reporter protein are each subunit of a split reporter protein. [3] The genetically modified non-human animal according to [2], wherein the split reporter protein is split luciferase. [4] The genetically modified non-human animal of [2], wherein the split reporter protein is a split fluorescent protein. [5] The genetically modified non-human animal of [1], wherein the first reporter protein and the second reporter protein are a combination of proteins that exhibit fluorescence resonance energy transfer (FRET) or bioluminescence resonance energy transfer (BRET). [6] The genetically modified non-human animal according to any one of [1] to [5], wherein the protein that binds to the membrane protein to which the ligand is bound is an arrestin or a part thereof. [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 part thereof. [8] The genetically modified non-human animal according to [7], wherein the GPCR is a P1 receptor. [9] The genetically modified non-human animal according to [8], wherein the P1 receptor is selected from the group consisting of adenosine A1 receptor, adenosine A2A receptor, adenosine A2B receptor, and adenosine A3 receptor.

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

[11] The genetically modified non-human animal according to

[10] , wherein the P1 receptor ligand is selected from the group consisting of adenosine, AMP, ADP, and ATP.

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

[13] The genetically modified non-human animal according to

[12] , wherein the P2 receptor is a P2Y receptor.

[14] The genetically modified non-human animal of

[13] , wherein the P2Y receptor is selected from the group consisting of P2Y1, P2Y2, P2Y4B, P2Y6, P2Y11, P2Y12, P2Y13, and P2Y14.

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

[12] to

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

[16] The genetically modified non-human animal according to

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

[17] The genetically modified non-human animal according to

[15] or

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

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

[15] to

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

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

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

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

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

[21] The genetically modified non-human animal according to

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

[22] The genetically modified non-human animal according to

[20] or

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

[23] A genetically modified non-human animal that is a disease model animal, any of [1] to

[22] .

[24] The genetically modified non-human animal of

[23] , wherein the disease is selected from the group consisting of cancer, acute inflammation, chronic inflammation, infectious disease, fibrosis, physical or chemical organ damage, and cell damage caused by drugs such as anticancer drugs.

[0012]

[25] An animal cell expressing a first fusion protein and a second fusion protein for detecting a purine receptor ligand present extracellularly, the first fusion protein comprises a membrane protein that binds to a purinergic receptor ligand and a first reporter protein; An animal cell, wherein the second fusion protein comprises a protein that binds to the membrane protein to which the ligand is bound and a second reporter protein.

[26] The animal cell of

[25] , wherein the first reporter protein and the second reporter protein are each subunit of a split reporter protein.

[27] The animal cell according to

[26] , wherein the split reporter protein is split luciferase.

[28] The animal cell of

[26] , wherein the split reporter protein is a split fluorescent protein.

[29] The animal cell of

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

[30] The animal cell of any one of

[25] to

[29] , wherein the protein that binds to the membrane protein to which the ligand is bound is arrestin or a part thereof.

[31] The animal cell of any one of

[25] to

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

[32] The animal cell of any one of

[25] to

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

[33] The animal cell of

[32] , wherein the P1 receptor is selected from the group consisting of adenosine A1 receptor, adenosine A2A receptor, adenosine A2B receptor, and adenosine A3 receptor.

[34] The animal cell of

[32] or

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

[35] The animal cell according to

[34] , wherein the P1 receptor ligand is selected from the group consisting of adenosine, AMP, ADP, and ATP.

[36] The animal cell of any one of

[25] to

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

[37] The animal cell of

[36] , wherein the P2 receptor is a P2Y receptor.

[38] The animal cell of

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

[39] The animal cell according to

[37] or

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

[40] The animal cell of

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

[41] The animal cell of

[39] or

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

[42] The animal cell of any one of

[39] to

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

[43] The animal cell of any one of

[25] to

[42] , wherein the animal is a mammal.

[44] The animal cell of any one of

[25] to

[43] , wherein the animal is a rodent.

[45] The animal cell of any one of

[25] to

[44] , wherein the animal is a mouse.

[0013]

[46] A detection kit for detecting extracellular purinergic receptor ligands, including any of the animal cells of

[25] to

[45] .

[0014]

[47] A method for producing a mouse model comprising: introducing into a genome a gene encoding a first fusion protein comprising a membrane protein that binds to an extracellular purinergic receptor ligand and a first reporter protein; and introducing into the genome a gene encoding a second fusion protein comprising a protein that binds to the membrane protein to which the ligand is bound and a second reporter protein; A method for producing a genetically modified animal cell for detecting a purine receptor ligand present outside the cell, comprising:

[48] ​​The method of

[47] , wherein the first reporter protein and the second reporter protein are each subunit of a split reporter protein.

[49] The method of

[48] , wherein the split reporter protein is split luciferase.

[50] The method of

[48] , wherein the split reporter protein is a split fluorescent protein.

[51] The method of

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

[52] The method according to any one of

[47] to

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

[53] The method according to any one of

[47] to

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

[54] The method of

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

[55] The method of

[54] , wherein the P1 receptor is selected from the group consisting of adenosine A1 receptor, adenosine A2A receptor, adenosine A2B receptor, and adenosine A3 receptor.

[56] The method according to

[54] or

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

[57] The method of

[56] , wherein the P1 receptor ligand is selected from the group consisting of adenosine, AMP, ADP, and ATP.

[58] The method of

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

[59] The method of

[58] , wherein the P2 receptor is a P2Y receptor.

[60] The method of

[59] , wherein the P2Y receptor is selected from the group consisting of P2Y1, P2Y2, P2Y4B, P2Y6, P2Y11, P2Y12, P2Y13, and P2Y14.

[61] The method of any one of

[58] to

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

[62] The method of

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

[63] The method for producing

[61] or

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

[64] The method of any one of

[61] to

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

[65] The method of any one of

[47] to

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

[66] The method according to any one of

[47] to

[65] , wherein the animal cells are mammalian cells.

[67] The method according to any one of

[47] to

[66] , wherein the animal cells are rodent cells.

[68] The method according to any one of

[47] to

[67] , wherein the animal cells are mouse cells.

[69] A method for producing any one of

[47] to

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

[0015]

[70] A method for producing a mouse model comprising: introducing into a genome a gene encoding a first fusion protein comprising a membrane protein that binds to an extracellular purinergic receptor ligand and a first reporter protein; and introducing into the genome a gene encoding a second fusion protein comprising a protein that binds to the membrane protein to which the ligand is bound and a second reporter protein; A method for producing a genetically modified non-human animal for detecting extracellular purinergic receptor ligands, comprising:

[71] The method of

[70] , wherein the first reporter protein and the second reporter protein are each subunit of a split reporter protein.

[72] The method of

[71] , wherein the split reporter protein is split luciferase.

[73] The method of

[71] , wherein the split reporter protein is a split fluorescent protein.

[74] The method of

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

[75] The method according to any one of

[70] to

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

[76] The method according to any one of

[70] to

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

[77] The method of

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

[78] The method of

[77] , wherein the P1 receptor is selected from the group consisting of adenosine A1 receptor, adenosine A2A receptor, adenosine A2B receptor, and adenosine A3 receptor.

[79] The method according to

[77] or

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

[80] The method according to any one of

[77] to

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

[81] The method of

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

[82] The method of

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

[83] The method of

[82] , wherein the P2Y receptor is selected from the group consisting of P2Y1, P2Y2, P2Y4B, P2Y6, P2Y11, P2Y12, P2Y13, and P2Y14.

