Inbred mice and method for producing inbred mice

Abstract

To provide an inbred mouse having the increased number of eosinophils to solve the problem of a conventional Th2 immune reaction activation model, where sufficient Th2 immune reaction mechanism analysis is disabled due to influence of another cytokine and complication of the analysis.SOLUTION: There is manufactured an inbred mouse that is obtained by mating non-inbred mice, is with postnatal 6-18 week-old, and has eosinophils in its peripheral blood, the number of which is equal to or larger than 200 / μL. Preferably, the non-inbred mice are not non-inbred ICR mice and are inbred mice at n-th (n=25 or more) generation of sib-mating.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to an inbred mouse having a higher number of eosinophils in peripheral blood than normal inbred mice, and a method for producing such an inbred mouse. relates to.

Background Art

[0002] As model animals with activated Th2 immune responses, parasite-infected animals and model animals with experimentally controlled cytokines are used. However, due to the complexity of the reaction process and the non-continuous activation of the Th2 immune response, sufficient results have not been obtained in the analysis of the Th2 activation mechanism using these models. Therefore, these models with activated Th2 immune responses are only used when studying how Th2 activation affects the living body. However, even regarding such usage methods, the period of Th2 activation in these models is temporary, about two weeks, and it is relatively difficult to measure the timing at which the Th2 immune response is optimal when conducting a predetermined treatment or analysis. From this point of view, these Th2 activation models have been considered insufficient.

[0003] On the other hand, there are several model mice showing eosinophilia. For example, IL-5 transgenic mice into which the IL-5 gene having proliferative activity against eosinophils has been introduced (see Non-Patent Document 1), and knockout mice of IL-10, which is a suppressive cytokine and is considered to cause suppression of Th2 cytokines (see Non-Patent Document 2), have been disclosed. These are model animals using known cytokine functions, and cannot be used for analyzing the Th2 activation mechanism, and can only be used for analyzing the eosinophil function itself.

[0004] On the one hand, the present inventor has disclosed a method for increasing eosinophils in mice by a single administration of a poorly soluble substance derived from a parasite (see Patent Document 1). Although such a method is excellent from the viewpoint of inducing immunopotentiation, there are problems in that it is necessary to adjust the poorly soluble substance derived from a parasite and there is variation in the content of the active ingredient. Therefore, there has been a demand for a simpler method for producing a model animal with increased eosinophils.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Non-Patent Documents

[0006]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0007] As described above, in the conventional Th2 immune response activation model, the mechanism of the sufficient Th2 immune response could not be analyzed due to the influence of other cytokines and the complexity of the analysis. Therefore, an object of the present invention is to provide an inbred mouse with an increased number of eosinophils.

Means for Solving the Problems

[0008] In immune system experiments, inbred mice such as C57BL and Blub / C are usually used. Outbred mice are not used because their genetic background is too large. Under these circumstances, the inventors of the present invention, while earnestly studying to solve the above problems, used outbred mice administered with the Th2 immune activation composition described in Patent Document 1 above, and focused on the number of peripheral blood eosinophils as an index of the activation of the Th2 immune response and conducted research. In this process, several outbred mice with a higher number of eosinophils in peripheral blood than normal outbred mice were found. Therefore, when inbred mice were produced using the outbred mice with a high number of peripheral blood eosinophils, inbred mice with the trait of a high number of peripheral blood eosinophils fixed were obtained, and the present invention was completed.

[0009] That is, the present invention is as follows. 〔1〕An inbred mouse obtained by mating outbred mice, having 200 / μL or more eosinophils in peripheral blood at 6 to 18 weeks of age. 〔2〕The inbred mouse according to the above 〔1〕, wherein the outbred mouse is an outbred ICR mouse. 〔3〕The inbred mouse according to the above 〔1〕 or 〔2〕, which is the nth generation (n = 25 or more) of sibling mating. 〔4〕The inbred mouse according to any one of the above 〔1〕 to 〔3〕, characterized in that the expression of IL-5 mRNA in the spleen has no significant difference compared to the expression of IL-5 mRNA in the spleen of normal outbred ICR mice. 〔5〕The inbred mouse according to any one of the above 〔1〕 to 〔4〕, wherein the fertilized egg is deposited under the accession number NITE P-03337. 〔6〕(a) Step A of mating outbred mice and selecting offspring mice having eosinophils in peripheral blood of 2SD values or more compared to the average value of eosinophils in peripheral blood of the mice before mating; (b) Step B of producing inbred mice by sibling mating the offspring mice selected in Step A for 20 generations or more; A method for producing inbred mice, comprising Steps A and B. The method for producing an inbred mouse according to [6] above, characterized in that the outbred mouse is an outbred ICR mouse or a DDY mouse. 〔8〕The method for producing an inbred mouse according to [6] or [7] above, wherein the number of eosinophils in peripheral blood is 200 / μL or more at 6 to 18 weeks of age after birth.

