Patent Description:
As an extremely excellent immunodeficient mouse, a NOG mouse is known (Patent Literature <NUM>). Humanized NOG mice are prepared by transplanting human cells or tissues into NOG mice. In order to reproduce the human innate immune system, it is necessary to develop a mouse, in which human granulocytes such as human neutrophils can circulate in the periphery for a long time. For the purpose, it is known that human hematopoietic stem cells are transplanted and human G-CSF protein is administered to prepare mice (Non Patent Literatures <NUM>, <NUM>); however, human neutrophils engrafted but transient and mouse neutrophils increases. A system dedicated to the human innate immune system cannot be obtained. This is a matter of great concern.

Eosinophils and basophils are the cells of the innate immune system involved in allergic diseases. There are mice in which human eosinophils and human basophils differentiate (Non Patent Literatures <NUM>, <NUM>). However, in any mouse, human eosinophils and human basophils differentiate only in small amounts. Rodents, in which sufficient amounts of human eosinophils and basophils are maintained for a long term, have not yet been obtained.

Patent Literatures <NUM>: International Publication No. <CIT>.

Further documents identified during the Search and Examination procedure are outlined below.

is a conference booklet from the Japanese Society of Hematology, disclosing abstracts of the posters to be presented. The abstract forPS2-<NUM>-<NUM> discloses a novel NOG mouse strain with human G-CSF knock-in targeting the G-CSF receptor locus to induce mature human neutrophils in their circulation.

Rongvaux et al. discloses development of humanized mice with human innate immune cells.

Inoue discloses the generation of various FcyR-deficientNOD lines.

An object of the present invention is to provide a humanized mouse in which the human innate immune system can be reproduced and maintained for a long term without activating the mouse innate immune system.

The present inventors knocked-in a human G-CSF cytokine known as a neutrophil-inducing factor in an immunodeficient mouse in order to promote engraftment of human neutrophils. At this time, to prevent a cross reaction between a human G-CSF and a mouse G-CSF receptor to activate the mouse innate immune system, a human G-CSF gene was knocked-in at a mouse G-CSF receptor locus to provide deficiency of the mouse G-CSF receptor.

As a result, the inventors have successfully produced a humanized mouse in which a human G-CSF is present in serum and the human hematopoietic system is reconstructed in an immunodeficient mouse.

The inventors further deleted a receptor that binds to the Fc region of an antibody in the humanized mouse in which a human G-CSF is present in serum and the human hematopoietic system is reconstructed in an immunodeficient mouse, to produce a humanized mouse in which human neutrophils much more differentiate than in the aforementioned mouse.

Furthermore, the inventors deleted a receptor that binds to the Fc region of an antibody in a hIL-<NUM>/hGM-CSF Tg mouse and a hIL-<NUM>/hGM-CSF/hIL-<NUM> Tg mouse in which small amounts of human eosinophils and basophils differentiate to produce a humanized mouse in which the eosinophils and basophils much more differentiate.

In a first aspect there is provided a rodent having a human G-CSF gene knock-in at a G-CSF receptor locus thereof, as defined in claim <NUM>.

In a second aspect, there is provided a rodent model for a human pathogen infectious disease, obtained by infecting the humanized rodent with a bacterium or virus, as set out in claim <NUM>.

The rodent described herein, which is a knock-in rodent prepared by knocking in a human G-CSF gene at a rodent's G-CSF receptor locus and transplanting human hematopoietic stem cells thereinto, is a humanized rodent in which the human immune system is reproduced and human neutrophils and monocytes circulate in the periphery. Such a rodent can be used in studies for human immune system. The mouse described herein is the first humanized mouse in which human neutrophils circulate in the peripheral blood for a long term and useful in studies for a congenital defense mechanism against human bacterial infection. The rodent further infected with a pathogen can be used as a rodent model for a human infectious disease.

A humanized mouse, in which human eosinophils and basophils are highly differentiated and maintained for a long term, can be used for reproducing a severe human allergic disease such as refractory asthma that has been difficult to treat.

The following passages are provided for illustration purposes only.

Described herein is a knock-in rodent having a human G-CSF (granulocyte-colony stimulating factor) gene knock-in. In the knock-in rodent described herein, a human G-CSF gene is knocked-in at a G-CSF receptor locus that a rodent originally has, and thus the G-CSF receptor that the rodent originally has is not expressed, or even if it is expressed, the receptor is deficient in the function. The knock-in rodent as described herein can be prepared in one step by knocking in the human G-CSF gene, while defunctionalizing the G-CSF receptor of a rodent.

The DNA sequence of the human G-CSF gene is represented by SEQ ID NO: <NUM> and the DNA sequence of the mouse G-CSF receptor gene is represented by SEQ ID NO: <NUM>.