[84] The method of any one of

[81] to

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

[85] The method of

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

[86] The method of

[84] or

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

[87] The method of any one of

[84] to

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

[88] The method of any one of

[70] to

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

[89] The method of any one of

[70] to

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

[90] The method of any one of

[70] to

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

[91] The method of any one of

[70] to

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

[92] A method for producing any one of

[70] to

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

[0016]

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

[23] or

[24] .

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

[23] or

[24] .

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

[23] or

[24] .

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

[23] or

[24] , and evaluating the efficacy of the preventive or therapeutic agent based on a change in the detected amount of a reporter protein before and after administration.

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

[23] or

[24] , and evaluating the toxicity of the preventive or therapeutic agent based on a change in the detected amount of a reporter protein before and after administration.

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

[23] or

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

[0017]

[99] A method for evaluating the effect of a medicinal molecule using a purine receptor ligand as an index, comprising the steps of administering the medicinal molecule to a genetically modified non-human animal of any one of [1] to

[24] , or adding the medicinal molecule to a genetically modified animal cell of any one of

[25] to

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

[0100] A method for evaluating the toxicity of a medicinal molecule using a purine receptor ligand as an index, comprising the steps of administering the medicinal molecule to a genetically modified non-human animal of any one of [1] to

[24] , or adding the medicinal molecule to a genetically modified animal cell of any one of

[25] to

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

[0101] A method for evaluating the effect of a medicinal molecule dependent on a purinergic receptor ligand, comprising the steps of administering the drug to a genetically modified non-human animal of any one of [1] to

[24] , or adding the medicinal molecule to a genetically modified animal cell of any one of

[25] to

[45] , and evaluating the effect of the medicinal molecule in the presence of a purinergic receptor ligand, as represented by the detected amount of a reporter protein.

[0102] A method for screening for a purinergic receptor ligand-dependent medicinal molecule, comprising the steps of administering a test substance to a genetically modified non-human animal of any one of [1] to

[24] , or adding the medicinal molecule to a genetically modified animal cell of any one of

[25] to

[45] , and screening for a substance of interest based on the effect of the medicinal molecule in the presence of a purinergic receptor ligand, which is represented by the detected amount of a reporter protein. [Effects of the Invention]

[0018] According to the present invention, it is possible to provide an evaluation system that is capable of detecting extracellular purinergic receptor ligands systemically, over time, and in a minimally invasive manner.

[0019] The present invention makes it possible to provide a genetically modified non-human animal that allows non-invasive time-dependent detection of purinergic receptor ligands throughout the body. By using the genetically modified non-human animal of the present invention, it becomes possible to identify the site of causation of a disease in which purinergic receptor ligands are thought to be involved and to investigate the timing of disease onset. Furthermore, since the genetically modified non-human animal of the present invention can be applied to drug screening using the mouse, it becomes possible to efficiently develop therapeutic agents for diseases in which purinergic receptor ligands are involved. [Brief explanation of the drawings]

[0020] [Figure 1] Figure 1 shows an overview of the P2Y11-split Luc (C-terminus) knock-in vector. [Figure 2] FIG. 2 shows an outline of the arrestin-split Luc (N-terminus) knock-in vector. [Figure 3] Figure 3 is a photograph showing the results of measuring luminescence signal intensity after 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. P2Y: P2Y11-split Luc (C-terminus) knock-in; Arrb: Arrestin-split Luc (N-terminus) knock-in; wt: wild-type. [Figure 4] Figure 4 shows the analytical data obtained by visualizing the luminescence signals detected in fibroblasts derived from P2Y11-split Luc (C-terminus) and Arrestin-split Luc (N-terminus) double knock-in mice treated with various concentrations of ATP and D-luciferin substrate. [Figure 5] Figure 5 is a graph showing the luminescence signal intensity detected when fibroblasts derived from P2Y11-split Luc (C-terminus) and Arrestin-split Luc (N-terminus) double knock-in mice were treated with various concentrations of ATP and D-luciferin substrate. [Figure 6]Figure 6 is a photograph showing the results of measuring the luminescence signal intensity after subcutaneous administration of a mixture of various concentrations 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. [Figure 7] Figure 7 is a graph showing the luminescence signal intensity after subcutaneous administration of a mixture of various concentrations 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. [Figure 8] Figure 8 is a photograph showing the results of noninvasive visualization of ATP leakage in the liver associated with hepatocellular damage caused by hydrodynamic injection in P2Y11-split Luc (C-terminus) (P2Y) and Arrestin-split Luc (N-terminus) (Arrb) double knock-in mice, measured by measuring luminescence signal intensity. [Figure 9] Figure 9 shows photographs of ATP leakage associated with liver canceration induced by hydrodynamic injection in P2Y11-split Luc (C-terminus) (P2Y) and Arrestin-split Luc (N-terminus) (Arrb) double knock-in mice. (A) ATP leakage was visualized noninvasively by measuring luminescence signal intensity, and (B) ATP leakage was visualized in excised livers by measuring luminescence signal intensity. DETAILED DESCRIPTION OF THE INVENTION

[0021] 1. Genetically modified non-human animals The present invention relates to a genetically modified non-human animal that expresses a first fusion protein and a second fusion protein for detecting extracellularly present purinergic receptor ligands.

[0022] (1-1) First fusion protein In the present invention, the "first fusion protein" comprises or consists of a membrane protein that binds to a purinergic receptor ligand and a first reporter protein.

[0023] In the first fusion protein, the membrane protein that binds to a purinergic receptor ligand and the first reporter protein may be linked directly or via a linker. The membrane protein that binds to a purinergic receptor ligand and the first reporter protein can be linked 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 that binds to a purinergic receptor ligand and the first reporter protein can be linked in this order from the N-terminus. Any conventionally known "linker" can be used, for example, a peptide linker can be used. The number and type of amino acids of the peptide linker are not particularly limited.

[0024] (1-1-1) Purinergic receptor ligand-binding membrane proteins In the present invention, a "membrane protein that binds to a purinergic receptor ligand" (hereinafter, sometimes simply referred to as a "membrane protein") refers to a protein that is present in a manner that is bound to or penetrates the cell membrane and is capable of binding to a purinergic receptor ligand present outside the cell. In the present invention, the membrane protein is not particularly limited as long as it is capable of binding to a purinergic receptor ligand present outside the cell, and suitable examples include purinergic receptors such as G protein-coupled receptors (hereinafter, referred to as "GPCRs") and ligand-gated ionotropic receptors, or parts thereof. Preferably, the membrane protein is a purinergic receptor, such as a GPCR.

[0025] GPCRs are primarily seven-transmembrane receptors, with seven α-helices spanning the cytoplasmic membrane, the N-terminal domain located extracellularly, and the C-terminal domain located intracellularly. When a ligand binds to the extracellular domain, GPCRs are activated, undergoing a conformational change, and the intracellular domain is phosphorylated by the action of G protein-coupled receptor kinases.

[0026] Purinergic receptor GPCRs include, but are not limited to, P1 receptors and P2 receptors. Preferably, the membrane proteins are P1 receptors and P2 receptors.

[0027] P1 receptors, which are purinergic 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.

[0028] P2 receptors, which are purinergic receptors, are GPCRs that use AMP, ADP, ATP, UTP, UDP, and UDP-glucose as ligands, and are preferably P2Y receptors. P2Y receptors include P2Y1 receptor, P2Y2 receptor, P2Y4B receptor, P2Y6 receptor, and P2Y 11 Receptor, P2Y 12 Receptor, P2Y 13 receptors, and P2Y 14 In the present invention, one or more receptors selected from these can be used as membrane proteins. More preferably, P2Y2 receptor, P2Y 11 receptors, especially P2Y 11 Receptors are preferred.