Advantages of the Invention

[0010] Since the number of eosinophils in the inbred mouse of the present invention is higher than that of a mouse with normal eosinophils, it is possible to analyze the function of eosinophils, and it can also be used as a model animal with increased eosinophils.

Brief Description of the Drawings

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Modes for Carrying Out the Invention

[0012] The inbred mice of the present invention are not particularly limited as long as they are inbred mice obtained by mating non-inbred mice and having an eosinophil count of 200 / μL or more in peripheral blood at 6 to 18 weeks of age after birth, and are also referred to as "the present inbred mice" hereinafter. Further, the method for producing the inbred mice of the present invention includes: (a) Step A of mating non-inbred mice and selecting offspring mice having an eosinophil count in peripheral blood that is 2SD (standard deviation) values or more compared to the average value of the eosinophil count in peripheral blood of the mice before mating; (b) Step B of brother-sister mating the offspring mice selected in Step A for 20 generations or more to produce inbred mice; It is not particularly limited as long as it is a method for producing inbred mice including Steps A and B, and is also referred to as "the method for producing the present inbred mice" hereinafter.

[0013] Here, in this specification, an inbred strain refers to a strain that has been subcultured for 20 or more generations by brother-sister mating. Such inbred mice are presumed to have 99% of their gene loci identical and are used as genetically completely identical. Also, a non-inbred strain refers to a strain that contains many heterozygous gene loci. Note that in this specification, the above-mentioned brother-sister mating means selecting a male and a female from among the offspring of the same litter for mating.

[0014] This inbred mouse is preferably of the nth generation (n = 25 or more) of brother-sister mating from the viewpoint of more fixed traits. Also, in brother-sister mating, a method of selecting mice with 300 or more eosinophils in peripheral blood from among the offspring can be mentioned.

[0015] Examples of non-inbred mice include ICR mice, DDY mice, etc., and ICR mice can be preferably mentioned. Furthermore, it is preferable that the non-inbred mouse has 200 / μL or more eosinophils in peripheral blood, or that the ICR mouse has eosinophil counts of 2SD or more compared to the average value of eosinophil counts in peripheral blood of normal ICR mice. Note that the average value of eosinophil counts in peripheral blood of normal ICR mice is 101.4 ± 59.3 / μL (n = 34).

[0016] Examples of this inbred mouse include those having 200 / μL or more eosinophils in peripheral blood at 6 to 18 weeks of age after birth, preferably 300 / μL or more, and more preferably 400 / μL or more. The eosinophil count in peripheral blood can be counted by known methods. Specifically, methods such as collecting blood and counting with an eosinophil counting plate (Hinkelmann method), or staining by percentage (relative to the white blood cell count) can be mentioned. Note that having 200 / μL or more eosinophils in peripheral blood at 6 to 18 weeks of age after birth means that it is sufficient if the eosinophil count in peripheral blood is 200 / μL or more at any time between 6 and 18 weeks of age after birth, but preferably the eosinophil count in peripheral blood is 200 / μL or more at all times between 6 and 18 weeks of age after birth.

[0017] As another aspect, fertilized eggs can include inbred mice deposited under accession number NITE P-03337. The fertilized eggs deposited under the accession number NITE P-03337 were deposited at the Patent Microorganisms Depositary, National Institute of Technology and Evaluation (NITE), 2-5-8 Kazusa Kamatari, Kisarazu-shi, Chiba 292-0818, Japan on December 17, 2020, and are distributable under certain conditions.