The knock-in (gene knock-in) is a genetic engineering technique for inserting a DNA sequence encoding a protein at a predetermined locus on a chromosome of an organism.

Human G-CSF can bind to a G-CSF receptor of a rodent. Accordingly, when a G-CSF receptor is expressed in a rodent having a human G-CSF gene knock-in, if the human G-CSF gene knock-in in a rodent is expressed within the body of the rodent, human G-CSF binds to the G-CSF receptor expressed in, e.g., neutrophil progenitor cells of the rodent, with the result that the neutrophils of the rodent may proliferate. However, the G-CSF receptor of the knock-in rodent as described herein is already defunctionalized, the human G-CSF expressed in a knock-in rodent will not bind to the rodent's G-CSF receptor.

In the blood of the knock-in rodent having a human G-CSF gene at a rodent's G-CSF receptor locus, the human G-CSF is present. If human hematopoietic stem cells (HSC) are transplanted into the knock-in rodent, the human G-CSF stimulates human hematopoietic stem cells within the body of the rodent, with the result that human neutrophils and monocytes differentiate, proliferate and circulate in the peripheral blood. More specifically, when human HSCs are transplanted into a rodent having a human G-CSF gene knock-in at a rodent's G-CSF receptor locus, the human hematopoietic system is reconstructed within the body of the rodent. In this sense, the rodent is a humanized rodent in which the human hematopoietic system is reconstructed. In the rodent, human neutrophils and monocytes circulate in the periphery.

Disclosed herein is a method for preparing a knock-in rodent having a human G-CSF gene, comprising knocking in a human G-CSF gene at a G-CSF receptor locus of a rodent.

Disclosed herein is a method for preparing a knock-in rodent having a human G-CSF gene at a G-CSF receptor locus thereof, wherein human HSCs are transplanted, and human neutrophils are differentiating and circulating in the periphery, by transplanting human HSCs into a knock-in rodent having a human G-CSF gene.

Described herein is a knock-in rodent having a human G-CSF gene at a rodent's G-CSF receptor locus, wherein human HSCs are transplanted, and human neutrophils are differentiating and circulating in the periphery.

As described herein, examples of the rodent include, but are not limited to, a mouse, a rat, a guinea pig, a hamster, a rabbit and a nutria. Of them, a mouse is preferable.

The nucleotide sequence of the human G-CSF gene is represented by SEQ ID NO: <NUM> and the nucleotide sequence of the mouse G-CSF receptor gene is represented by SEQ ID NO: <NUM>.

A humanized rodent, as described herein. in which the human neutrophils circulate in the periphery is a rodent that does not eliminate human cells such as human neutrophils by the immune system, and more specifically, a rodent in which the immune response to humans is inactivated. As such a rodent, a rodent having a lowered or deficient immune function and inactivated in immune response to humans is mentioned. For example, an immunodeficient rodent, can be used.

The immunodeficient rodent is a rodent having lowered or deficient immune function, and more specifically, a rodent which lacks a part or whole of T cells, B cells, NK cells, dendritic cells and macrophages. The immunodeficient rodent can be prepared by irradiating the whole body with X-rays. Alternatively, a rodent genetically deficient in immune function can be used.

Examples of the immunodeficient mouse that can be used include a nude mouse, a NOD/SCID mouse, a Rag2 knockout mouse, a SCID mouse administered with an asialo-GM1 antibody or TMβ1, and a mouse irradiated with X-rays. Also, knockout mice (hereinafter referred to as dKO (double knockout) mice) obtained by crossing a NOD/SCID mouse or a Rag2 knockout mouse with an IL-2Ry knockout mouse, can be used. For example, a dKO mouse (Rag2 KO, IL-2Rnull) can be used. The dKO mouse having a genetic background of Balb/c is referred to as a Balb/c dKO mouse; a dKO mouse having a genetic background of NOD is referred to as a NOD dKO mouse. The genetic background of a mouse is not limited to these. Not only C57BL/<NUM>, C3H, DBA2 and IQI strains but also a strain having a SCID mutation and an IL-2Ry knockout, or a Rag2 knockout and an IL-2R γ knockout mutation, as well as a deficiency of Jak3 protein responsible for signal transduction downstream of common γ chain of an IL-<NUM> receptor, have the similar phenotype to IL2Rγnull. Because of this, a knockout mouse obtained by crossing a Rag2 knockout mouse with a Jak3 knockout; a knockout mouse obtained by crossing a SCID mutation and a Jak3 knockout; and an inbred mouse, a non-inbred mouse and a crossbreeding (F1 hybrid) mouse obtained by crossing them, may be used.