[0029] In the present invention, known membrane proteins that bind to purinergic receptor ligands can be used, and known proteins registered in public databases such as NCBI and GenBank can be used. For example, the A1 receptor, which belongs to the P1 receptor family, is registered as NP_000665.1 for human origin and NP_001008533.1 for mouse origin. The P2Y2 receptor is registered as NP_002555.3 for human origin and NP_001289275.1 for mouse origin. 11 The receptor of human origin 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, the P2Y receptor represented by the amino acid sequence of SEQ ID NO: 9 is used. 11 Receptors can be used as membrane proteins that bind purinergic receptor ligands.

[0030] In the present invention, analogs and mutants of "membrane proteins that bind to purinergic receptor ligands" can also be used as long as they are capable of binding to extracellular purinergic receptor ligands. Analogs and mutants of "membrane proteins that bind to purinergic receptor ligands" refer to (i) proteins that have an amino acid sequence in which 1 to 50, e.g., 1 to 20 or 1 to 10, amino acids have been deleted, substituted, added, or inserted in the amino acid sequence of the "membrane protein that binds to purinergic receptor ligands," and that are capable of binding to extracellular purinergic receptor ligands, or (ii) proteins that have an amino acid sequence that shares 80% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 99% or more sequence identity with the amino acid sequence of the "membrane protein that binds to purinergic receptor ligands," and that are capable of binding to extracellular purinergic receptor ligands. The sequence identity of amino acid sequences can be calculated using known methods, such as BLAST (Basic Local Alignment Search Tool at the National Center for Biological Information), using the default settings. The sequence identity of an amino acid sequence may be calculated based on the entire protein capable of binding to a purinergic receptor ligand, or may be calculated based on a binding domain having binding activity to a purinergic receptor ligand. For example, if the sequence homology of a binding domain in a receptor that binds to a specific purinergic receptor ligand is high, the sequence homology of the specific binding domain is considered to be high even if the homology of the entire membrane protein is not high because the sequence homology of a portion other than the binding domain, such as a transmembrane domain, is low.

[0031] Furthermore, in the present invention, the terms "those parts" and "parts thereof" used in connection with membrane proteins refer to proteins that consist of a portion of the amino acid sequence of a membrane protein and are capable of binding to at least an extracellular purinergic receptor ligand. Examples of such proteins include those that consist of at least a portion of the amino acid sequence of a membrane protein, such as a domain required for binding to an extracellular purinergic receptor ligand (e.g., a binding domain in a receptor that binds to a specific purinergic receptor ligand) or a domain required for binding to a cell membrane (e.g., a transmembrane domain). Each domain of a membrane protein can be determined based on amino acid sequence information, gene information, and the like registered in public databases such as NCBI and GenBank.

[0032] (1-1-2) First reporter protein In the present invention, the term "first reporter protein" refers to a protein that functions as a reporter protein together with a second reporter protein in a second fusion protein described below. "Functioning as a reporter protein together" means that the first reporter protein and the second reporter protein only function as a reporter protein when they come into close proximity or bind to each other, and serve as an indicator of the expression or localization of a specific molecule. Examples of such proteins that "function as a reporter protein together" include split reporter proteins and combinations of proteins that produce fluorescence resonance energy transfer (FRET) or bioluminescence resonance energy transfer (BRET).

[0033] A "split reporter protein" is a reporter protein split into two or more subunits. When the subunits are separated, they do not function as a reporter protein. However, when the two or more split subunits are brought into close proximity or bound to each other, the reporter protein can be reconstituted and function. A "subunit-split reporter protein" refers to a protein originally composed of two or more subunits that can be split into individual 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, 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. Other examples include, but are not limited to, split fluorescent proteins such as split GFP, split YFP, and split CYP. While previously reported split reporter proteins can be used, it is also possible to artificially split specific reporter proteins.

[0034] "FRET" refers to the phenomenon in which, when two fluorescent proteins, the fluorescence wavelength of a first fluorescent protein (donor) and the excitation light wavelength of a second fluorescent protein (acceptor) overlap in spectrum, are brought into close proximity, the energy absorbed by the excitation of the first fluorescent protein is used to excite the second fluorescent protein, causing the second fluorescent protein to emit fluorescence. Furthermore, "BRET" refers to the phenomenon in which, when a bioluminescent protein is used instead of the first fluorescent protein (donor) in FRET, and the bioluminescent protein and the second fluorescent protein are brought into close proximity, the energy of the bioluminescent protein is used to excite the second fluorescent protein, causing the second fluorescent protein to emit fluorescence. Examples of bioluminescent proteins include luciferase.

[0035] Therefore, the "first reporter protein" in the present invention can be one subunit in a split reporter protein, or one fluorescent protein in a combination of fluorescent proteins capable of generating FRET, or any protein in a combination of a bioluminescent protein and a fluorescent protein capable of generating BRET.

[0036] (1-2) Second fusion protein In the present invention, the "second fusion protein" comprises or consists of a protein capable of binding to the membrane protein bound to a purinergic receptor ligand present outside the cell (hereinafter, sometimes simply referred to as "a protein capable of binding to a membrane protein bound to a ligand") and a second reporter protein.

[0037] In the second fusion protein, the protein capable of binding to the membrane protein bound to the ligand and the second reporter protein may be linked directly or via a linker. The protein capable of binding to the membrane protein bound to the ligand and the second reporter protein can be linked 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 capable of binding to the membrane protein bound to the ligand can be linked in this order from the N-terminus. Any conventionally known "linker" can be used, for example, a peptide linker can be used. The number and type of amino acids of the peptide linker are not particularly limited.

[0038] (1-2-1) Proteins that can bind to ligand-bound membrane proteins In the present invention, "a protein capable of binding to the membrane protein bound to an extracellular purinergic receptor ligand" refers to a protein capable of specifically or selectively binding to the above-mentioned membrane protein bound to an extracellular purinergic receptor ligand. In the present invention, such a protein is not particularly limited, and membrane protein activation regulators, antibodies, receptor kinases, transcription factors, etc., or parts thereof, can be suitably used. In the present invention, when the membrane protein is a GPCR, arrestins, G proteins, and GPCR kinases, which are GPCR activation regulators, can be suitably used.

[0039] "Arrestins" are activation regulators that bind to the intracellular domain of GPCRs that are activated by ligand binding to the extracellular domain and are phosphorylated by the action of G protein-coupled receptor kinases, resulting in desensitization of the GPCR. Four subtypes of arrestins are known in mammals: arrestin-1, arrestin-2 (also known as "β-arrestin-1"), arrestin-3 (also known as "β-arrestin-2"), and arrestin-4 (also known as "X-arrestin"). In the present invention, arrestin-2 (β-arrestin-1) and arrestin-3 (β-arrestin-2) are preferably used, and it is particularly preferred to use arrestin-2 (β-arrestin-1) and arrestin-3 (β-arrestin-2) in combination. By using arrestin-2 (β-arrestin-1) and arrestin-3 (β-arrestin-2) in combination, both signal transduction mediated by these two subtypes can be detected.