[0018] It is preferable that the expression of IL-5 mRNA in the spleen of this inbred mouse has no significant difference compared to the expression of IL-5 mRNA in the spleen of normal non-inbred ICR mice. Here, "no significant difference" means no statistical significant difference. For example, a significant difference means that the p-value is 0.05 or less by a T-test.

[0019] Hereinafter, the present invention will be described more specifically by way of examples, but the technical scope of the present invention is not limited to these illustrations.

[0020] [Example 1] (Production of mice with high eosinophil-producing ability) In the process of elucidating the mechanism of Th2 activation, the present inventor previously attempted to create a new model different from the conventional models for activating Th2 immune responses. In that process, the experiment was advanced by focusing on the number of peripheral blood eosinophils. At that time, several mice with a much higher number of eosinophils than ICR normal control mice (control mice) were found among the ICR mice used. The number of eosinophils in the offspring mice obtained by mating these ICR mice was measured, and mice showing an eosinophil count equal to or higher than the value of the average ± 2SD of the number of eosinophils in the peripheral blood of commercially available ICR mice (Japan SLC) were judged as positive individuals. Brother-sister mating by the positive individual selection method was repeated 20 times to produce inbred mice, and such inbred mice were named "Yama mice".

[0021] [Example 2] (Number of eosinophils in peripheral blood of Yama mice) The eosinophil count in the peripheral blood of Yama mice prepared in Example 1 was examined. From the perspective of animal welfare, and considering the burden of blood sampling and the trend of eosinophil counts from preliminary experiments, the determination of eosinophil counts was performed at 6 weeks of age.

[0022] The eosinophil count was measured by the following method. A part of the blood collected by orbital bleeding from Yama mice (n = 40) and ICR normal control mice (control mice) (n = 34) at 6 or 18 weeks of age was diluted 10-fold with Hinkelmann solution (0.5% yellow eosin, 0.5% phenol, 0.5% formalin), gently stirred, and then the eosinophil count (Number of eosinophils) was counted using an eosinophil counting chamber (TATAI). The results are shown in Figure 1.

[0023] As is clear from Figure 1, the eosinophil count (eosinophils / μL) in peripheral blood at 6 weeks of age was maintained at a value more than 5.5 times higher in Yama mice compared to control mice, and more than 3.5 times higher at 18 weeks of age. Subsequently, sibling mating was continued for 25 generations, and going back approximately 10 generations, 100% of the individuals were above the normal ICR average value + 2SD (positive), and it was considered that the expression of the trait was more stable than in the initial stage of mating. On the other hand, although a decrease in eosinophil count was observed as age progressed, due to the initially high values, it showed a significantly higher value compared to the normal control group at 18 weeks of age. Although the eosinophil count decreased from 6 weeks to 18 weeks of age, this is a tendency also seen in normal mice, indicating a state closer to the living body and that the immune mechanism is functioning normally. Also, the fact that the eosinophil count remained high at 18 weeks of age is useful as a Th2 activation model for analyzing the continuous Th2 activation mechanism.

[0024] [Example 3] (Eosinophil count in bone marrow tissue) To examine whether the number of eosinophils increases in bone marrow tissue, histopathological observation was performed. The histopathological specimens were prepared by the following procedure. That is, 6-week-old Yama mice and control mice were each anesthetized with a mixture of ketamine hydrochloride and xylazine hydrochloride, and then euthanized by whole blood collection from the heart using a heparinized syringe. Thereafter, the spleen, mesenteric lymph nodes, liver, and jejunum from the base of the duodenum were sampled, fixed with Carnoy's solution, and paraffin sections (histopathological specimens) of 4 μm were prepared. The above histopathological specimens were stained with hematoxylin-eosin (HE) by a conventional method and then observed with an optical microscope. The results are shown in Figure 2. Also, a graph showing the ratio of the number of eosinophils among all bone marrow cells in the above histopathological specimens is shown in Figure 3.

[0025] As shown by the left arrow (black triangle) in Figure 2, eosinophils in the bone marrow were significantly increased in Yama mice. Also, as shown in Figure 3, the proportion of cells showing positivity in total bone marrow cells was more than twice as high as that of control mice.

