Pancreatitis model animal and production method therefor

Abstract

To provide a pancreatitis model animal and its production method.SOLUTION: A rodent has all of the following (1) to (3) qualities: (1) a function of SLC29A3 deteriorates; (2) a function of TLR7 deteriorates; and (3) (i) a peptide containing an amino acid sequence shown by a sequence number 1, (ii) a peptide containing an amino acid sequence having an identify of 80% or more with an amino acid sequence shown by the sequence 1, or (iii) a peptide containing an amino acid sequence in which one or more amino acids of the amino acid sequence shown by the sequence number 1 have addition, substitution or deletion, and (i) a peptide containing an amino acid sequence shown by a sequence number 2, (ii) a peptide containing an amino acid sequence having an identity of 80% or more with an amino acid sequence shown by the sequence number 2, or (iii) a peptide containing an amino acid sequence in which one or more amino acids of the amino acid sequence shown by the sequence number 2 have addition, substitution or deletion are expressed.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a pancreatitis model animal and a method for producing the same. [Background technology]

[0002] Although the onset of acute and chronic pancreatitis is believed to be caused by excessive alcohol intake or gallstones, the detailed molecular basis of onset has not been clarified. In addition, the cause of autoimmune pancreatitis remains unknown. As model animals for analyzing the onset mechanism of pancreatitis, commonly known models include bile duct ligated mice and rats, and models in which bile acids or drugs such as caerulein are retrogradely injected into the pancreatic duct. However, there have been no reports of model animals that spontaneously develop pancreatitis.

[0003] TLR7 and TLR8 are RNA sensors localized in endolysosomes. Structural analysis has revealed that they bind to nucleosides and oligoribonucleotides at two distinct sites. SLC29A3 is also localized in endolysosomes and is a transporter responsible for transporting nucleosides between the cytoplasm and the cytoplasm. Patent Document 1 discloses that accumulation of nucleosides in endolysosomes due to genetic mutations in SLC29A3 leads to constitutive activation of TLR7 and TLR8. Patent Document 1 also discloses that mouse TLR7 is constitutively activated in mice lacking SLC29A3 alone, and that the mice exhibit pathology similar to that of human SLC29A3 disorders. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] JP 2019-85370 A Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present invention is to provide a novel pancreatitis model animal. [Means for solving the problem]

[0006] The present inventors discovered that expression of human TLR8 in mice with reduced functions of SLC29A3 and TLR7 caused spontaneous onset of pancreatitis, and thus completed the present invention.

[0007] That is, the present invention includes the following inventions. [1] A rodent that has all of the following characteristics (1) to (3). (1) Decreased function of SLC29A3 (2) TLR7 function is impaired (3) (i) a peptide comprising the amino acid sequence shown in SEQ ID NO: 1; (ii) a peptide comprising an amino acid sequence having 80% or more identity to the amino acid sequence shown in SEQ ID NO: 1, or (iii) a peptide comprising an amino acid sequence having one or more amino acid additions, substitutions or deletions of the amino acid sequence shown in SEQ ID NO:1; and (i) a peptide comprising the amino acid sequence shown in SEQ ID NO: 2; (ii) a peptide comprising an amino acid sequence having 80% or more identity to the amino acid sequence shown in SEQ ID NO: 2, or (iii) a peptide comprising an amino acid sequence having one or more amino acid additions, substitutions or deletions of the amino acid sequence shown in SEQ ID NO: 2; It expresses [2] A rodent described in [1], which is a pathological model of pancreatitis. [3] A rodent according to [1] or [2], wherein the decrease in function is due to a genetic mutation. [4] A rodent according to any one of [1] to [3], which is double deficient in SLC29A3 and TLR7. [5] A rodent according to any one of [1] to [4], wherein TLR8 is expressed specifically in monocytes and / or macrophages. [6] The rodent according to [5], wherein the TLR8 is human TLR8. [7] The rodent according to any one of [1] to [6], wherein the rodent is a mouse. [8] The rodent according to any one of [2] to [7], wherein the pancreatitis is acute pancreatitis, chronic pancreatitis or autoimmune pancreatitis. [9] A cell derived from a rodent according to any one of [1] to [8].

[10] Reducing the function of SLC29A3 and TLR7; comprising a step of expressing TLR8, A method for producing a rodent having all of the following properties (1) to (3). (1) Decreased function of SLC29A3 (2) TLR7 function is impaired (3) (i) a peptide comprising the amino acid sequence shown in SEQ ID NO: 1; (ii) a peptide comprising an amino acid sequence having 80% or more identity to the amino acid sequence shown in SEQ ID NO: 1, or (iii) a peptide comprising an amino acid sequence having one or more amino acid additions, substitutions or deletions of the amino acid sequence shown in SEQ ID NO:1; and (i) a peptide comprising the amino acid sequence shown in SEQ ID NO: 2; (ii) a peptide comprising an amino acid sequence having 80% or more identity to the amino acid sequence shown in SEQ ID NO: 2, or (iii) a peptide comprising an amino acid sequence having one or more amino acid additions, substitutions or deletions of the amino acid sequence shown in SEQ ID NO: 2; It expresses Effect of the Invention

[0008] According to the present invention, it is possible to provide a pancreatitis model animal and a method for producing the same. [Brief description of the drawings]

[0009] [Figure 1] FIG. 1 shows the phenotypic findings of slc29a3 KO mice and slc29a3 / tlr7 dKO mice. [Diagram 2] FIG. 2 shows a schematic diagram for establishing slc29a3 / tlr7 dKO-huTLR8-Lyz2-Cre mice. [Diagram 3] FIG. 3 shows the results of a survey of the phenotype (survival rate and body weight change) of slc29a3 / tlr7 dKO-huTLR8-Lyz2-Cre mice. [Figure 4] FIG. 4 shows the results of a survey of the phenotype of slc29a3 / tlr7 dKO-huTLR8-Lyz2-Cre mice (food residue in the jejunum and ileum, undigested feces in the large intestine, and appendix weight). [Diagram 5] FIG. 5 shows the results of biochemical tests and pancreatic weight examination of slc29a3 / tlr7 dKO-huTLR8-Lyz2-Cre mice. [Figure 6] Figure 6 shows the results of the pancreatic pathological findings of slc29a3 / tlr7 dKO-huTLR8-Lyz2-Cre mice. "HE" shows the results of counterstaining with hematoxylin and eosin. "CD11b" shows the results of immunohistochemical staining using CD11b-positive macrophages as an antigen. [Figure 7] FIG. 7 shows the results of elevation of pancreatitis-specific chemokines in serum. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] Hereinafter, an embodiment of the present invention (hereinafter, referred to as the present embodiment) will be described. Note that the present invention is not limited to the following embodiment, and can be modified in various ways within the scope of the present invention.