[0040] In the present invention, known proteins capable of binding to a membrane protein bound to a ligand can be used, 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, human-derived β-arrestin-1 and β-arrestin-2 are registered as NP_004032.2 and NP_004304.1, respectively, and mouse-derived β-arrestin-2 are registered as NP_796205.1 and NP_001258287.1, respectively. These 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 NO: 10 and SEQ ID NO: 11 can be used in the present invention as proteins capable of binding to a membrane protein bound to a ligand.

[0041] In the present invention, analogs and mutants of "proteins capable of binding to a membrane protein bound to a ligand" can also be used as long as they are capable of binding to a membrane protein bound to a ligand. Analogs and mutants of "proteins capable of binding to a membrane protein bound to a ligand" refer to (a) proteins that have an amino acid sequence in which 1 to 50, e.g., 1 to 20, or 1 to 10 amino acids have been deleted, substituted, added, or inserted in the amino acid sequence of the "protein capable of binding to a membrane protein bound to a ligand," and that are capable of binding to a membrane protein bound to a ligand, or (b) proteins that have an amino acid sequence that shares 80% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 99% or more sequence identity with the amino acid sequence of the "protein capable of binding to a membrane protein bound to a ligand," and that are capable of binding to a membrane protein bound to a ligand. The sequence identity of the amino acid sequence can be calculated as described above, and may be calculated based on the entire protein capable of binding to a membrane protein bound to a ligand, or may be calculated based on the binding domain that can bind to a membrane protein bound to a ligand.

[0042] Furthermore, in the present invention, the terms "portion thereof" and "part thereof" used in connection with a protein capable of binding to a membrane protein bound to a ligand refer to a protein consisting of a portion of the amino acid sequence of the protein and capable of binding to at least the membrane protein bound to the ligand. Examples of such proteins include those consisting of at least a portion of the amino acid sequence of a protein capable of binding to a membrane protein bound to a ligand, such as a domain necessary for binding to the membrane protein bound to the ligand. For example, when the protein capable of binding to a membrane protein bound to a ligand is an antibody, examples of the portion include partial fragments of the antibody that have binding activity to the membrane protein bound to the ligand, such as Fab, F(ab')2, and scFV.

[0043] (1-2-2) Second reporter protein In the present invention, the term "second reporter protein" refers to a protein that functions as a reporter protein together with the first reporter protein in the first fusion protein described above. The "second reporter protein" in the present invention can be a subunit other than the first reporter protein in a split reporter protein, a fluorescent protein other than the first reporter protein in a combination of fluorescent proteins capable of generating FRET, or a protein other than the first reporter protein in a combination of a bioluminescent protein and a fluorescent protein capable of generating BRET.

[0044] (1-3) Genetically modified non-human animals In the present invention, "genetic modification" is used in the broadest sense and refers to any manipulation of modifying a nucleic acid sequence possessed by a host, including the introduction of a gene or partial sequence of a gene from a heterologous organism, or a nucleic acid sequence having a function such as a promoter, into a host organism. In other words, genetic modification also includes deleting the full length or partial sequence of a gene possessed by a host organism, or replacing it with a heterologous gene having the same function as the gene possessed by the host organism. Genetic modification also includes the use of genetic modification techniques to perform amino acid substitutions on a nucleotide sequence encoding a specific protein, or the creation of a nucleotide sequence encoding a fusion protein by linking it to a nucleotide sequence encoding another protein.

[0045] In the present invention, the "non-human animal" is not intended to be particularly limited, but is preferably a non-human mammal. For example, rodents such as mice, rats, and hamsters, non-human primates such as monkeys and chimpanzees, other mammals such as rabbits, sheep, cows, and pigs, as well as birds, amphibians, reptiles, and fish can be used as the non-human animal of the present invention. Rodents are particularly preferred, and mice are most preferred.

[0046] The genetically modified non-human animal of the present invention has cells that express the first fusion protein and the second fusion protein, and preferably has cells that express the first fusion protein and the second fusion protein throughout its body.

[0047] In the genetically modified non-human animals of the present invention, when a purinergic receptor ligand present extracellularly in a cell expressing a first fusion protein and a second fusion protein binds to the membrane protein in the first fusion protein, a protein in the second fusion protein capable of binding to the membrane protein bound to the ligand binds to the first fusion protein. This causes the first reporter protein in the first fusion protein and the second reporter protein in the second fusion protein to come into close proximity or bind, and together function as a reporter protein. By detecting this reporter protein, the location, amount, etc. of the purinergic receptor ligand present extracellularly in the genetically modified non-human animals of the present invention can be evaluated.

[0048] The method for detecting extracellular purinergic receptor ligands using the genetically modified non-human animals of the present invention will be described later.

[0049] (1-3-1) Method for producing genetically modified non-human animals The genetically modified non-human animals of the present invention can be produced using conventionally known genetic modification techniques by introducing genes encoding the first and second fusion proteins into a non-human animal. As used herein, the term "gene" refers to DNA, RNA, or a DNA / RNA hybrid, and is not particularly limited in form as long as it encodes a specific protein.

[0050] The gene encoding the first fusion protein can be obtained by linking a gene encoding a membrane protein that binds to a purinergic receptor ligand and a gene encoding a first reporter protein, either directly or via a nucleotide sequence encoding a linker. The gene encoding the membrane protein that binds to a purinergic receptor ligand may be cloned from an arbitrary gene library using gene information registered in the above-mentioned publicly available database, or may be chemically produced by gene synthesis technology. The gene encoding the first reporter protein is commercially available, or may be chemically produced by gene synthesis techniques together with a gene encoding a membrane protein that binds to a purinergic receptor ligand.

[0051] In addition, a gene encoding a second fusion protein can be obtained by linking a protein capable of binding to a membrane protein bound to a ligand to a gene encoding a second reporter protein, either directly or via a base sequence encoding a linker. The gene encoding the protein capable of binding to the ligand-bound membrane protein may be cloned from an arbitrary gene library using the gene information registered in the above-mentioned public database, or may be chemically produced by gene synthesis technology. The gene encoding the second reporter protein can be commercially available, or it can be chemically produced by gene synthesis techniques together with a gene encoding a protein capable of binding to the ligand-bound membrane protein.

[0052] In addition to the above genes, the gene encoding the first fusion protein and the gene encoding the second fusion protein may each contain, as necessary, regulatory sequences for transcription and translation (e.g., promoter sequences, enhancer sequences, splicing acceptor sequences, terminator sequences, polyA sequences, etc.), selection marker genes (e.g., neomycin resistance gene, hygromycin resistance gene, puromycin resistance gene, diphtheria toxin A gene, herpes simplex virus thymidine kinase gene, etc.), tag sequences for isolation and purification, etc.

[0053] The method for introducing the gene encoding the first fusion protein and the gene encoding the second fusion protein into a non-human animal is not particularly limited, and can be carried out using any known method, such as microinjection of a DNA vector (plasmid vector, cosmid vector, and non-plasmid vector such as bacterial artificial chromosome (BAC) or yeast artificial chromosome (YAC)) or a viral vector (e.g., retroviral vector) into the pronucleus of a fertilized egg, as well as electroporation or lipofection of cells such as embryonic stem cells, spermatogonial stem cells, and induced pluripotent stem cells (iPS cells). The gene encoding the first fusion protein and the gene encoding the second fusion protein may be incorporated into the same vector, or may be incorporated into separate vectors.