In order to exclude an effect by immune cells such as NK cells observed in the mouse, a SCID mouse administered with an asialo GM1 antibody is used as mentioned above. Other than this embodiment, there is another mouse to be used described herein. This is a genetically modified immunodeficient mouse lacking the IL-<NUM> receptor γ chain due to introduction of a mutation in an IL-<NUM> receptor γ chain gene and having a SCID mutation of a gene responsible for reconstruction of antigen receptor genes of a T cell and a B cell at both allele loci. Examples of such a mouse include mice such as a NOD mouse derived from a NOD/SCID mouse and having a knock-out of common γ chain of IL-<NUM> receptor (NOD/SCID/γcnull (NOD/Shi-scid, IL-2Rγ KO mouse)), a NSG mouse (NOD/Scid/IL2Rγnull (NOD. Cg-PrkdcscidIL2rgtm1Wjl/SzJ)) and a NCG mouse (NOD-Prkdcem26Cd52IL2rgem26Cd22/NjuCrl). Further, a genetically modified immunodeficient mouse NOJ mouse (NOD/Scid/Jak3null (NOD. Cg-PrkdcscidJak3tm1card)) deficient in Jak3 due to introduction of a mutation in a Jak3 gene and having a SCID mutation of a gene responsible for reconstruction of an antigen receptor gene of a T cell and a B cell in both allele loci, can be used. Hereinafter, animals (mice) deficient in function of a Prkdc gene and a gene product thereof due to e.g., a scid mutation; and losing a normal function of an IL2Rγ gene product due to deletion or mutation of an IL2Rγ gene or loss-of-function of a gene present downstream of signal transduction and a gene product thereof will be referred to as NOG mice ("NOG mouse" is a registered trademark) and can be used as a host. Since lymphocytes are not present in these mice, the NOG mice have neither the NK activity nor dendritic cell function. A method for producing NOG mice is described in <CIT>. A method for producing NSG mice is described in <NPL>. A method for producing NCG mice is described in<NPL>. A method for producing the NOJ mice is described in <NPL>.

A method for knocking in the human G-CSF gene is not limited, and for example, a gene knock-in can be made by homologous recombination and genome editing. The following example disclosing the preparation of the humanized rodent described herein is present for illustration purposes only.

Homologous recombination refers to a phenomenon where recombination of two DNA molecules occurs via the same nucleotide sequence within a cell and is a method frequently used for recombination of an organism having huge genomic DNA. A plasmid (called a targeting vector) is constructed by connecting foreign DNA in such a manner that the sequence of a target gene site is divided at the center. More specifically, the human G-CSF gene is isolated. A construct is prepared by sandwiching DNA of the gene between homologous sequences of the upstream part and the downstream part of a rodent's G-CSF receptor. The construct is inserted into a vector known in the technical field to prepare a targeting vector, which is then introduced into an immunodeficient rodent's ES cell (embryonic stem cell). Owing to homologous recombination, the rodent's G-CSF receptor gene DNA is exchanged with the same sequence part on the targeting vector. Since the foreign DNA sandwiched is integrated into a target gene, i.e., a G-CSF receptor gene, the gene loses function. In this manner, ES cells are established, injected in a rodent's embryo or blastocyst and transplanted into the uterus of a rodent having a pseudo-pregnancy with a chimera embryo to prepare a chimera mouse. The chimera mouse thus obtained is crossed with the aforementioned immunodeficient rodent to obtain F1 individuals having heterologous human G-CSF gene knock-ins at a rodent's G-CSF receptor locus. The individuals having heterologous knock-ins are crossed to successfully obtain an immunodeficient rodent having heterologous human G-CSF gene knock-ins at a rodent's G-CSF receptor locus.

At this time, as a vector for use in preparing a targeting vector, it is possible to use a vector that can be expressed in rodent's ES cells and can be used for transformation. For example, a plasmid derived from Escherichia coli, a retrovirus, a lentivirus, an adeno-associated virus and vaccinia virus, can be used.

A promoter may be linked to a site upstream of the human G-CSF gene. For example, a CMV promoter can be used.

A targeting vector can be introduced into an ES cell by a method known in the technical field such as an electroporation method, a calcium phosphate co-precipitation method, a lipofection method, a microinjection method and a particle gun method.

The vector may contain a selective marker gene. Examples of the marker gene include a hygromycin resistance gene, a neomycin resistance gene and a puromycin resistance gene. The marker gene may be removed after a homologous recombinant is selected and can be removed by use of the Cre-loxP system and Flp-frt system.

In place of ES cells, stem cells such as rodent's iPS cells can be used.