[0011] The present invention relates to a rodent having all of the following characteristics (1) to (3): (1) Decreased function of SLC29A3 (2) TLR7 function is impaired (3) (i) a peptide comprising the amino acid sequence shown in SEQ ID NO: 1; (ii) a peptide comprising an amino acid sequence having 80% or more identity to the amino acid sequence shown in SEQ ID NO: 1, or (iii) a peptide comprising an amino acid sequence having one or more amino acid additions, substitutions or deletions of the amino acid sequence shown in SEQ ID NO:1; and (i) a peptide comprising the amino acid sequence shown in SEQ ID NO: 2; (ii) a peptide comprising an amino acid sequence having 80% or more identity to the amino acid sequence shown in SEQ ID NO: 2, or (iii) a peptide comprising an amino acid sequence having one or more amino acid additions, substitutions or deletions of the amino acid sequence shown in SEQ ID NO: 2; is expressed. In this specification, "a peptide comprising the amino acid sequence shown in SEQ ID NO: 1, a peptide comprising an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 1, or a peptide comprising an amino acid sequence in which one or more amino acids of the amino acid sequence shown in SEQ ID NO: 1 have been added, substituted or deleted" may be expressed as "a peptide comprising the amino acid sequence shown in SEQ ID NO: 1, etc." The same applies to SEQ ID NOs: 2 to 3 and 5. The rodent of the present invention is used as a pancreatitis model animal, and may be a model animal that spontaneously develops severe pancreatitis. The rodent of the present invention is useful as a pathological model of pancreatitis and can be used in research on acute / chronic pancreatitis and autoimmune pancreatitis for which no fundamental treatment has been found, etc. In other words, this model animal is useful as a model animal for analyzing the mechanism of pancreatitis onset.

[0012] SLC29A3 is a transporter that transports nucleosides between the cytoplasm and the endolysosome, and it is known that mutations in SLC29A3 cause SLC29A3 disorders. In this specification, "SLC29A3" is used to include the SLC29A3 gene and the SLC29A3 protein.

[0013] As used herein, the term "gene" is used to encompass not only DNA but also mRNA, cDNA, and cRNA.

[0014] In mice lacking SLC29A3 alone, mouse TLR7 is constitutively activated and exhibits the pathology of SLC29A3 deficiency, whereas in SLC29A3 / TLR7 double-deficient mice, the pathology of SLC29A3 deficiency almost disappears. TLR7 is one of the Toll-like receptors (TLRs) that form one of the pathogen sensor families. In this specification, "TLR7" is used to include the TLR7 gene and TLR protein.

[0015] The rodent of the present invention has a property of having reduced functions of SLC29A3 and TLR7. As used herein, "reduced function" or "has reduced function" means that the function of the gene and / or protein is reduced compared to when it is normally expressed, and includes a state in which the function is completely absent.

[0016] The method for reducing the function of SLC29A3 and TLR7 in the rodent of the present invention is not particularly limited, and may be a method commonly used in the present technical field, such as gene knockout (including gene destruction, inhibition of expression, interference with expression, etc.), gene knockdown (including reduction of expression, decreased expression, etc.), gene silencing (including gene suppression, reduction of expression, decreased expression, etc.), etc. Therefore, in the present invention, a decrease in the function of SLC29A3 and TLR7 may mean that a change in the expression of SLC29A3 and TLR7 at the gene level due to gene knockout, gene knockdown, gene silencing, etc. has occurred, resulting in a change in the function of SLC29A3 and TLR7 at the protein level.

[0017] Examples of gene knockout include gene targeting, which modifies endogenous genes using homologous recombination, and genome editing using artificial DNA cleavage enzymes such as ZFN and TALEN, or site-specific nucleases such as CRISPR / Cas. Among these, genome editing using site-specific nucleases such as CRISPR / Cas is preferred because of its high site specificity.

[0018] As used herein, "knockout" is sometimes used synonymously with "deficiency," and "double knockout" is sometimes used synonymously with "double deficiency."

[0019] Examples of gene knockdown include the antisense method, which uses an antisense strand of a target mRNA, and the RNAi method, which uses siRNA or microRNA.

[0020] Examples of gene silencing include transcriptional gene silencing, which occurs by halting mRNA synthesis through histone modifications or changes in the three-dimensional structure of heterochromatin, and post-transcriptional gene silencing, which occurs by destroying target mRNA through RNAi methods.

[0021] The reduction in the functions of SLC29A3 and TLR7 can also be achieved by amino acid mutations. In this specification, the term "mutation" mainly refers to a mutation artificially introduced by site-directed mutagenesis or the like, but may also refer to a similar mutation that exists in nature. Therefore, in the present invention, the reduced function of SLC29A3 and TLR7 may also mean that a function-reducing mutation has occurred in the protein, causing a change in the function of SLC29A3 and TLR7 at the protein level.

[0022] Whether a rodent has the property of reduced function of SLC29A3 and TLR7 can be confirmed by methods well known to those skilled in the art, for example, by comparison with the expression levels and functional analysis of SLC29A3 and TLR7 in wild-type rodents.

[0023] The rodent of the present invention has reduced function of SLC29A3 and TLR7, as well as expressing a peptide, etc. comprising the amino acid sequence shown in SEQ ID NO: 1 and a peptide, etc. comprising the amino acid sequence shown in SEQ ID NO: 2. In the present invention, the property of expression of a peptide, etc. comprising the amino acid sequence shown in SEQ ID NO: 1 and a peptide, etc. comprising the amino acid sequence shown in SEQ ID NO: 2 can be rephrased as the property of expression of TLR8. The amino acid sequence shown in SEQ ID NO: 1 and the amino acid sequence shown in SEQ ID NO: 2 are both sequences containing a sequence necessary for ligand binding of TLR8, and the ranges of sequences included are partially overlapping. Thus, in this specification, "expressing a peptide, etc. containing the amino acid sequence shown in SEQ ID NO: 1 and a peptide, etc. containing the amino acid sequence shown in SEQ ID NO: 2" does not necessarily require that they are expressed separately, but may be in a state in which the ligand binding ability of TLR8 is ultimately exerted. For example, a peptide, etc. containing an amino acid sequence containing an amino acid sequence shown in SEQ ID NO: 1 and an amino acid sequence containing an amino acid sequence shown in SEQ ID NO: 2 may be expressed, or a peptide, etc. containing a peptide, etc. containing an amino acid sequence shown in SEQ ID NO: 1 and a peptide, etc. containing an amino acid sequence shown in SEQ ID NO: 2 may be expressed. One or more peptides, etc. containing the amino acid sequence shown in SEQ ID NO: 1 and a peptide, etc. containing the amino acid sequence shown in SEQ ID NO: 2 may each be expressed. The peptides, etc. containing the amino acid sequence shown in SEQ ID NO: 1 and the peptides, etc. containing the amino acid sequence shown in SEQ ID NO: 2 are not particularly limited as long as the resulting expressed amino acid sequence exhibits the ligand binding ability of TLR8, and may contain any other sequence. An example of any other sequence is a sequence derived from TLR8. An example of a sequence containing such any other sequence is not particularly limited, but may be, for example, a sequence shown in SEQ ID NO: 3 or 5. That is, the rodent of the present invention may have a property in which a peptide, etc. containing an amino acid sequence shown in SEQ ID NO: 3 or 5 is expressed, in addition to the property of reduced function of SLC29A3 and TLR7. Having a property in which a peptide, etc. containing an amino acid sequence shown in SEQ ID NO: 3 or 5 is expressed can be rephrased as having a property in which TLR8 is expressed. One or more peptides, etc. containing an amino acid sequence shown in SEQ ID NO: 3 or 5 may be expressed. When the amino acid sequence shown in SEQ ID NO:1 and / or the amino acid sequence shown in SEQ ID NO:2 contains mutated amino acids, the amount and position of the mutations are not particularly limited as long as the resulting expressed amino acid sequence exhibits the ligand-binding ability of TLR8, and may be set independently of each other.