[0026] [Example 4] (Immunohistochemical Observation of Bone Marrow Tissue) Immunohistochemical observations were also performed to determine whether an increase in eosinophils was observed in the bone marrow tissue. The prepared bone marrow sections were immunostained with an anti-ECP (Eosinophil cationic protein) antibody, which is a marker for eosinophils. For the immunostaining, after removing paraffin with xylene, antigen activation was performed with 0.05% trypsin (37 °C, 30 minutes). Subsequently, endogenous peroxidase was inactivated with 3% hydrogen peroxide solution (room temperature, 10 minutes), and blocking was performed with a blocking solution (10% skim milk and 2% BSA dissolved in PBS) (room temperature, 30 minutes). Rabbit Anti-Mouse Eosinophil Cationic Protein (ECP) IgG (Aviscera Bioscience Inc.) was diluted 400-fold and dropped onto the sections, and the sections were left standing at room temperature for 1 hour. Subsequently, the sections were washed three times with PBS, and EnVision+System-HRP Labelled Polymer Anti-Rabbit (Dako) was dropped onto the sections and left standing at room temperature for 1 hour. After washing three times with PBS, color development was performed with DAB Substrate (Roche), and observations were made using an optical microscope. The results are shown in Figure 4. Also, the number of cells showing positive for the anti-ECP antibody was measured. The measurement was performed by measuring the total number of bone marrow cells and the number of positive cells in five fields at 100-fold magnification for each individual in each group. Then, the ratio of the number of cells showing positive to the total number of bone marrow cells was calculated. The results are shown in Figure 5.

[0027] As shown by the arrow (black triangle) in Figure 4, it was also observed by immunohistochemical observation that the production of eosinophils had increased. Furthermore, as is clear from Figure 5, the ratio of the number of cells showing positive to the total bone marrow cells was the same as the value in Figure 3 of Example 3 above, and the increase in eosinophils in the bone marrow was also confirmed by immunostaining.

[0028] [Example 5] (Expression of IL-5 mRNA) As conventional mice with high eosinophil counts, genetically modified mice that secrete the cytokine IL-5, which is an eosinophil growth factor, genetically modified mice in which the secretion of IL-10, which is a Th2 activation inhibitor, is suppressed, mice infected with parasites, and Th2 immune-activated mice by sensitization with an antigen or the like have been used. In these models, although the eosinophil count is high, an increase in IL-4 or IL-5 is observed, and concerns about the influence of the presence or absence of cytokines and the complexity of the treatment process on the experiment have not been eliminated. Furthermore, due to parasite infection or antigen sensitization, various other influences are a concern in addition to the increase in eosinophil count. Therefore, the expression of IFN-γ, IL-4, and IL-5 mRNAs in Yama mice was examined.

[0029] RNA extraction, cDNA synthesis, and mRNA detection by quantitative PCR were performed according to the following procedures. That is, RNA was manually extracted from the spleen, mesentery lymph node, or marrow of 6-week-old mice (n = 3) of the 21st generation using the RNeasyTM Kit (QIAGEN). Subsequently, 1 μg of random primer (Invitrogen) was added to 10 μl of the extracted RNA, and after reacting at 70°C for 10 minutes, it was immediately placed on ice. Then, 5×RT buffer, dNTP, 0.1 M DTT, and RNasin (Promega) were added to make it 19 μL, and 1 μL of SuperScriptII (Invitrogen) was added to make it a total of 20 μL, and cDNA was synthesized by reacting at 42°C for 2 minutes, then at 42°C for 50 minutes, and at 70°C for 15 minutes. Furthermore, a primer set (Applied Biosystems) for amplifying the mRNAs of IFN-γ, IL-4, and IL-5, the cDNA synthesized above, and the TaqManTM Gene Expression Assays kit (Applied Biosystems) were used to perform real-time PCR using the Step OneTM Real-Time PCR System (Applied Biosystems) to quantify the mRNA amounts of IFN-γ, IL-4, and IL-5. The PCR conditions were 1 cycle of 2 minutes at 50°C and 10 minutes at 95°C, followed by 50 cycles of 15 seconds at 95°C and 1 minute at 60°C. In addition, a real-time PCR method using a primer set (Applied Biosystems) for amplifying the housekeeping gene 18s rRNA was performed as a control mouse, and the values of the mRNA amounts of IFN-γ, IL-4, and IL-5 were standardized by the calculated values of the 18s rRNA amounts. The results are shown in Figure 6.