[0054] Alternatively, the gene encoding the first fusion protein and the gene encoding the second fusion protein may each be inserted into a host chromosome. In a non-limiting embodiment, each gene can be inserted into a host chromosome by random integration at any desired position, or by using zinc finger nucleases (US 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 (US Patent Nos. This can be achieved by modifying the target site using techniques such as those disclosed in U.S. Patent Nos. 8,420,782, 8,440,431, 8,440,432, and 8,450,471) or by modifying the target site using CRISPR-Cas9 technology (U.S. Patent Nos. 8,697,359, 8,795,965, and 8,771,945). In a non-limiting embodiment of the present invention, the insertion sites on the chromosome of the gene encoding the first fusion protein and the gene encoding the second fusion protein are not particularly limited, and examples thereof include the Rosa26 gene, Hippo gene, and TIGRE gene, which are known as stable expression regions for foreign genes. These genes can be operably inserted into the predetermined sites using homologous recombination or the like. As used herein, the term "operable" means that the inserted genes encoding the first fusion protein and the second fusion protein are under the control of a transcriptional regulatory sequence on the host chromosome, or under the control of a transcriptional regulatory sequence possessed by each gene, so that the first fusion protein and the second fusion protein are expressed, respectively.

[0055] The genetically modified non-human animals of the present invention may be produced by introducing both a gene encoding the first fusion protein and a gene encoding the second fusion protein into a single individual (cell), or by mating animals into which a gene encoding the first fusion protein and a gene encoding the second fusion protein have been introduced, respectively, to produce offspring, and then selecting from the offspring those carrying both genes.

[0056] The genetically modified non-human animals of the present invention are heterozygous or homozygous for each of the gene encoding the first fusion protein and the gene encoding the second fusion protein, and are preferably homozygous for both genes.

[0057] The genetically modified non-human animals of the present invention can be screened based on the detectable amount of the reporter protein, and genetically modified non-human animals having a desired detectable amount of the reporter protein can be obtained. The genetically modified non-human animals having a predetermined detectable amount of the reporter protein thus obtained can be used in the methods for evaluating and screening purinergic receptor ligand-dependent medicinal molecules described below.

[0058] (1-3-2) Disease model animals The genetically modified non-human animals of the present invention can be used as disease model animals. The "disease" is preferably a disease characterized by extracellular purine receptor ligands, such as cancer, acute inflammation, chronic inflammation, infectious disease, fibrosis, physical or chemical organ damage, and cell damage caused by anticancer drugs.

[0059] In the present invention, "cancer" refers to a malignant neoplasm, which may be either metastatic or non-metastatic. Non-limiting examples of carcinomas originating 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, colorectal cancer, colon cancer, bladder cancer, and ovarian cancer. Non-limiting examples of sarcomas originating from non-epithelial tissues (stroma) such as muscle include osteosarcoma, chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma, liposarcoma, and angiosarcoma. Further, non-limiting examples of hematopoietic cancers include malignant lymphomas, including Hodgkin's lymphoma and non-Hodgkin's lymphoma; leukemias, including acute or chronic myelocytic leukemia, and acute or chronic lymphatic leukemia; and multiple myeloma. In the present invention, "cancer" also includes "neoplasms." Neoplasms result in the formation of tumors, which are characterized in part by angiogenesis. Neoplasms refer to any newly formed pathological tissue tumor, and can be benign, such as hemangioma, glioma, and teratoma, or malignant, such as carcinoma, sarcoma, glioma, astrocytoma, neuroblastoma, and retinoblastoma.

[0060] The genetically modified non-human animals of the present invention can be generated as cancer model animals bearing cancer tissue. "Cancer tissue" refers to tissue containing at least one cancer cell. Therefore, it refers to all cell types that contribute to the formation of a tumor mass, including cancer cells and endothelial cells, such as cancer tissue containing cancer cells and blood vessels. A tumor mass refers to a foci of tumor tissue, and "tumor" generally refers to benign or malignant neoplasms.

[0061] Cancer model animals can be produced in the genetically modified non-human animals of the present invention by conventionally known techniques such as administering carcinogens, expressing cancer genes through genetic modification, or transplanting cancer cells.

[0062] The disease model animals of the present invention can be used in the detection of the site and time of disease onset described below, monitoring the course of disease, evaluating medicinal molecules effective in preventing or treating disease, evaluating the toxicity and side effects of drugs, and screening methods.

[0063] 2. Genetically modified animal cells The present invention also relates to genetically modified animal cells that express a first fusion protein and a second fusion protein for detecting a purinergic receptor ligand present extracellularly. In the present invention, the "first fusion protein" and the "second fusion protein" are as defined above.

[0064] In the present invention, "animal cells" refers to cells derived from animals belonging to the phylum Vertebrates and cells derived from invertebrates (animals other than animals belonging to the phylum Vertebrates), and is not particularly limited. Preferably, in the present invention, "animal cells" refers to cells derived from animals belonging to the phylum Vertebrates. The phylum Vertebrates includes the classes Agnathostomata and Gnathostomata, which in turn includes the classes Mammalia, Aves, Amphibia, and Reptilia. More preferably, in the present invention, "animal cells" refers to cells derived from animals belonging to the class Mammalia, known as mammals, and is not particularly limited, but is particularly preferably cells derived from mice, rats, humans, monkeys, pigs, dogs, sheep, goats, and the like.

[0065] In the genetically modified animal cells of the present invention, when an extracellular purinergic receptor ligand binds to the membrane protein in the first fusion protein, a protein in the second fusion protein capable of binding to the membrane protein bound to the ligand binds to the extracellular purinergic receptor ligand. This causes the first reporter protein in the first fusion protein and the second reporter protein in the second fusion protein to come into close proximity or bind to each other, and together function as a reporter protein. By detecting this reporter protein, the presence or amount of an extracellular purinergic receptor ligand in the genetically modified animal cells of the present invention can be evaluated.

[0066] The method for detecting extracellular purine receptor ligands using the genetically modified animal cells of the present invention will be described later.

[0067] (2-1) Method for producing genetically modified animal cells The genetically modified animal cells of the present invention can be produced using conventionally known genetic modification techniques, and can be produced by introducing a gene encoding the first fusion protein described above and a gene encoding the second fusion protein described above into an animal cell.

[0068] The gene encoding the first fusion protein and the gene encoding the second fusion protein can be introduced into animal cells using known techniques as appropriate, and for example, DNA vectors (plasmid vectors, cosmid vectors, and non-plasmid vectors such as bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs)) or viral vectors (e.g., retroviral vectors), electroporation, lipofection, etc. can be used. The gene encoding the first fusion protein and the gene encoding the second fusion protein may be incorporated into the same vector, or may be incorporated into separate vectors.

[0069] Alternatively, the gene encoding the first fusion protein and the gene encoding the second fusion protein may each be inserted into a host chromosome. In a non-limiting embodiment, insertion of each gene into a host chromosome can be achieved by modifying any desired position by random integration, or by modifying a target position using zinc finger nuclease, TALEN, or CRISPR-Cas9 technology. In a non-limiting embodiment of the present invention, the insertion positions of the genes encoding the first fusion protein and the second fusion protein on the chromosome are not particularly limited, and examples include the Rosa26 gene, Hippo gene, and TIGRE gene, and the genes can be operably inserted into predetermined positions using homologous recombination or the like.

[0070] The genetically modified animal cells of the present invention are heterozygous or homozygous for each of the gene encoding the first fusion protein and the gene encoding the second fusion protein, and are preferably homozygous for both genes.

[0071] Alternatively, the genetically modified animal cells of the present invention can be prepared by isolating them from the above-mentioned genetically modified non-human animal of the present invention.