[0024] As used herein, "amino acid" is used in its broadest sense and includes not only naturally occurring amino acids but also unnatural amino acids such as amino acid variants and derivatives. Amino acids include, but are not limited to, naturally occurring proteinogenic L-amino acids; D-amino acids; chemically modified amino acids such as amino acid variants and derivatives; naturally occurring non-proteinogenic amino acids such as norleucine, β-alanine, ornithine; and chemically synthesized compounds having properties known in the art that are characteristic of amino acids. Unnatural amino acids include, but are not limited to, α-methyl amino acids (such as α-methylalanine), D-amino acids, histidine-like amino acids (such as 2-amino-histidine, β-hydroxy-histidine, homohistidine, α-fluoromethyl-histidine, α-methyl-histidine), amino acids with an extra methylene in the side chain ("homo" amino acids), and amino acids in which the carboxylic acid functional group in the side chain is replaced with a sulfonic acid group (such as cysteic acid).

[0025] As used herein, when it is stated that "one or more amino acid deletions, substitutions, or additions are present," the number of amino acids to be deleted, substituted, etc. is not particularly limited, as long as the resulting amino acid sequence retains the ligand binding ability of TLR8. Here, "multiple" means an integer of 2 or more, and is several, for example, 2 to 50, preferably 2 to 40, more preferably 2 to 30, and even more preferably 2 to 20. The positions of deletions, substitutions, or additions in each amino acid sequence are not particularly limited, as long as the resulting amino acid sequence retains the ligand binding ability of TLR8.

[0026] As used herein, "having Y% or more identity with the amino acid sequence shown in SEQ ID NO:X" means that when the amino acid sequences of the two peptides are aligned to maximize identity, the ratio of the number of common amino acid residues to the total number of amino acids shown in SEQ ID NO:X is Y% or more. In this embodiment, the identity is not particularly limited as long as the resulting expressed amino acid sequence retains the ligand binding ability of TLR8, but can be, for example, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more. In this embodiment, for example, having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 1 means that when the amino acid sequence shown in SEQ ID NO: 1 is compared with an amino acid sequence containing mutated amino acids and aligned to maximize identity, the ratio of the number of common amino acid residues to the total number of amino acids in the amino acid sequence shown in SEQ ID NO: 1 is 80% or more.

[0027] The origin of TLR8 is not particularly limited, but examples include human TLR8 (huTLR8), monkey TLR8, and the like, with human TLR8 being particularly preferred.

[0028] As used herein, "TLR8" is used to encompass both the TLR8 gene and the TLR8 protein.

[0029] The method of expressing a target sequence such as TLR8 in the rodent of the present invention is not particularly limited, and may be a method commonly used in the technical field, such as knock-in. Examples of knock-in include gene knock-in, which introduces a target gene via homologous recombination, safe harbor transgene knock-in, which introduces a target gene into a safe harbor site via homologous recombination, genome editing, microhomology-mediated end joining (MMEJ), etc., which introduces a target gene via homologous recombination using an artificial DNA cleavage enzyme such as ZFN or TALEN, a site-specific nuclease such as CRISPR / Cas, and a donor sequence. Here, in this specification. "Gene knock-in" does not necessarily mean knock-in of the entire "gene", and may be appropriately interpreted as synonymous with "knock-in of a target sequence". In addition, in this specification, "knock-in" may be used synonymously with "introduction (including insertion)". Here, the sequence (target sequence) to be introduced by knock-in may be a nucleic acid sequence that expresses a peptide containing the amino acid sequence shown in SEQ ID NO: 1, a peptide containing the amino acid sequence shown in SEQ ID NO: 2, a peptide containing the amino acid sequence shown in SEQ ID NO: 3, or a peptide containing the amino acid sequence shown in SEQ ID NO: 5, and such nucleic acid sequences can be produced by those skilled in the art using known techniques.

[0030] Known safe harbor sites used for safe harbor transgene knock-in include human AAVS1 and mouse ROSA26, etc. When a mouse is used to produce the rodent of this embodiment, the ROSA26 region can be used.

[0031] The mouse ROSA26 region is a region containing the exons of three non-coding genes present on mouse chromosome 6, and is known as a safe site for the introduction of exogenous sequences because exogenous sequences introduced into this region can be stably expressed. The target sequence can be introduced using a vector plasmid having an arm complementary to the restriction enzyme recognition site (XbaI recognition site) located between the exons. The vector plasmid for introducing the target sequence into the ROSA26 region may be a commercially available one, for example, a vector plasmid available from Addgene.

[0032] When using the mouse ROSA26 region, a vector plasmid carrying a target sequence for a recombinase, which is part of the conditional knock-in system described below, may be used. An example of such a vector plasmid is the CAG-STOP-eGFP-ROSA26TV vector.

[0033] MMEJ is one of the DNA repair mechanisms of eukaryotes, and is a mechanism that repairs the double-stranded break by binding complementary sequences (5 to 25 base pairs) between the two ends of the break. When introducing an exogenous sequence using the MMEJ repair mechanism, for example, by introducing a donor vector carrying a recognition sequence of a site-specific nuclease and a target sequence together with a site-specific nuclease (TALEN, CRISPR / Cas, etc.), the chromosomal target site and the cut end of the donor vector are complementary bonded at the time of double-stranded break (DSB) by the site-specific nuclease, and the exogenous target sequence can be knocked in. When TALEN is used as the site-specific nuclease, it is called the TAL-PITCh method, and when CRISPR / Cas is used as the site-specific nuclease, it is called the CRIS-PITCh method (Nature Communications volume 5, Article number: 5560 (2014)).

[0034] The rodent of the present invention preferably has the property that (i) a peptide or the like comprising the amino acid sequence shown in SEQ ID NO: 1 and a peptide or the like comprising the amino acid sequence shown in SEQ ID NO: 2; (ii) a peptide or the like comprising the amino acid sequence shown in SEQ ID NO: 3; or (iii) a peptide or the like comprising the amino acid sequence shown in SEQ ID NO: 5 are specifically expressed in myeloid (myeloid) cells. The rodent of the present invention preferably has the property that a peptide or the like comprising the amino acid sequence shown in SEQ ID NO: 1 and a peptide or the like comprising the amino acid sequence shown in SEQ ID NO: 2; (ii) a peptide or the like comprising the amino acid sequence shown in SEQ ID NO: 3; or (iii) a peptide or the like comprising the amino acid sequence shown in SEQ ID NO: 5 are specifically expressed in monocytes, macrophages, neutrophils, or dendritic cells. The peptide or the like comprising the amino acid sequence shown in SEQ ID NO: 1 and a peptide or the like comprising the amino acid sequence shown in SEQ ID NO: 2; (ii) a peptide or the like comprising the amino acid sequence shown in SEQ ID NO: 3; or (iii) a peptide or the like comprising the amino acid sequence shown in SEQ ID NO: 5 may be specifically expressed in one type of cell among monocytes, macrophages, neutrophils, or dendritic cells, or may be specifically expressed in any two or more types of cells.