The genome editing is a method for modifying a target gene by use of a site specific nuclease. As a genome editing method, the following methods, which vary depending on the type of the nuclease to be used can be mentioned: ZFN (zinc finger nuclease) method (<NPL>), TALEN (Tale nuclease) method (<NPL>), methods using the CRISPR/Cas system such as CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 (Crispr Associated protein <NUM>) (<NPL>) and CRISPR/Cas3. A method using a modified nuclease such as nickase modified Cas is also included in these methods. Of them, a method using the CRISPR/Cas9 system is preferred. In the CRSPR/Cas9 system, for example, a targeting vector containing guide RNA (crRNA, tracrRNA), which contains a complementary sequence for a target sequence of a rodent's G-CSF receptor gene, whose function is desired to destroy by cutting, a nuclease Cas9, and human G-CSF gene DNA to be knocked in as donor DNA, is injected into a fertilized egg of an immunodeficiency rodent gene. In the fertilized egg, the rodent's G-CSF receptor gene is deficient and, in place of the receptor gene, the human G-CSF gene is knocked in.

When a rodent's G-CSF receptor gene is destroyed by genome editing, a target sequence in the gene is selected and a guide RNA sequence may be designed so as to contain a complementary sequence for the sequence. The nucleotide length of the guide RNA is preferably <NUM> or more. The genome editing by CRSPR/Cas9 can be carried out by a commercially available CRISPR/Cas9 tool.

The strain of a knock-in mouse prepared having a human G-CSF gene knock-in at a G-CSF receptor locus of a NOG mouse is officially expressed as NOD. Cg-PrkdcscidIl2rgtm1SugCsf3rtm1(CMV-CSF3)/Jic and abbreviated as hG-CSF KI.

By transplanting human hematopoietic stem cells into the knock-in rodent prepared having a human G-CSF gene knock-in at a rodent's G-CSF receptor locus, a humanized rodent in which human hematopoietic stem cells are engrafted and human neutrophils circulate in the periphery, can be obtained.

It is preferable that the knock-in rodent is exposed to radiation before transplantation. At this time, it is preferable that radiation is applied at a dose of <NUM> to <NUM> Gy, preferably <NUM> to <NUM> Gy, and further preferably <NUM> Gy.

Human hematopoietic stem cells (CD34+ cells) are preferably transplanted via a vein, for example, a tail vein. The number of the human hematopoietic stem cells to be transplanted varies depending on the body weight of a rodent. In the case of a mouse, for example <NUM><NUM> to <NUM><NUM> human hematopoietic stem cells, preferably <NUM><NUM> to <NUM><NUM> human hematopoietic stem cells, and further preferably, <NUM> to <NUM> × <NUM><NUM> human hematopoietic stem cells may be transplanted. A humanized rodent in which human neutrophils circulate in the periphery can be obtained <NUM> to <NUM> weeks, preferably <NUM> to <NUM> weeks, further preferably <NUM> to <NUM> weeks after transplantation.

The humanized rodent having a human G-CSF gene knock-in at a rodent's G-CSF receptor locus prepared in step <NUM>, and transplanted with human HSCs, in which human neutrophils circulate in the periphery, has the following characteristics.

The human G-CSF is present in blood and a rodent G-CSF receptor in cells is deficient. Because of this, the rodent's neutrophil count is low or zero.

Human neutrophils differentiate, increase and circulate in the peripheral blood. The ratio of human neutrophils <NUM> weeks after transplantation is <NUM> to <NUM>%. The human neutrophils have an ability to produce reactive oxygen species.

Human neutrophils and monocytes differentiate, increase, and circulate in the peripheral blood. The ratio of human monocytes <NUM> weeks after transplantation is <NUM> to <NUM>%.

Human cytokines derived from the bone marrow cells are present in the blood. Examples of the cytokines include IL-<NUM>, IL-<NUM>, IL-<NUM>, MCP-<NUM>, MIP1a, MIP-1b and IFNg.

In the knock-in rodent described herein transplanted with human hematopoietic stem cells and having the above characteristics, the human innate immune system can be reproduced.

Human neutrophils are increased by administering, for example, a TLR2 ligand, zymosan, with the result that the human cytokine concentration increases. Human neutrophils are increased by administering, for example, a bacterium such as Escherichia coli, with the result that emergency granulopoiesis, which occurs at the time of bacterial infection of a human, is reproduced. Human cytokine concentration is also increased by bacterial administration.

In the humanized rodent having a human G-CSF gene knock-in at a rodent's G-CSF receptor locus and transplanted with human hematopoietic stem cells (HSC), in which human neutrophils circulate in the periphery, the human innate immune system can be reproduced. Thus, the humanized rodent can be used in studies for human immunology.