[0072] 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. That is, cells contained in the living body of the above-mentioned genetically modified non-human animal of the present invention and genetically modified animal cells of the present invention transplanted into the living body of an animal also fall under the genetically modified animal cells of the present invention.

[0073] In the genetically modified animal cells of the present invention, an extracellular purinergic receptor ligand binds to a membrane protein in the first fusion protein that binds to the purinergic receptor ligand, activating the membrane protein, which then causes a protein in the second fusion protein that binds to the activated membrane protein to bind to the membrane protein. This brings the first reporter protein in the first fusion protein and the second reporter protein in the second fusion protein into close proximity or bind to each other, functioning together as a reporter protein. By detecting and measuring this reporter protein, the amount of the extracellular purinergic receptor ligand and its effect can be evaluated.

[0074] The genetically modified animal cells of the present invention can be provided in a detection kit for detecting extracellular purinergic receptor ligands. In addition to the cells, the kit can include instructions for cell culture methods and reporter protein detection methods, as well as a calibration curve showing the range of the detected amount of reporter protein and the amount of extracellular purinergic receptor ligand. Furthermore, if the reporter protein is an enzyme, the kit can also include an appropriate substrate.

[0075] Methods for detecting and evaluating extracellular purinergic receptor ligands using the genetically modified animal cells of the present invention will be described later.

[0076] 3. Various evaluation methods using genetically modified animals and genetically modified animal cells (3-1) Detection of extracellular purinergic receptor ligands In one aspect, the genetically modified animals and genetically modified animal cells of the present invention can be used in a method for detecting extracellular purinergic receptor ligands. In the present invention, "detecting" means qualitatively distinguishing and / or quantitatively measuring, and "detecting a purinergic receptor ligand" means either qualitatively distinguishing the presence or absence of a purinergic receptor ligand or quantitatively measuring the concentration of the purinergic receptor ligand outside the cells, or both.

[0077] In the present invention, detection of a purinergic receptor ligand can be achieved by detecting a reporter protein. 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 membrane protein in the first fusion protein, a protein in the second fusion protein capable of binding to the membrane protein bound to the ligand binds to the purinergic receptor ligand. This causes the first reporter protein in the first fusion protein and the second reporter protein in the second fusion protein to come into close proximity or bind to each other, and together they function as a reporter protein. By detecting this reporter protein, the purinergic receptor ligand present outside the cell can be detected.

[0078] The method for detecting a reporter protein can be appropriately selected depending on the reporter protein used and can be performed using a common technique. For example, when a fluorescent protein is used as the reporter protein, detection can be performed by irradiating the reporter with light of the excitation wavelength of the fluorescent protein and detecting the emitted fluorescence. Specific examples of such fluorescent proteins include GFP, CYP, and YFP. When an enzyme is used as the reporter protein, detection 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 reaction product. When the reaction product is a fluorescent substance, detection can be performed by irradiating the reporter with light of the excitation wavelength of the fluorescent substance and detecting the emitted fluorescence. When the reaction product emits light, detection can be performed by detecting the emission. Alternatively, when the reaction product is a dye, detection can be performed by measuring the absorbance of the dye. More specifically, when luciferase is used as the reporter protein, detection can be performed by administering or adding a cell membrane-permeable luciferin as a substrate, causing a luciferin-luciferase reaction, and detecting the emission of the produced protein.

[0079] For example, according to the present invention, ATP can be used as a purinergic receptor ligand, and extracellular ATP can be detected using the genetically modified animals and genetically modified cells of the present invention.

[0080] (3-2) Evaluation and screening of medicinal molecules In another aspect, the genetically modified animals and genetically modified animal cells of the present invention can be used in a method for evaluating the effect of a medicinal molecule using an extracellular purinergic receptor ligand as an indicator.

[0081] In this method, the test substance is administered to a genetically modified animal of the present invention or added to the culture medium in which genetically modified animal cells of the present invention are cultured, and if the reporter protein is an enzyme, a substrate for the enzyme is further administered or added as necessary to detect the reporter protein, and the amount detected is compared with the amount detected before administration or addition, thereby evaluating the efficacy of the test substance.

[0082] The "test substance" is not particularly limited, but examples include low molecular weight compounds, amino acids, nucleic acids, lipids, sugars, extracts of natural products, etc., and natural compound libraries, synthetic compound libraries, metabolite libraries, existing drug libraries, etc. can be used.

[0083] The test substance and substrate (if necessary) can be administered to the genetically modified animal of the present invention by any means, including, but not limited to, injection, such as intravenous injection, intradermal injection, subcutaneous injection, intramuscular injection, or intraperitoneal injection. The test substance and substrate may be administered to the genetically modified animal of the present invention simultaneously or separately, and the administration means may be the same or different.

[0084] The test substance and the substrate (if necessary) may be added simultaneously or separately to the medium for culturing the genetically modified animal cells of the present invention.

[0085] When the amount of reporter protein detected is reduced when the test substance is administered or added compared to before administration or addition, the test substance can be evaluated as having the effect of suppressing the production of a purine receptor ligand. On the other hand, when the amount of reporter protein detected is increased compared to before administration or addition, the test substance can be evaluated as having the effect of enhancing the production of a purine receptor ligand, and medicinal molecules having each effect can be screened based on this evaluation.

[0086] When a test substance has cytotoxicity or organ damage as its primary or side effect, the toxicity of the test substance can be evaluated by detecting purinergic receptor ligands leaked from damaged tissues or secreted by immune cells present in damaged tissues. When administered to genetically modified animals, it is possible to non-invasively identify the organs exhibiting toxicity and also to capture changes over time.

[0087] More specifically, ATP can be used as an example of a purinergic receptor ligand, and according to this method, the efficacy of enhancing or suppressing ATP production can be evaluated using ATP as an indicator, and medicinal molecules having such effects can be screened.

[0088] (3-3) Evaluation and screening of purinergic receptor ligand-dependent medicinal molecules In another aspect, the genetically modified animals and genetically modified animal cells of the present invention can be used in methods for evaluating the effects of purinergic receptor ligand-dependent medicinal molecules.

[0089] In the present invention, the term "purinergic receptor ligand-dependent medicinal molecule" refers to a medicinal molecule that is activated or inactivated depending on the presence or absence or amount of a purinergic receptor ligand present outside the cell.

[0090] In this method, a test substance is administered to a genetically modified animal of the present invention or added to a medium for culturing genetically modified animal cells of the present invention, and the efficacy of the test substance can be evaluated in the presence of a predetermined amount of an extracellular purinergic receptor ligand.

[0091] In this method, the amount of purinergic receptor ligand present extracellularly in the genetically modified animal of the present invention or the genetically modified animal cells of the present invention can be confirmed by detecting a reporter protein. The reporter protein detection only needs to clarify the amount of extracellular purinergic receptor ligand at least during the action of the test substance, and can be carried out either before administration or addition of the test substance, or after administration or addition of the test substance, or both. Preferably, the reporter protein detection can be carried out before administration or addition of the test substance.

[0092] In this method, a genetically modified animal of the present invention that has been pre-screened based on the amount of a predetermined reporter protein detected can be used. Alternatively, the amount of a purinergic receptor ligand present extracellularly in a genetically modified animal of the present invention or a genetically modified animal cell of the present invention may be adjusted by administering a purinergic receptor ligand to the genetically modified animal of the present invention or by adding the ligand to the medium in which the genetically modified animal cell of the present invention is cultured.

[0093] 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. The reporter protein can be detected as described above depending on the reporter protein used.