[0035] Myeloid cell, preferably monocyte and / or macrophage and / or neutrophil-specific expression of peptides, etc. comprising the amino acid sequence shown in SEQ ID NO:1 and peptides, etc. comprising the amino acid sequence shown in SEQ ID NO:2; (ii) peptides, etc. comprising the amino acid sequence shown in SEQ ID NO:3; or (ii) peptides, etc. comprising the amino acid sequence shown in SEQ ID NO:5, can be achieved by, but is not limited to, a method commonly used in the art, for example, conditional knock-in. Conditional knock-in is a method for controlling knock-in (expression) of a target gene (target sequence) in a tissue-, cell-, site-, or time-specific manner.

[0036] An example of conditional knock-in is conditional knock-in using a recombinase.

[0037] As an example of conditional knock-in using recombinase, there is a technique in which a non-human animal having a gene region in which all or part of a gene of interest (sequence of interest) is sandwiched between target sequences of a recombinase, which is a DNA recombinase enzyme, is mated with a non-human animal that expresses a recombinase that recognizes the target sequence in a tissue-, cell-, site-, or time-specific manner to obtain a non-human animal in which a gene of interest (sequence of interest) is expressed in a tissue-, cell-, site-, or time-specific manner. For example, an individual in which a sequence of interest is introduced into a vector plasmid carrying a target sequence of the recombinase can be mated with an individual that expresses the recombinase in a macrophage-specific manner to obtain an individual in which a sequence of interest is expressed in a macrophage-specific manner. Examples of systems based on such techniques include the Cre / loxP system and the FLP / FRT system.

[0038] The Cre / loxP system utilizes the site-specific recombination system of bacteriophage P1. For example, the DNA recombination enzyme Cre recombinase acts on a 34-base DNA sequence derived from the genome of bacteriophage P1, called the loxP sequence, to cause site-specific recombination between loxP sequences.

[0039] Examples of target sequences for recombinase in the Cre / loxP system include loxP, lox511, lox2272, lox66, lox71, loxM2, lox5171, FRT, FRT11, FRT71, attp, att, FRT, rox, etc., and any one of them may be used, or any two or more of them may be used in combination.

[0040] In the FLP / FRT system, for example, FLP recombinase acts on a sequence derived from budding yeast, called an FRT sequence, to cause site-specific recombination between FRT sequences.

[0041] Target sequences for the recombinase in the FLP / FRT system include, for example, FRT, FRT11, FRT71, attp, att, FRT, etc., and any one of them may be used, or any two or more of them may be used in combination.

[0042] In conditional knock-in using recombinase, the manipulation of the target gene (target sequence) can be controlled in a tissue-, cell-, site-, or time-specific manner by selecting a promoter for expressing the recombinase according to the purpose. Examples of the promoter include tissue- or cell-specific promoters. Examples of the tissue- or cell-specific promoter include myeloid (bone marrow) cell-specific promoters, bone cell-specific promoters, kidney cell-specific promoters, cardiac cell-specific promoters, muscle cell-specific promoters, and endothelial cell-specific promoters. Examples of the myeloid (bone marrow) cell-specific promoters include immune cell-specific promoters. Examples of the immune cell-specific promoters include B cell-specific promoters, T cell-specific promoters, neutrophil-specific promoters, macrophage-specific promoters, dendritic cell-specific promoters, and monocyte-specific promoters.

[0043] In one aspect, the promoter can be a developmentally-regulated promoter, such as a promoter that is active only during the embryonic stage of development or a promoter that is active only during the mature cell stage.

[0044] In one embodiment, the promoter can be selected according to the type of cell. Thus, depending on the promoter selected, it can find use in eukaryotic cells, rodent cells, pluripotent cells, mouse cells, rat cells, hamster cells, guinea pig cells, fibroblasts, CHO cells, human pluripotent cells, human ES cells, human adult stem cells, developmentally restricted human progenitor cells, human iPS cells, human cells, etc.

[0045] Conditional genetic engineering using these recombinases can also be used for conditional knockout using recombinases based on similar principles.

[0046] In conditional knock-in using a recombinase, the recombinase may be inserted in-frame into a genetic region that is expressed in a tissue-, cell-, site-, or time-specific manner. An individual in which a recombinase has been inserted in-frame into such a genetic region that is expressed in a tissue-, cell-, site-, or time-specific manner can express the recombinase in a tissue-, cell-, site-, or time-specific manner. Thus, for example, an individual in which a recombinase has been inserted in-frame into a genetic region that is expressed in a macrophage-specific manner can be crossed with an individual in which a target sequence for the recombinase has been introduced into a vector plasmid carrying the target sequence for the recombinase, thereby obtaining an individual that expresses the target sequence in a macrophage-specific manner.

[0047] An example of an individual in which a recombinase has been inserted in-frame into a gene region that is expressed in a tissue-, cell-, site-, or time-specific manner is the Lyz2-Cre mouse. The Lyz2-Cre mouse is known to be a mouse in which Cre recombinase has been inserted into the gene region of M-lysozyme, a lysosomal enzyme that is highly expressed in myeloid cells such as macrophages and monocytes, and a commercially available mouse may be used as appropriate, for example, a mouse available from The Jackson Laboratory may be used.

[0048] Whether a rodent has the property of expressing TLR8 can be confirmed by methods well known to those skilled in the art, for example, by analysis of TLR8 expression in the rodent.

[0049] An individual having the property of expressing a peptide, etc. comprising the amino acid sequence shown in SEQ ID NO: 1 and a peptide, etc. comprising the amino acid sequence shown in SEQ ID NO: 2 in addition to the property of reducing the functions of SLC29A3 and TLR7 can be obtained, for example, by mating an individual having the property of expressing a peptide, etc. comprising the amino acid sequence shown in SEQ ID NO: 1 and a peptide, etc. comprising the amino acid sequence shown in SEQ ID NO: 2. Here, an individual having a property of reduced function of SLC29A3 and TLR7, and having a property of conditionally expressing a peptide or the like comprising the amino acid sequence shown in SEQ ID NO: 1 and a peptide or the like comprising the amino acid sequence shown in SEQ ID NO: 2, is for example, (i) an individual having reduced SLC29A3 and TLR7 functions and an individual having the property of expressing a peptide, etc. comprising the amino acid sequence shown in SEQ ID NO:1 and a peptide, etc. comprising the amino acid sequence shown in SEQ ID NO:2, obtained by mating an individual having reduced SLC29A3 and TLR7 functions with an individual having the property of expressing a peptide, etc. comprising the amino acid sequence shown in SEQ ID NO:1 and a peptide, etc. comprising the amino acid sequence shown in SEQ ID NO:2; (ii) an individual obtained by mating an individual having reduced functions of SLC29A3 and TLR7 with an individual that conditionally expresses recombinase, such as a Lyz2-Cre mouse; It can be obtained by crossbreeding using The above-mentioned "peptide etc. comprising the amino acid sequence shown in SEQ ID NO:1 and peptide etc. comprising the amino acid sequence shown in SEQ ID NO:2" may be "peptide etc. comprising the amino acid sequence shown in SEQ ID NO:3", "peptide etc. comprising the amino acid sequence shown in SEQ ID NO:5", or "TLR8".