If the rodent is infected with a bacterium, the rodent can be used in studies for the congenital defense mechanism of humans against bacterial infection.

Described herein is a rodent model for a human infectious disease prepared by infecting a humanized rodent having a human G-CSF gene knock-in at a rodent's G-CSF receptor locus and transplanted with human hematopoietic stem cells (HSC), in which human neutrophils are circulating in the periphery, with a bacterium or virus. Examples of the bacterium and virus include those pathogenic to humans, such as pathogenic Escherichia coli, enterohemorrhagic Escherichia coli, Salmonella spp. , Campylobacter jejunilcoli, Staphylococcus aureus, Vibrio parahaemolyticus, Clostridium botulinum, Bacillus cereus, Clostridium welch, norovirus, hepatitis A virus and hepatitis E virus. A rodent model for a human infectious disease can be prepared by infecting the humanized rodent having a human G-CSF gene knock-in at a rodent's G-CSF receptor locus, in which human neutrophils are circulating in the periphery with these pathogens. The rodent model can be used in study for the defense mechanism of humans against an infectious disease and further for developing novel therapies and therapeutic drugs.

Described herein is a rodent having a human G-CSF gene knock-in at a rodent's G-CSF receptor locus and further deficient in Fcer1g and Fcgr2b, obtained by crossing the aforementioned rodent having a human G-CSF gene knock-in with a rodent deficient in Fcer1g and Fcgr2b, which are constituent molecules of a receptor that binds to the Fc region of an antibody.

Human neutrophils differentiate, increase and circulate in the peripheral blood by transplanting human hematopoietic stem cells (CD34+ cells) to the rodent. Human hematopoietic stem cells (CD34+ cells) may be transplanted in accordance with the method mentioned above.

Examples of the rodent include a rodent having a declined immune function or no immune function and inactivated immune response to humans. Examples of the immunodeficient rodent are the same as mentioned above. Of them, a mouse, particularly a NOG mouse, is preferable.

Examples of the mouse include a hG-CSF KI/FcR KO mouse, which is obtained by crossing a hG-CSF KI mouse with an FcR KO mouse (<NPL>. ) deficient in Fcerlg and Fcgr2b, which are constituent molecules of a receptor that binds to the Fc region of an antibody. Note that, the hG-CSF KI/FcR KO mouse can be prepared by deleting an Fcerlg gene and Fcgr2b gene by genome editing.

The mouse has a human G-CSF gene knock-in at a G-CSF receptor locus of a NOG mouse and is deficient in Fcerlg and Fcgr2b.

Human neutrophils differentiate, increase and circulate in the peripheral blood by transplanting human hematopoietic stem cells (CD34+ cells) into the mouse. The ratio of the human neutrophils <NUM> weeks after transplantation is <NUM> to <NUM>%. The human neutrophils have an ability to produce reactive oxygen species.

Described herein is a rodent model for a human infectious disease obtained by infecting the above rodent with a bacterium or virus. Examples of the bacterium or virus are the same as mentioned above.

Described herein is a rodent having human IL-<NUM> and GM-CSF genes inserted in the genome and deficient in Fcerlg and Fcgr2b, obtained by crossing a rodent having human IL-<NUM> and GM-CSF genes inserted in the genome with a rodent deficient in Fcer1g and Fcgr2b, which are constituent molecules of a receptor that binds to the Fc region of an antibody.

Human eosinophils differentiate, increase and circulate in the peripheral blood by transplanting human hematopoietic stem cells (CD34+ cells) into the rodent.

Examples of the immunodeficient rodent are the same as mentioned above. Of them, a mouse, particularly, a NOG mouse, is preferable.

In the case of a mouse, a hIL-<NUM>/hGM-CSF Tg, FcR KO mouse is mentioned, which is obtained by crossing a hIL-<NUM>/hGM-CSF Tg mouse (<NPL>. ) with an FcR KO mouse. Note that, a hIL-<NUM>/hGM-CSF Tg, FcR KO mouse can be prepared by deleing an Fcerlg gene and Fcgr2b gene by genome editing.

Human eosinophils differentiate, increase, and circulate in the peripheral blood by transplanting human hematopoietic stem cells (CD34+ cells) into the mouse. The ratio of human eosinophils <NUM> weeks after transplantation is <NUM> to <NUM>%.

Described herein is a rodent having human IL-<NUM>, GM-CSF and IL-<NUM> genes inserted in the genome and deficient in Fcer1g and Fcgr2b, obtained by crossing a rodent having human IL-<NUM>, GM-CSF and IL-<NUM> gene inserts in the genome with a rodent deficient in Fcerlg and Fcgr2b, which are constituent molecules of a receptor that binds to the Fc region of an antibody.