[0030] As is clear from Figure 6, there was no significant difference in the expression of IL-5 mRNA related to eosinophils between control mice and Yama mice in any of the spleen, mesentery lymph node, or marrow. Therefore, it became clear that eosinophils were increased in Yama mice regardless of the increased expression of IL-5.

[0031] In addition, regarding IFN-γ and IL-4, which are indicators of Th-1 and Th-2 immune responses, both IFN-γ and IL-4 increased in the spleen, and no significant difference was observed in the mesenteric lymph nodes and bone marrow. Normally, if it shifts to TH-1, the expression of IFN-γ increases and the expression of IL-4 decreases, and if it shifts to TH-2, the expression of IFN-γ decreases and the expression of IL-4 increases. Therefore, the results in Figure 5 suggest that the balance between TH-1 and Th-2 tends not to be disrupted.

[0032] [Example 6] (Th2 Immune Response in Yama Mice) Using an experimental ulcerative colitis model in which the Th2 immune response is considered to be suppressive, changes in body weight, length of the large intestine, and histopathological changes were examined.

[0033] For 10-week-old Yama mice and ICR normal control mice (control mice) after birth, 5% dextran sulfate sodium (DSS) was dissolved in drinking water to make an ulcerative colitis model, and they were allowed to drink freely. The body weight was measured over time with the start of DSS administration as 0 day, and then after 6 days, they were anesthetized with a mixture of ketamine hydrochloride and xylazine hydrochloride, and then euthanized by whole blood collection from the heart using a heparinized syringe. The large intestine from the colon to the rectum was sampled, the length of the large intestine was measured, fixed with Carnoy's solution, and then 4-μm paraffin sections (large intestine histopathological specimens) were prepared. After subjecting the above histopathological specimens to the conventional hematoxylin-eosin (HE) staining, they were observed under an optical microscope. Figure 7 shows the results of body weight measurement, Figure 8 shows the results of measuring the length of the large intestine, and Figure 9 shows the results of observing the histopathological specimens under an optical microscope.

[0034] In normal control mice, weight loss and shortening of the large intestine length were observed due to colitis, but neither weight loss nor shortening of the large intestine length was observed in Yama mice. Histopathologically, necrosis of the mucosal epithelium and infiltration of inflammatory cells into the surrounding tissues were observed in normal control mice, but neither necrosis of the mucosal epithelium nor infiltration of inflammatory cells was seen in Yama mice. From these results, it was confirmed that in Yama mice, not only eosinophilia but also little eosinophil infiltration at the lesion site occurred, so eosinophilia was not the cause of the lesion but was caused by the activation of the Th2 immune response itself. That is, Yama mice are considered to be Th2 activation model mice.

Industrial Applicability

[0035] This inbred strain of mice can be used to elucidate diseases in which the number of eosinophils is thought to be involved in the exacerbation of symptoms, such as cirrhosis, lung adenocarcinoma, or atopic diseases, to conduct pharmacological effect tests for these diseases, and to elucidate the mechanism of eosinophilia.

Claims

1. An inbred mouse obtained by mating non-inbred ICR mice, having an eosinophil count of 200 / μL or more in peripheral blood at 6 to 18 weeks of age after birth.

2. The inbred mouse according to Claim 1, which is the nth generation (n = 25 or more) of sibling mating.

3. The inbred mouse according to Claim 1 or 2, characterized in that the expression of IL-5 mRNA in the spleen has no significant difference compared to the expression of IL-5 mRNA in the spleen of normal non-inbred ICR mice.

4. The inbred mouse according to any one of Claims 1 to 3, wherein the fertilized egg is deposited under the accession number NITE P-03337.

5. (a) Step A of mating non-inbred ICR mice or DDY mice and selecting offspring mice having an eosinophil count in peripheral blood that is 2 SD values or more compared to the average value of the eosinophil count in peripheral blood of the mice before mating; (b) Step B of producing inbred mice by sibling mating the offspring mice selected in Step A for 20 generations or more; A method for producing an inbred mouse, comprising Step A and Step B.

6. A method for producing the inbred mouse according to Claim 5, having an eosinophil count of 200 / μL or more in peripheral blood at 6 to 18 weeks of age after birth.

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