[0050] The rodent of the present invention is a pathological model of pancreatitis. Here, the phrase "rodent is a pathological model of pancreatitis" means that the rodent is a rodent that exhibits the pathological condition of pancreatitis, and is used to mean both rodents that actually exhibit the pathological condition of pancreatitis and rodents that have the properties to exhibit the pathological condition of pancreatitis. In this specification, the term "pathological model of pancreatitis" may be used synonymously with the term "animal model of pancreatitis".

[0051] In this specification, the term "pancreatitis" broadly includes acute pancreatitis, chronic pancreatitis, hereditary pancreatitis, autoimmune pancreatitis, etc. It is known that acute pancreatitis progresses to chronic pancreatitis, and chronic pancreatitis is highly likely to be complicated with exocrine pancreatic insufficiency and diabetes mellitus.

[0052] The fact that a rodent exhibits a pathological condition of pancreatitis can be confirmed by one or more of the phenotype of the rodent, pancreatic pathological findings, pancreatitis-specific chemokines in serum, etc. These confirmations can be performed by techniques well known to those skilled in the art.

[0053] The pathology of pancreatitis can be confirmed by phenotypes such as vomiting, diarrhea, fatty stools, weight loss, fever, pancreatic enlargement, enlarged appendix, retention of food residue in the jejunum or ileum, undigested feces in the large intestine, peripancreatic fluid retention, necrotic retention, pancreatic pseudocysts, encapsulated necrosis, inflammation and fibrosis of pancreatic tissue, atrophy and fibrosis of the pancreatic parenchyma, pancreatic necrosis, loss of pancreatic weight, peripancreatic fat necrosis, pancreatic stones, reduction of blood or urinary pancreatic enzymes due to deterioration of pancreatic function, irregular or dilated pancreatic ducts and branches, pseudocysts, etc. When the pathology of pancreatitis becomes severe, it can cause the death of the individual.

[0054] The pathology of pancreatitis can also be confirmed by serum biochemical tests that examine pancreatitis-specific chemokines in serum, etc. In serum biochemical tests, the pathology of pancreatitis can also be confirmed by, for example, decreased blood sugar (serum glucose), decreased total protein, decreased albumin, increased amylase, increased lipase concentration, etc., which indicate pancreatic abnormalities.

[0055] Measurement values ​​indicative of the pathological condition of pancreatitis in serum biochemical tests may include, for example, blood glucose levels of 75 to 150 mg / dL, total protein levels of 4.5 to 6.5 g / dL, albumin levels of 3.0 to 4.5 g / dL, amylase levels of 1550 to 2500 IU / L, and lipase levels of 13 to 55 U / L.

[0056] The pathology of pancreatitis can also be confirmed in pancreatic pathological findings, for example, by destruction of pancreatic tissue (acinar cell region, etc.) which can be confirmed by immunohistochemical staining.

[0057] Diseases for which treatment methods can be investigated using the rodent of the present invention include, but are not limited to, acute pancreatitis such as interstitial edematous pancreatitis and necrotizing pancreatitis, chronic pancreatitis, autoimmune pancreatitis, pancreatic cysts, and the like.

[0058] As used herein, "rodent" includes all members of the rodent phylogenetic family and all future progeny. As used herein, "progeny" refers to all subsequent generations that are descendants of any given generation, whether by phylogenetic development, genetic engineering techniques, molecular biological techniques, or breeding.

[0059] The rodents used in the present invention are not particularly limited, and examples thereof include mice, rats, hamsters, and guinea pigs. From the viewpoint of ease of handling, mice and rats are preferred, and mice are particularly preferred.

[0060] One embodiment of the rodent of the present invention is an SLC29A3 / TLR7dKO-huTLR8-Lyz2-Cre mouse, which has reduced SLC29A3 and TLR7 functions and expresses human TLR8 specifically in monocytes and / or macrophages.

[0061] SLC29A3 / TLR7dKO-huTLR8-Lyz2-Cre mice can be obtained, for example, by crossing a huTLR8 mouse expressing human TLR8 with an SLC29A3 / TLR7 dKO mouse to obtain progeny mice, and then crossing the resulting mice with Lyz2-Cre mice.

[0062] The huTLR8 mouse is a mouse expressing huHTR8. The huTLR8 mouse can be produced, for example, by safe harbor transgene knock-in, which involves the introduction of a gene of interest via homologous recombination using the ROSA26 region (Gt(ROSA)26Sor) present on mouse chromosome 6 as a safe harbor site.

[0063] The SLC29A3 / TLR7 dKO mouse is a mouse in which the functions of both SLC29A3 and TLR7 are reduced. The SLC29A3 / TLR7 dKO mouse can be produced, for example, by genome editing using CRISPR / Cas9 as a site-specific nuclease, without being particularly limited thereto.

[0064] Lyz2-Cre mice are mice that express Cre recombinase specifically in myeloid (myeloid) cells. Commercially available Lyz2-Cre mice may be used, for example, B6.129P2-Lyz2tm1(cre)Ifo / J (Strain #004781, Common Name: LysMcre) available from The Jackson Laboratory.

[0065] In one embodiment, Reducing the function of SLC29A3 and TLR7; comprising a step of expressing TLR8, There is also provided a method for producing a rodent having all of the following properties (1) to (3): (1) Decreased function of SLC29A3 (2) TLR7 function is impaired (3) (i) a peptide comprising the amino acid sequence shown in SEQ ID NO: 1; (ii) a peptide comprising an amino acid sequence having 80% or more identity to the amino acid sequence shown in SEQ ID NO: 1, or (iii) a peptide comprising an amino acid sequence having one or more amino acid additions, substitutions or deletions of the amino acid sequence shown in SEQ ID NO:1; and (i) a peptide comprising the amino acid sequence shown in SEQ ID NO: 2; (ii) a peptide comprising an amino acid sequence having 80% or more identity to the amino acid sequence shown in SEQ ID NO: 2, or (iii) a peptide comprising an amino acid sequence having one or more amino acid additions, substitutions or deletions of the amino acid sequence shown in SEQ ID NO: 2; It expresses

[0066] Also, in one aspect, Reducing the function of SLC29A3 and TLR7; comprising a step of expressing TLR8, Methods for inducing pancreatitis in rodents are also provided.

[0067] The "step of reducing the function of SLC29A3 and TLR7" in the method for producing a rodent or the method for inducing pancreatitis in a rodent of this embodiment (hereinafter sometimes referred to as the "method of the present invention") may include an operation for imparting to the rodent produced by the method of the present invention the property of "reduced function of SLC29A3 and TLR7." The "step of reducing the functions of SLC29A3 and TLR7" may include, for example, producing an individual having a property in which the functions of SLC29A3 and TLR7 are reduced. The "step of expressing TLR8" in the method of the present invention may include an operation for imparting the property of "TLR8 expression" to the rodent produced by the method of the present invention. The "step of expressing TLR8" may include, for example, producing an individual having the property of expressing TLR8. For the preparation of an individual having reduced functions of SLC29A3 and TLR7, the description of "Method for reducing functions of SLC29A3 and TLR7" in this embodiment can be referred to, and for the preparation of an individual having the property of expressing TLR8, the description of "Method for expressing a target sequence such as TLR8" in this embodiment can be referred to.