Human eosinophils and human basophils differentiate, increase, and circulate in the peripheral blood by transplanting human hematopoietic stem cells (CD34+ cells) into the rodent.

Examples of the immunodeficient rodent are the same as mentioned above. Of them, a mouse, particularly, a NOG mouse, is preferable. The rodent having human IL-<NUM>, GM-CSF and IL-<NUM> gene inserts in the genome secretes human IL-<NUM>, GM-CSF and IL-<NUM> cytokines. After human hematopoietic stem cells are transplanted into the rodent, human cells such as eosinophils, basophils and mast cells can differentiate.

In the case of a mouse, a hIL-<NUM>/hGM-CSF/hIL-<NUM> Tg, FcR KO mouse is mentioned, which is obtained by crossing a hIL-<NUM>/hGM-CSF/hIL-<NUM> Tg mouse (<NPL>)) with an FcR KO mouse. Note that, a hIL-<NUM>/hGM-CSF/hIL-<NUM> Tg, FcR KO mouse can be prepared by deleting an Fcerlg gene and Fcgr2b gene by genome editing.

Human basophils differentiate, increase, and circulate in the peripheral blood by transplanting human hematopoietic stem cells (CD34+ cells) into the mouse. The ratio of human basophils <NUM> weeks after transplantation is <NUM> to <NUM>%.

Described herein is a rodent model for a human infectious disease obtained by infecting the above rodent with a bacterium or virus. Examples of the bacterium and virus are the same as mentioned above.

The relationships between mouse strains and the ratios of human granulocytes in human leukocytes are shown in <FIG>.

The following examples are not necessarily according to the invention and are present for illustration purposes only.

A KI targeting vector was prepared by placing a human G-CSF gene directly downstream of a systemic expression promoter, i.e., cytomegalovirus (CMV) promoter, and sandwiching it between homologous sequences, that is, a sequence (<NUM> Kb) upstream of a mouse G-CSF receptor region and a sequence (<NUM> Kb) downstream thereof. The structure of the targeting vector is shown in <FIG>.

The targeting vector prepared as mentioned above was introduced in NOG-mouse ES cells by electroporation to establish homologous recombination ES cells. Thereafter, chimera mice were prepared and crossed with NOG mice to prepare F1 mice. A G-CSF receptor gene was analyzed by a PCR method. The results of the gene analysis are shown in <FIG>. The mouse strain established is officially expressed as NOD. Cg-PrkdcscidIl2rgtm1SugCsf3rtm1(CMV-CSF3)/Jic and abbreviated as hG-CSF KI.

Blood was taken from <NUM> to <NUM> weeks-old hG-CSF KI mice (hetero, homo) and NOG mice by use of heparin and plasma was separated by a centrifugal operation. The level of human G-CSF cytokine was measured by a Human G-CSF Quantikine ELISA Kit (R&D systems). The results are shown in <FIG>. The levels of the human G-CSF cytokine in KI/+ hetero mice and KI/KI homo mice were as high as <NUM>µg/ml and <NUM>µg/ml in concentration, respectively.

Blood was taken from <NUM> to <NUM> weeks-old hG-CSF KI mice and NOG mice by use of heparin. White blood cells were separated by lysing red blood cells and stained with an anti-mouse G-CSF receptor antibody. Thereafter, expression thereof was measured by a flow cytometer. The results are shown in <FIG>. G-CSFR was not expressed in the granulocytes of KI/KI homo mice. From this, it was demonstrated that a loss in expression is due to gene disruption.

Blood was taken from <NUM> to <NUM> weeks-old hG-CSF KI mice (hetero, homo) and NOG mice by use of heparin. White blood cells were separated by lysing red blood cells and stained with an anti-mouse Gr1 antibody. Thereafter, mouse neutrophil count was analyzed by flow cytometry. The results are shown in <FIG>. The mouse neutrophil count significantly increased in KI/+ and extremely low in KI/KI. From this, it was demonstrated that a decrease of mouse neutrophil count is due to defect of a mouse G-CSF receptor in the KI/KI mouse.

Radiation was applied to <NUM> to <NUM> weeks-old hG-CSF KI mice at a dose of <NUM>, <NUM>, <NUM>, and <NUM> Gy. The mice were observed for a month or more and the death rate was determined. The results of the radiation sensitivity test are shown in <FIG>. The hG-CSF KI mice had high radiation sensitivity compared to the NOG mice. About <NUM>% of the mice died at a dose of <NUM> Gy and about <NUM>% of mice died even at a dose of <NUM> Gy. From this, a dose of <NUM> Gy was employed as an optimal irradiation condition.