[0094] When a change in efficacy is observed depending on the presence or absence or amount of a purinergic receptor ligand present outside the cell, the test substance can be evaluated as a purinergic receptor ligand-dependent medicinal molecule, and further, purinergic receptor ligand-dependent medicinal molecules can be screened based on this evaluation.

[0095] More specifically, ATP can be mentioned as an example of a purinergic receptor ligand, and this method makes it possible to evaluate the effects of ATP-dependent medicinal molecules and to screen for ATP-dependent medicinal molecules.

[0096] (3-4) Monitoring disease pathology and screening of therapeutic molecules for disease prevention or treatment In yet another embodiment, the genetically modified animal of the present invention can be used in a method for detecting the site and time of disease onset or for monitoring the pathological condition of a disease. In this method, by detecting the reporter protein in the disease model animal, the site and severity of disease onset, as well as the time of onset, can be identified for a disease characterized by extracellularly present purinergic receptor ligands, and by identifying these over time, the progression of the pathological condition of the disease can be monitored.

[0097] In another embodiment, the genetically modified animals of the present invention can be used in methods for evaluating preventive or therapeutic agents for diseases, or for screening for effective preventive or therapeutic agents.

[0098] In this method, the test substance is administered to the above-mentioned disease model animal, and if the reporter protein is an enzyme, a substrate for the enzyme is further administered to detect the reporter protein. The amount of the reporter protein detected is compared with the amount detected before administration, thereby evaluating the efficacy of the test substance.

[0099] If the amount of reporter protein detected after administration of the test substance is reduced compared to before administration, the test substance can be evaluated as being effective in preventing or treating a disease, and medicinal molecules having each effect can be screened based on this evaluation.

[0100] The "test substance" is as described above, and the test substance and substrate (if necessary) can be administered by any means as described above. The reporter protein can be detected as described above depending on the reporter protein used.

[0101] More specifically, ATP can be used as an example of a purinergic receptor ligand, and according to this method, the effects of medicinal molecules effective for the prevention or treatment of cancer can be evaluated using ATP as an indicator, making it possible to screen for medicinal molecules effective for the prevention or treatment of cancer. Furthermore, this method can be used to screen for medicinal molecules effective for the prevention or treatment of diseases other than cancer, as well as side effects caused by other drugs. Non-limiting examples of diseases and side effects other than cancer include acute inflammation, chronic inflammation, infection, fibrosis, physical or chemical organ damage, and cell damage caused by anticancer drugs, etc., and ATP can be used as an indicator to evaluate the effects of medicinal molecules effective for the prevention or treatment of these diseases and side effects.

[0102] In this specification, unless a term is explained by specifying a limitation indicating a quantity such as "one" or "multiple," the terms described in this specification are not construed as being particularly limited in quantity, but are understood to be terms having the meaning of "one or multiple."

[0103] It will be understood by those skilled in the art that any combination of one or more aspects described in this specification is also included in the present invention, as long as there is no technical contradiction based on the technical common sense of those skilled in the art. All prior art documents cited herein are hereby incorporated by reference. This specification includes the contents disclosed in the specification and / or drawings of Japanese Patent Application No. 2019-225404, which is the priority document of this application. [Example]

[0104] Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0105] Example 1: Generation of P2Y11-split Luc (C-terminus) and Arrestin-split Luc (N-terminus) double knock-in mice 1.1. Construction of P2Y11-split Luc (C-terminus) knock-in vector A splicing acceptor, polyA addition signal, and FLAG tag were added to the sequence combining the human P2Y11 gene and the C-terminal region of the luciferase gene to create the "P2Y11-split Luc (C-terminus)" expression cassette (SEQ ID NO: 1). This sequence was cloned into the pZDonor-mRosa26 vector (SIGMA-Aldrich Inc. #D9196), a homologous recombination vector for knock-in into the mouse Rosa gene region, using methods known to those skilled in the art, to construct the P2Y11-split Luc (C-terminus) knock-in vector (Figure 1).

[0106] 1.2. Construction of arrestin-split Luc (N-terminus) knock-in vector The sequence consisting of the mouse arrestin-2 (β-arrestin-1) gene (referred to as "mArrb1a" in Figure 2) and the N-terminal region of the luciferase gene, and the sequence consisting of the mouse arrestin-3 (β-arrestin-2) gene (referred to as "mArrb2a" in Figure 2) and the N-terminal region of the luciferase gene were each tagged with a Myc or His tag, connected with a 2A peptide, and combined with a mouse β-actin promoter to form the "Arrestin-split Luc (N-terminus)" expression cassette (SEQ ID NO: 2). This sequence was cloned into the pZDonor-mRosa26 vector (SIGMA-Aldrich Inc. #D9196) for homologous recombination to construct the Arrestin-split Luc (N-terminus) knock-in vector (Figure 2).

[0107] 1.3. DNA microinjection of P2Y11-split Luc (C-terminus) and Arrestin-split Luc (N-terminus) knock-in vectors into mouse fertilized eggs The P2Y11-split Luc (C-terminus) knock-in vector or the Arrestin-split Luc (N-terminus) knock-in vector was mixed with ZFN mRNA (Sigma-Aldrich Inc. #M4574) targeting the mouse Rosa gene and injected into mouse pronuclear stage fertilized eggs. After incubation at 37°C overnight, embryos developed to the 2-cell stage were transferred into the uterus of 0.5-day-old pseudopregnant ICR recipient females to generate live offspring. The knock-in allele was detected by PCR, and founder mice were selected. To detect the knock-in allele in the P2Y11-split Luc (C-terminus) knock-in mouse, 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. To detect the knock-in allele in the Arrestin-split Luc (N-terminus) knock-in mouse, primers mR1247Fq (SEQ ID NO: 3) and cmv-R (SEQ ID NO: 7) and mArrb2a-F2 (SEQ ID NO: 8) and mR3334R (SEQ ID NO: 6) were used.

[0108] 1.4.Generation of P2Y11-split Luc (C-terminus) and Arrestin-split Luc (N-terminus) double knock-in mice The resulting founder mice were mated with C57BL / 6N mice after sexual maturity, and the transmission of the knock-in allele to the offspring was confirmed by PCR using genomic DNA extracted from the offspring's tissues as a template. By crossing the established P2Y11-split Luc (C-term) knock-in mice with the Arrestin-split Luc (N-term) knock-in mice, P2Y11-split Luc (C-term) and Arrestin-split Luc (N-term) double knock-in mice were generated.

[0109] Example 2: In vivo ATP signal detection in P2Y11-split Luc (C-terminus) and Arrestin-split Luc (N-terminus) double knock-in mice P2Y11-split Luc (C-terminus) and Arrestin-split Luc (N-terminus) double knock-in mice were subcutaneously administered 50 μL of a mixture of ATP (final concentration 1 mM) and 15 mg / mL VivoGlo Luciferin (Promega #P1043). 10–20 minutes after ATP administration, luminescence signal intensity was measured using an IVIS Spectrum CT (PerkinElmer). Measurement conditions were Exposure time = 180 sec, Binning = Medium, F / Stop = 1, and luminescence signal was analyzed in physical quantities (photons / sec).

[0110] As a result, luminescence signals were detected upon ATP administration in P2Y11-split Luc (C-terminus) and Arrestin-split Luc (N-terminus) double knock-in mice, but this luminescence signal was not detected in mice with only one allele or wild-type mice (Fig. 3). These results confirmed that ATP binding to P2Y11 recruits Arrestin, and that these mice function as reaction-specific reporter mice capable of obtaining luminescence signals by reconstituting split-luciferase.