[0068] Furthermore, the step of reducing the functions of SLC29A3 and TLR7 and the step of expressing TLR8 are not particularly limited, and may be performed simultaneously or separately, so long as the rodent produced has the properties of reduced SLC29A3 function, reduced TLR7 function, and expression of TLR8. For example, an expression cassette designed to simultaneously achieve knockout and knock-in may be used, or an individual having the properties of reduced SLC29A3 and TLR7 function and an individual having the property of expressing TLR8 may be prepared separately. When performed at separate times, either step may be performed first, without particular limitation, so long as the rodent to be produced has the properties of reduced SLC29A3 function, reduced TLR7 function, and expression of TLR8.

[0069] In one aspect, the production method or the method of inducing pancreatitis in a rodent of the present invention may further include a step of breeding individual animals. The breeding of individual animals is not particularly limited, but may be, for example, breeding an individual having a property of reduced function of SLC29A3 and TLR7 with an individual having a property of expressing TLR8, which have been produced in advance. The individual animals used for breeding may be produced by various genetic manipulations described in this embodiment.

[0070] Such animal populations may be used for breeding together through a series of crosses, backcrosses, and selections. Typically, crosses and backcrosses are accomplished by mating sibling or parental strains to offspring, depending on the purpose of each particular step in the breeding process. In certain cases, it may be necessary to generate multiple offspring to generate a single offspring that contains each knockout construct and / or transgene at the appropriate chromosomal location. Furthermore, crosses or backcrosses may be required for several generations to ultimately obtain the desired genotype.

[0071] In one embodiment, a rodent produced by the method of the present invention is also provided. The rodent produced by the method of the present invention for producing a rodent and the rodent produced by the method of the present invention for inducing pancreatitis in a rodent are rodents having at least all of the following properties (1) to (3): (1) Decreased function of SLC29A3 and TLR7 (2) TLR7 function is impaired (3) TLR8 is expressed Such properties can be confirmed by techniques known in the art, such as protein analysis and functional assays. Furthermore, rodents produced by the rodent production method of the present invention and rodents produced by the method of inducing pancreatitis in a rodent of the present invention have the above-mentioned properties and are therefore useful as pathological models of pancreatitis. The use of rodents as a pathological model of pancreatitis can be confirmed by the phenotype of the rodent, pancreatic pathological findings, pancreatitis-specific chemokines in serum, and the like.

[0072] In one embodiment, a cell derived from the rodent of the present invention is provided. Examples of the cell include embryonic cells, juvenile-derived cells, adult-derived cells, and immune cells isolated from organs such as the pancreas, bone marrow, and spleen. The isolation of the cells is not particularly limited and may be performed by a method well known to those skilled in the art. From the viewpoint of use in transplantation, myeloid (myeloid) cells, including bone marrow cells, are preferred. The transplantation of bone marrow cells is not particularly limited and may be performed by a method well known to those skilled in the art.

[0073] An individual transplanted with cells derived from the rodent of the present invention may exhibit the pathological condition of pancreatitis, and thus the cells derived from the rodent of the present invention are useful for producing an individual exhibiting a pathological model of pancreatitis and for research on the same.

[0074] In one aspect, a cell having genetic information for achieving the properties of the rodent of the present invention is provided. Such a cell can be produced by genetic manipulation using a vector plasmid carrying genetic information for achieving the properties of the rodent of the present invention. The genetic information for achieving the properties of the rodent of the present invention can refer to the contents described in this embodiment, and such genetic information can be loaded into the vector plasmid by a method well known to those skilled in the art. Furthermore, genetic manipulation using such a vector plasmid can be performed by a method well known to those skilled in the art.

[0075] The sequence shown in SEQ ID NO:1 is the following sequence. YLNLSSTSLRKINAAWFKNMPHLKVLDLEFNYLVGEIASGAFLTMLPRLEILDLSFNYIKGSYPQHINISRNFSKLLSLRALHLRGYVFQELREDDFQPLMQLPNLSTINLGINFIKQIDFKLFQNFS NLEIIYLSENR

[0076] The sequence shown in SEQ ID NO:2 is the following sequence. YHFTRPLIKPQCAAYGKALDLSLNSIFFIGPNQFENLPDIACLNLSANSNAQVLSGTEFSAIPHVKYLDLTNNRLDFDNASALTELSDLEVLDLSYNSHYFRIAGVT HHLEFIQNFT

[0077] The sequence shown in SEQ ID NO:3 is the following sequence. YLNLSSTSLRKINAAWFKNMPHLKVLDLEFNYLVGEIASGAFLTMLPRLEILDLSFNYIKGSYPQHINISRNFSKLLSLRALHLRGYVFQELREDDFQPLMQLPNLSTINLGINFIKQIDFKLFQNFSNLEIIYLSENRISP LVKDTRQSYANSSSFQRHIRKRRSTDFEFDPHSNFYHFTRPLIKPQCAAYGKALDLSLNSIFFIGPNQFENLPDIACLNLSANSNAQVLSGTEFSAIPHVKYLDLTNNRLDFDNASALTELSDLEVLDLSYNSHYFRIAGVT

[0078] The sequence shown in SEQ ID NO:4 is the following sequence.

[0079] The sequence shown in SEQ ID NO:5 is the following sequence.

[0080] The sequence shown in SEQ ID NO:6 is the following sequence. gaagttatgggcgcgcccaacagaaacatggaaaacatgttccttc

[0081] The sequence shown in SEQ ID NO:7 is the following sequence. cgcggccgcggcgcgccttagtattgcttaatggaatcgacatac

[0082] The sequence shown in SEQ ID NO: 8 is the following sequence: aattcatgtcacttgcactgggaaaacaaaacaaagatcctcctcaccctattacccacattactatttttaggttttttaggggttaaaaaagatcaaaacactaatgaactttaagtcctgtgaagggtaaaacctcagatagtaacaaaagcttccaacccctcctcaaacaaaaaaccccaagtctttaactttgatccagttttcagatgctgatatccataaatggatacagttatgaattgctaattctggtctcttcactagcaaaaagcaaagcagctcagcagtacaatttcccaggaaagcaagcaaggtttctttccagcctgagcagccatcac taagtgcagttccctgcagccaacagcattaatggacgctgcactgctgctgtccttccctggagacagcagccagcactactcaagcttctcacgtagcaaccagagctccagagccagcagctgctgccgccttgtatactcactcctgtgatccaacacaggagcaaccttttc tttaccccacccccacttcttaacacacttttttttgggggggggggggaacaagtgctccatgctggaaggattggaactatgcttttagaaaggaacaatcctaaggtcacttttaaattgaggtctttgatttgaaaatcaacaaataccaaattccaaatattcgttttaa

[0083] The sequence shown in SEQ ID NO:9 is the following sequence. ccagatgactacctatcctc

[0084] The sequence shown in SEQ ID NO:10 is the following sequence. gagctgcagtggagtaggcg

[0085] The sequence shown in SEQ ID NO:11 is the following sequence. gtgcagcaatcgtcgactacagg