Radiation was applied to <NUM> to <NUM> weeks-old hG-CSF KI mice at a dose of <NUM> Gy. At the following day, <NUM> to <NUM> × <NUM><NUM> human CD34+ cells (HSC: hematopoietic stem cells) were transplanted via the tail vein. Twelve weeks after transplantation, the peripheral blood was taken and the ratios of human leukocyte fractions, i.e., human neutrophils and monocytes, were analyzed by a flow cytometer. The results are shown in <FIG>. It was found that the ratios of CD66b+ granulocytes and CD33+ monocytes in human leukocytes (CD45) increased in the G-CSF KI mice, and that most of the granulocytes consisted of CD16+ neutrophils. The fractions of neutrophils and monocytes in the NOG mice were extremely low.

The ratio and count of human CD45+ cells (human leukocytes) shown in <FIG> were analyzed over time, at <NUM>, <NUM> and <NUM> weeks after transplantation. The results are shown in <FIG>. The ratio and count of the cells both were high in KI mice <NUM> weeks after humanization but no significant difference was observed thereafter.

The ratio and count of the human CD66b+CD16+ cells (human neutrophils) shown in <FIG> were analyzed over time, at <NUM>, <NUM> and <NUM> weeks after transplantation. The results are shown in <FIG>. The ratio and count both were significantly high in the KI mice during the whole period after humanization.

The ratio and count of the human CD33+CD14+ cells (human monocytes) shown in <FIG> were analyzed over time, at <NUM>, <NUM> and <NUM> weeks after transplantation. The results are shown in <FIG>. The ratio and count both were significantly high in KI mice during the whole period after humanization.

Blood was taken from hG-CSF KI mice <NUM> weeks after humanization and stained with a human neutrophil marker. Neutrophil fractions were separated by a cell sorter and subjected to Giemsa staining to observe morphology of the cells. The results are shown in <FIG>. Many cells having neutrophil-like morphology having multilobed nuclei were observed.

Blood was taken from hG-CSF KI mice <NUM> weeks after humanization. After white blood cells were isolated and stimulated in vitro with LPS (<NUM>µg/ml) or PMA (<NUM> ng/ml), ROS production ability was analyzed by flow cytometry. The results are shown in <FIG>. When neutrophils were stimulated with LPS, <NUM> to <NUM>% of them were ROS positive. When neutrophils were stimulated with PMA, <NUM> to <NUM>% of them were ROS positive. It was found that the human neutrophils have an ROS producing ability.

Blood was taken from the hG-CSF KI mice and NOG mice <NUM> to <NUM> weeks after humanization. After plasma was separated, levels of various human cytokines were determined by Cytokine Beads Array (CBA: BD Bioscience). The results are shown in <FIG>. Productions of IL-<NUM>, IL-<NUM>, IL-<NUM>, MCP-<NUM>, MIP1a, MIP-1b and IFNg significantly increased in G-CSF KI mice.

To the hG-CSF KI mice and NOG mice <NUM> weeks after humanization, <NUM>/<NUM>µL of a zymosan solution was intraperitoneally administered. Four hours later, blood was taken and subjected to flow cytometric analysis for human neutrophils. The results are shown in <FIG>. The ratio of human neutrophils before administration of zymosan was about <NUM>% and increased up to about <NUM>% <NUM> hours after administration.

The flow-cytometry analysis results of human CD45-positive white blood cells, monocytes, neutrophils, T cells and B cells in the experiment shown in <FIG> are collectively shown in <FIG>. In the hG-CSF KI mice, the ratio of human monocytes decreased and the ratio of human neutrophils increased after administration of zymosan. Also in the NOG mice, the human neutrophils increased but the ratio was as low as about <NUM>%.

In the experiment shown in <FIG>, plasma was taken and levels of various human cytokines in the plasma were determined by Cytokine Beads Array (CBA: BD Bioscience). The results are shown in <FIG>. In hG-CSF KI mice, productions of human IL-<NUM>, IL-1b, IL-<NUM>, MCP-<NUM>, MIP1a, MIP-1b and TNFa remarkably increased after administration of zymosan, demonstrating that innate immune response via human neutrophils, monocytes/macrophages increases compared to those in NOG mice.

An Escherichia coli solution (<NUM> × <NUM><NUM> cfu) was intraperitoneally administered to the hG-CSF KI mice and NOG mice <NUM> weeks after humanization. Four hours later, blood was taken and human neutrophils, monocytes and B cells were analyzed by flow cytometry. The results are shown in <FIG>. The human neutrophils significantly increased after administration of Escherichia coli. Emergency granulopoiesis occurring at the time of bacterial infection of a human was reproduced.