[0111] Example 3: Establishment of fibroblasts derived from P2Y11-split Luc (C-terminus) and Arrestin-split Luc (N-terminus) double knock-in mice and in vitro ATP signal detection 3.1. Establishment of fibroblasts from P2Y11-split Luc (C-terminus) and Arrestin-split Luc (N-terminus) double knock-in mice Skin tissue was collected from P2Y11-split Luc (C-terminus) and Arrestin-split Luc (N-terminus) double knock-in mice, and then cut into small pieces using surgical scissors in culture medium. A cover glass was placed on the dermal side of the culture dish so that it was in contact with the bottom, and the tissue was cultured in D-MEM medium supplemented with 10% FBS and 1x NEAA. Fibroblasts that had been confirmed to migrate and proliferate were recovered by trypsinization, and fibroblast cell lines were established by repeated passage.

[0112] 3.2. In vitro ATP concentration detection Fibroblasts derived from P2Y11-split Luc (C-terminus) and Arrestin-split Luc (N-terminus) double knock-in mice were seeded onto a 24-well culture plate. The following day, ATP (final concentrations: 0-1000 μM) and VivoGlo Luciferin (Promega #P1043) were added, and luminescence signals were detected using an IVIS Spectrum CT (PerkinElmer). The measurement conditions were exposure time = 180 sec, binning = medium, F / Stop = 1, and luminescence signals were analyzed in physical quantities (photons / sec).

[0113] As a result, concentration-dependent signal intensity was detected at 0 μM, 10 μM, 50 μM, 100 μM, 200 μM, 400 μM, 600 μM, 800 μM, and 1000 μM of ATP, and the obtained regression equation was y = 437.91x + 10840 (R 2 =0.9727) (Figures 4 and 5).

[0114] [Example 4] In vivo imaging of known concentrations of ATP P2Y11-split Luc (C-terminus) and Arrestin-split Luc (N-terminus) double knock-in mice were subcutaneously administered 50 μL of a mixture of ATP (final concentrations: 0 mM, 1 mM, 2 mM, 4 mM, 8 mM) and 15 mg / mL VivoGlo Luciferin (Promega #P1043). 10 to 20 minutes after ATP administration, 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 in physical quantities (photons / sec). As a result, ATP concentration-dependent signal intensity was detected, and the resulting regression equation was y = 1E + 06x + 3E + 06 (R 2 = 0.9964), which confirmed that the mouse functioned as a concentration-dependent reporter mouse (Fig. 6, Fig. 7).

[0115] [Example 5] ATP visualization by inflammatory stimulation We performed hydrodynamic injections on P2Y11-split Luc (C-terminus) and Arrestin-split Luc (N-terminus) double knock-in mice to visualize ATP leakage in the liver following hepatocellular damage. Normal saline equivalent to 10% of the mouse's body weight was administered via the tail vein. The day after the hydrodynamic injection, 150 mg / kg of VivoGlo Luciferin (Promega #P1043) was administered intraperitoneally. Luminescence signal intensity was measured using an IVIS Spectrum CT (PerkinElmer). The measurement conditions were exposure time = 60 sec, binning = medium, and F / Stop = 1, and the luminescence signal was analyzed in photons / sec.

[0116] As a result, a higher luminescence signal was detected in the livers of the hydrodynamic injection group compared to the group (-) that did not undergo hydrodynamic injection, confirming that these mice can serve as reporter mice for non-invasive visualization of ATP leakage in response to inflammatory stimuli (Figure 8).

[0117] [Example 6] Visualization of intracellular ATP by carcinogenesis induction We used P2Y11-split Luc (C-terminus) and Arrestin-split Luc (N-terminus) double knock-in mice to induce liver carcinogenesis using hydrodynamic DNA delivery, and compared and visualized ATP levels in normal tissue and tumor sites. Specifically, a mutant Kras expression vector and gRNA-Cas9 expression vectors targeting p53, p16, and Smad4 were mixed at 3-5 μg per mouse and administered intravenously in saline at a volume approximately 10% of the mouse's body weight. Mutant Kras expression was achieved using the expression vector pMacII, as described in The Journal of Biological Chemistry (2011) 286, 20109-20116. The sequence encoding mutant Kras (G12D) shown in SEQ ID NO: 12 was cloned into the expression vector pMacII. The construct expressed mutant Kras (G12D) under the control of a CMV enhancer and mouse beta-actin promoter. gRNA-Cas9 expression vectors targeting each target gene were cloned into pGENA22 (Horizon Discovery) with partial sequences of the p53, p16, and Smad4 genes as gRNA sequences. Three gRNA-Cas9 expression vectors were created for each target gene, and the three vectors were mixed and administered. The gRNA sequences for p53 were shown in SEQ ID NOs: 13, 14, and 15. The gRNA sequences for p16 were shown in SEQ ID NOs: 16, 17, and 18. The gRNA sequences for Smad4 were shown in SEQ ID NOs: 19, 20, and 21. Changes in tissue ATP concentration associated with liver cancer were analyzed by intraperitoneal administration of 150 mg / kg VivoGlo Luciferin (Promega #P1043), and luminescence signal intensity was measured using an 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 in physical quantities (photons / sec).

[0118] As a result, in individuals in which tumor formation and growth in the liver was observed following administration of a mutant Kras expression vector and gRNA-Cas9 expression vectors targeting the p53, p16, and Smad4 genes, strong luminescence signals were detected by non-invasive visualization (Figure 9(A)). Furthermore, after liver removal, ex vivo analysis detected high luminescence signals in areas where canceration was observed and at the sites of tumor formation compared with normal tissue or normal areas, confirming that ATP concentrations were high in areas where cancer cells were proliferating (Figure 9(B)).

Claims

1. A method for evaluating the efficacy of a preventive or therapeutic agent for cancer or an inflammatory disease, comprising: A genetically modified non-human animal that expresses a first fusion protein and a second fusion protein, the first fusion protein comprises a membrane protein that binds to a purinergic receptor ligand and a first reporter protein; the method comprises detecting a purine receptor ligand present outside a cell using a genetically modified non-human animal, wherein the second fusion protein comprises a protein that binds to the membrane protein to which the ligand is bound and a second reporter protein; and the method comprises administering the prophylactic or therapeutic agent to the genetically modified non-human animal and evaluating the efficacy of the prophylactic or therapeutic agent based on a change in the detected amount of a reporter protein that indicates the detected amount of the purine receptor ligand before and after the administration, the membrane protein is P2Y11, the purinergic receptor ligand is ATP; The method, wherein the first reporter protein and the second reporter protein are each subunit of a split reporter protein.

2. A method for screening a preventive or therapeutic agent for cancer or an inflammatory disease, comprising: A genetically modified non-human animal that expresses a first fusion protein and a second fusion protein, the first fusion protein comprises a membrane protein that binds to a purinergic receptor ligand and a first reporter protein; the method comprises detecting a purinergic receptor ligand present outside a cell using a genetically modified non-human animal, wherein the second fusion protein comprises a protein that binds to the membrane protein to which the ligand is bound and a second reporter protein; and the method comprises administering a test substance to the genetically modified non-human animal and screening for a substance effective in the prevention or treatment of a disease based on a change in the detected amount of a reporter protein that indicates the detected amount of the purinergic receptor ligand before and after the administration, the membrane protein is P2Y11, the purinergic receptor ligand is ATP; The method, wherein the first reporter protein and the second reporter protein are each subunit of a split reporter protein.

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