[0086] The sequence shown in SEQ ID NO:12 is the following sequence. ccaagtcggacaattcagtaagagc

[0087] The sequence shown in SEQ ID NO:13 is the following sequence. atgcttcttgatggttactcg

[0088] The sequence shown in SEQ ID NO:14 is the following sequence. gaacagttggccaatctctc

[0089] The sequence shown in SEQ ID NO:15 is the following sequence. tgcctctgaggacaatgtataccacagctccaatgctgtctacagagccctgatagcgtaaagcactgaggaagcgagtaaccatcaagaagctgaccaggaagccctgctgggaaactacta

[0090] The sequence shown in SEQ ID NO:16 is the following sequence. ccagcatggacgagagatgtcttc

[0091] The sequence shown in SEQ ID NO:17 is the following sequence. gcaccattgaagcgatcctctgg

[0092] The sequence shown in SEQ ID NO:18 is the following sequence. gagggtatgccgccaaatctaaagaatc

[0093] The sequence shown in SEQ ID NO:19 is the following sequence. ctgatgtctagatagcgcaattgc

[0094] The sequence shown in SEQ ID NO:20 is the following sequence. gcattgcagactagctaaaggcag

[0095] The sequence shown in SEQ ID NO:21 is the following sequence. gtcggccaggctgactccatag EXAMPLES

[0096] The present invention will be described in detail below with reference to examples, but the present invention is not limited thereto. Those skilled in the art can modify the present invention in various aspects without departing from the meaning of the present invention, and such modifications are also included in the scope of the present invention.

[0097] Establishment of SLC29A3 / TLR7dKO-huTLR8-Lyz2-Cre mice (Production of huTLR8 mice) The huTLR8 conditional knock-in mouse was generated by inserting human TLR8 cDNA into the CAG-STOP-eGFP-ROSA26TV vector. The cloning site AscI of the vector was cleaved with a restriction enzyme, and human TLR8 cDNA was inserted using an InFusion Kit (TAKARA, Japan). The following sequences were used as cloning primers. Lowercase letters represent the homology arms of the vector, and uppercase letters represent a part of the human TLR8 cDNA sequence.

[0098] [Table 1]

[0099] The constructed vector was introduced into the ES cell line JM8.A3 (KOMP, USA) by electroporation. Culture medium: Gibco knockout DMEM (ThermoFisher SCIENTIFIC, USA) / 15% FBS, 2 mM L-Glutamine (ThermoFisher SCIENTIFIC, USA), 1 mM MEM NEAA (ThermoFisher SCIENTIFIC, USA), 100 uM 2‐ME, 10 3 Geneticin (G418: ThermoFisher SCIENTIFIC, USA) was added to Units ESGRO Supplement (MILLIPORE, USA) at 150ug / ml to select non-vector-introduced ES cells. Single colonies were picked up, and genomic DNA was purified from a portion of the colony. After digestion with the restriction enzyme EcoRI, homologous recombinants were detected by Southern hybridization. A detection probe was used to detect a wild type of 15.6Kb and a homologous recombinant of 5.9Kb. The following sequence was used as the detection probe.

[0100] [Table 2]

[0101] The homologous recombinant ES cell clone was transferred into wild-type C57BL / 6 mouse fertilized eggs by microinjection, and the fertilized eggs were transplanted into pseudopregnant mice to generate chimeric mice. The chimeric mice obtained were mated with wild-type mice to transmit the gene in the germline, and huTLR8flx / wt heterozygous mice were selected from the offspring. huTLR8flx / flx was obtained by internal crossing of heterozygous mice. The following wild-type and huTLR8flx primers were used as typing primers to detect wild-type 350bp and huTLR8flx 1526bp, respectively.

[0102] [Table 3]

[0103] (Production of SLC29A3 / TLR7 dKO mice) SLC29A3 knockout mice and TLR7 knockout mice were generated using the CRISPR / CAS system. The target sequences of gRNA in each gene were searched using CRISPRdirect (http: / / crispr.dbcls.jp / ), and the following gRNA target sequences were used to generate SLC29A3 knockout mice and TLR7 knockout mice.

[0104] [Table 4]

[0105] These gRNA target sequences were cloned into the pKLV-U6gRNA(BbsI)-PGKpuro2ABFP Vector (Addgene plasmid 50946), and gRNA was synthesized using the same vector as a template with the MEGAshortscript T7 Transcription Kit (ThermoFisher Scientific, USA). In addition, hCAS9 mRNA was synthesized using the mMESSAGE mMACHIN T7 ULTRA Transcription Kit (ThermoFisher Scientific, USA) with the hCAS9 sequence in pX458 (Addgene plasmid 48138) as a template.

[0106] SLC29A3 and TLR7 knockout mice were generated by injecting both gRNAs targeting the SLC29A3 and TLR7 genes, hCAS9 mRNA, and single-stranded DNA that serves as a Repair Donor for the gRNA for the SLC29A3 gene into C57BL / 6 embryos. The following sequence was used for the single-stranded DNA that serves as a Repair Donor for the gRNA for the SLC29A3 gene. Lowercase letters represent the Homology Arm, and uppercase letters represent the knock-in sequence.

[0107] [Table 5]

[0108] After injection, the embryos were transferred back into the oviducts of pseudopregnant female mice, and the resulting offspring were typed by PCR to identify SLC29A3 / TLR7 double knockout (dKO) mice. Specifically, PCR was performed using the following SLC29A3 gene typing primer and TLR7 gene typing primer, and the PCR products were analyzed using a DNA / RNA analysis microchip electrophoresis device MultiNA (SHIMAZU, Japan). Mice with the allele in which the repair donor was knocked in were selected. One SLC29A3 / TLR7 dKO mouse was then mated with a wild-type C57BL / 6 mouse, and the resulting F1 mice were used to finally produce SLC29A3 knockout mice and SLC29A3 / TLR7 dKO mice.

[0109] [Table 6]

[0110] (Preparation of Lyz2-Cre mice) The Lyz2-Cre mice used were B6.129P2-Lyz2tm1(cre)Ifo / J (strain #004781, common name: LysMcre) obtained from The Jackson Laboratory. The following sequences were used as typing primers to detect the wild-type allele of 429 bp and the knock-in allele of 800 bp.

[0111] [Table 7]

[0112] (Production of SLC29A3 / TLR7dKO-huTLR8-Lyz2-Cre mice) SLC29A3 / TLR7dKO mice were crossed with huTLR8flx / flx mice, and the offspring were intercrossed to obtain SLC29A3- / -,TLR7- / -,huTLR8flx / flx mice (SLC29A3 / TLR7dKO-huTLR8f / f). Similarly, we crossed SLC29A3 / TLR7dKO mice with Lyz2-CreKi / Ki mice, and obtained SLC29A3- / -, TLR7- / -, Lyz2-CreKi / Ki mice by intercrossing the offspring (SLC29A3 / TLR7dKO-Lyz2-CreKi / Ki). All offspring obtained by mating male or female SLC29A3 / TLR7dKO / huTLR8f / f with female or male SLC29A3 / TLR7dKO / Lyz2-CreKi / Ki were SLC29A3- / -, TLR7- / -, huTLR8flx / wt, Lyz2-CreKi / wt mice (SLC29A3 / TLR7dKO-huTLR8-Lyz2-Cre).