In the experiment shown in <FIG>, plasma was taken, levels of various mouse and human cytokines in the plasma were determined by Cytokine Beads Array (CBA: BD Bioscience). The quantitative results of mouse cytokines are shown in <FIG> and the quantitative results of human cytokines are shown in <FIG>. No difference was observed between the NOG mice and the hG-CSF KI mice with respect to the mouse cytokines. Productions of human cytokines, IL-<NUM>, IL-1b, IL-<NUM>, MCP-<NUM>, MIP1a and TNFa significantly increased in G-CSF KI mice. From these, the G-CSF KI mice are considered as humanized mice in which emergency granulopoiesis after bacterial infection and innate immune response accompanied with increased human cytokines can be analyzed and useful as a model for developing a therapeutic drug for sepsis induced by bacterial infection.

FcR KO mice deficient in Fcer1g and Fcgr2b, which are constituent molecules of a receptor that binds to the Fc region of an antibody, have been already established (<NPL>). The FcR KO mice were crossed with hG-CSF KI mice to newly establish hG-CSF KI, FcR KO mice. When human hematopoietic stem cells were transplanted into hG-CSF KI, FcR KO mice, it was found that the ratio of human neutrophils in human leukocytes of mice peripheral blood is about <NUM> to <NUM>% in the hG-CSF KI mice, whereas the ratio is <NUM> to <NUM>% (<NUM> times higher) in the hG-CSF KI, FcR KO mice (<FIG>). Neutrophils differentiated from hematopoietic stem cells in the bone marrow in a G-CSF dependent manner, more differentiated in response to stimulation such as infection and quickly migrate to the site stimulated to successfully remove bacteria.

hIL-<NUM>/hGM-CSF Tg mice that the present inventors already reported (<NPL>. ) were crossed with the FcR KO mice to newly establish hIL-<NUM>/hGM-CSF Tg, FcR KO mice. When human hematopoietic stem cells were transplanted to hIL-<NUM>/hGM-CSF Tg, FcR KO mice, it was found that the ratio of human eosinophils in peripheral blood is about <NUM> to <NUM>% in hIL-<NUM>/hGM-CSF Tg mice, whereas the ratio is <NUM> to <NUM>% (three times higher) in hIL-<NUM>/GM-CSF Tg, FcR KO mice (<FIG>).

Similarly, hIL-<NUM>/hGM-CSF/hIL-<NUM> Tg mice (<NPL>)) were crossed with FcR KO mice to newly establish hIL-<NUM>/hGM-CSF/hIL-<NUM> Tg, FcR KO mice. When human hematopoietic stem cells were transplanted into hIL-<NUM>/hGM-CSF/hIL-<NUM> Tg, FcR KO mice, it was found that the ratio of human eosinophils in human leukocytes of mouse peripheral blood is about <NUM> to <NUM>% in the hIL-<NUM>/hGM-CSF Tg mice, whereas the ratio in the hIL-<NUM>/hGM-CSF/hIL-<NUM>/FcR KO mice is <NUM> to <NUM>% (about twice as high) (<FIG>). Eosinophils differentiate from hematopoietic stem cells in the bone marrow and are increased by a cytokine such as IL-<NUM>. Highly invasion in tissue was observed in response to inflammation via an allergen at a local site such as a lung tissue.

When human hematopoietic stem cells were transplanted to hIL-<NUM>/hGM-CSF/hIL-<NUM> Tg mice and hIL-<NUM>/hGM-CSF/hIL-<NUM> Tg, FcR KO mice and the ratio of human basophils in human leukocytes of mouse peripheral blood was determined, it was found that the ratio in hIL-<NUM>/hGM-CSF/hIL-<NUM> Tg mice is about <NUM> to <NUM>%, whereas the ratio is <NUM> to <NUM>% (about twice as high) in hIL-<NUM>/hGM-CSF/hIL-<NUM> Tg, FcR KO mice (<FIG>). Basophils differentiate from hematopoietic stem cells in the bone marrow and are increased by a cytokine such as IL-<NUM>. Basophils migrate to a local site during allergic inflammation and exacerbate inflammation.

Claim 1:
A rodent having a human G-CSF gene knock-in at a G-CSF receptor locus thereof, which is an immunodeficient rodent deficient in G-CSF receptor function by knock-in of the human G-CSF gene at the G-CSF receptor locus thereof, wherein the human G-CSF is expressed and the rodent G-CSF receptor is not expressed, and further deficient in Fcer1g and Fcgr2b, which are constituent molecules of a receptor that binds to an Fc region of an antibody, obtained by further deleting the Fcer1g and Fcgr2b in the rodent.