[0113] Characteristics of SLC29A3 / TLR7dKO-huTLR8-Lyz2-Cre mice (phenotype) As the mice aged, inflammation and fibrosis in pancreatic tissue progressed, and impaired nutrient absorption from the intestine led to significant weight loss and death. The weight of the individual at 5-6 weeks of age was set as 100%, and the weight was measured over time using an FZ-300iWP (A&D Japan) electronic balance. A weight loss of 21% or more was set as the endpoint of individual death, and the survival rate was calculated. The mesentery was dissected from the jejunum cut from the gastroduodenal junction using dissection scissors, and the ileum, appendix, colon, and rectum were completely dissected and cut just above the anus. The ileum-appendix junction and appendix-colon junction were cut, and the weight of the appendix was measured using an electronic balance. Serum biochemistry tests were sent to Nagahama Life Science Laboratory (Oriental Yeast Co., Ltd., Japan). Total protein (TP) was quantified by the biuret colorimetric method, albumin by the BCG colorimetric method, serum glucose by the HK-G-PDH method, amylase by the BG5P substrate method, and lipase by the DGGMR substrate method.

[0114] (Pancreatic pathology findings) The pancreas removed from the mouse was fixed in 4% neutral buffered formalin solution (WAKO, Japan), embedded in paraffin using an ASP200 automatic embedding machine (LEICA Biosystems, Germany), blocks were prepared using an EG1150H (LEICA Biosystems, Germany), and thin-sectioned specimens were prepared using an SM2000R (LEICA Biosystems, Germany). According to the standard method, the thin-sectioned specimens were deparaffinized, dexylene-treated, stained with Mayer's hematoxylin (Muto Chemical), washed with water, stained with eosin, and cleared and mounted. In addition, the thin-sectioned specimens were deparaffinized, dexylene-treated, and endogenous peroxidase was removed according to standard methods. Anti-mouse CD11b antibody (ThermoFisher SCIENTIFIC, Japan) was applied as the primary antibody at 4℃ overnight, and Histofine Simple Stain Mouse MAX-PO (Rat) was applied as the secondary antibody at room temperature for 30 minutes. The attached DBA substrate kit was applied at room temperature for 10 minutes, and the specimens were washed with purified water and then mounted. The prepared specimens were image-analyzed using EBOS FL Auto (life technologies, Japan).

[0115] (Pancreatitis-specific chemokines in serum) CXCL9 and CXCL16, which have been reported as pancreatic specific chemokines, were measured by ELISA using DuoSet MouseCXCL9 and MouseCXCL16 (R&D Systems, USA). Both chemokines were measured using sandwich ELISA. Primary antibodies were immobilized on 96-well ELISA plates overnight at 4℃, blocked with 5-fold diluted Blocking-One (Nacalai Tesque, Japan) for 30 minutes at room temperature, and serum collected from wild-type mice and SLC29A3 / TLR7dKO-huTLR8-Lyz2-Cre mice was allowed to react for 2 hours at room temperature. Biotin-labeled secondary antibodies were then allowed to react for 2 hours at room temperature, and HRP-labeled streptavidin was allowed to react for 30 minutes at room temperature. The color was developed using the ELISA POD substrate TMB kit (Nacalai Tesque, Japan), and the color development was stopped with 2N sulfuric acid. The absorbance at 450 nm was measured using a GloMax EXPLORER plate reader (Promega, USA). The attached standard substance was treated in the same manner to prepare a calibration curve, and the serum concentration was quantified.

[0116] (Phenotype of individuals receiving bone marrow cells) Bone marrow cells were collected from the femur and tibia of 10-15 week-old SLC29A3 / TLR7dKO-huTLR8-Lyz2-Cre mice and suspended in FBS-free RPMI medium. Wild-type C57BL / 6 mice (Japan SLC, Japan) were irradiated with 9.5 Gy using MBR-1520R-4 (HITACHI, Japan), and the collected bone marrow cells were transferred by intravenous injection. To prevent opportunistic infections, the mice were allowed to drink 2 mg / ml Neomycin (Sigma-Aldrich, USA) water ad libitum for 2 weeks. Bone marrow transplanted mice showed a gradual weight loss from 4-6 weeks after transplantation, and pathological analysis showed severe pancreatitis similar to that of SLC29A3 / TLR7dKO-huTLR8-Lyz2-Cre mice.

[0117] At around 7 weeks of age, monocytes and macrophages began to infiltrate into the pancreatic exocrine cell area, and by around 40 weeks of age, most of the adenocytes had disappeared and become fibrotic, causing severe hypoglycemic symptoms and resulting in the death of almost all individuals.

Claims

1. A rodent having all of the following characteristics (1) to (3): (1) Decreased function of SLC29A3 (2) Decreased function of TLR7 （３） (i) a peptide comprising the amino acid sequence shown in SEQ ID NO:1; (ii) a peptide comprising an amino acid sequence having 80% or more identity to the amino acid sequence shown in SEQ ID NO: 1, or (iii) a peptide comprising an amino acid sequence having one or more amino acids added, substituted or deleted from the amino acid sequence shown in SEQ ID NO:1; and (i) a peptide comprising the amino acid sequence shown in SEQ ID NO: 2; (ii) a peptide comprising an amino acid sequence having 80% or more identity to the amino acid sequence shown in SEQ ID NO: 2, or (iii) a peptide comprising an amino acid sequence having one or more amino acids added, substituted or deleted from the amino acid sequence shown in SEQ ID NO: 2; It expresses

2. The rodent of claim 1 , which is a pathological model of pancreatitis.

3. The rodent of claim 1 , wherein the reduced function is due to a genetic mutation.

4. The rodent of claim 1 , which is double deficient in SLC29A3 and TLR7.

5. The rodent of claim 1 , wherein TLR8 is specifically expressed in monocytes and / or macrophages.

6. The rodent of claim 5 , wherein the TLR8 is human TLR8.

7. The rodent of claim 1 , wherein the rodent is a mouse.

8. The rodent of claim 2 , wherein the pancreatitis is acute pancreatitis, chronic pancreatitis or autoimmune pancreatitis.

9. A cell derived from a rodent according to any one of claims 1 to 8.

10. Reducing the function of SLC29A3 and TLR7; comprising a step of expressing TLR8; A method for producing a rodent having all of the following properties (1) to (3): (1) Decreased function of SLC29A3 (2) Decreased function of TLR7 （３） (i) a peptide comprising the amino acid sequence shown in SEQ ID NO:1; (ii) a peptide comprising an amino acid sequence having 80% or more identity to the amino acid sequence shown in SEQ ID NO: 1, or (iii) a peptide comprising an amino acid sequence having one or more amino acids added, substituted or deleted from the amino acid sequence shown in SEQ ID NO:1; and (i) a peptide comprising the amino acid sequence shown in SEQ ID NO: 2; (ii) a peptide comprising an amino acid sequence having 80% or more identity to the amino acid sequence shown in SEQ ID NO: 2, or (iii) a peptide comprising an amino acid sequence having one or more amino acids added, substituted or deleted from the amino acid sequence shown in SEQ ID NO: 2; It expresses

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