Genetically Modified Mice and Transplantation Methods

Abstract

The present invention provides a mouse model of human mycobacterial infection that develops one or more granulomas containing human immune cells. (a) mouse RAG gene knockout; (b) mouse IL2rg gene knockout; (c) replacement of the mouse IL-3 gene with the human IL-3 gene, wherein the human IL-3 gene is present at the mouse IL-3 locus; and (d) replacement of the mouse GM-CSF gene with the human GM-CSF gene, wherein the human GM-CSF gene is present at the mouse GM-CSF locus. Genetically modified mice, including

Description

Technical Field

[0001] Field of the Invention The present invention relates to the field of genetically modified non-human animals, and in particular, immunodeficient mice having RAG gene knockout, Il2rgIl2rg gene knockout, and humanization of IL-3 and GM-CSF genes, and optionally humanization of the TPO gene; RAG / Il2rgIl2rg knockout mice having humanization of the TPO gene; genetically modified mice transplanted with human hematopoietic cells; and transplanted mice infected with human pathogens such as Salmonella typhi or Mycobacterium tuberculosis.

Background Art

[0002] Background The use of genetically modified mice, modified and transplanted mice, and their use in the modeling of human diseases, for example for the purpose of drug testing, is known in the art. Attempts have been made to use genetically modified mice to model the human immune system. An overview of the field is provided in Manz (2007) Human-Hemato-Lympoid-System Mice: Opportunities and Challenges, Immunity, 26:537-541 (Non-Patent Document 1), which is incorporated herein by reference.

[0003] To date, genetically modified mice that exhibit infectivity to specific human pathogens, such as Salmonella typhi, have not been produced. Even in the case of pathogenic infectious diseases for which mouse models exist, the model may not be able to appropriately model specific human pathologies. For example, in the mouse model of Mycobacterium tuberculosis, well-defined granulomas or granulomas containing human immune cells could not be formed. In order to study the effects of specific pathogens on humans and to test drugs for effectiveness in treating humans infected with specific pathogens, it would be useful to have non-human animals, such as genetically modified mice, that are more susceptible to infection by such pathogens, such as Salmonella typhi, and / or that are more rigorous models of human pathologies, such as more rigorous models of human infections with Mycobacterium tuberculosis.

[0004] Generally, there is a need for genetically modified mice that can support the maintenance and proliferation of human hematopoietic stem cells, and for mice suitable for transplantation that can model or approximate part of the human blood-lymphatic system, for example in response to human pathogens.

Prior Art Documents

Non-Patent Documents

[0005]

Non-Patent Document 1

Summary of the Invention

[0006] Summary Genetically modified non-human animals are provided. The non-human animals include mice that contain knockout of one or more endogenous genes and one or more humanized genes (i.e., substitution of the endogenous gene locus with a human analog or homolog of the endogenous gene).

[0007] Genetically modified mice with an immune system removed or impaired (e.g., by irradiation), as well as mice transplanted with human hematopoietic cells or human hematopoietic stem and progenitor cells (HSPC) are provided. Genetically modified mice containing human cells derived from human hematopoietic cells or HSPC are provided, as well as mice containing a human blood-lymphatic system.

[0008] Genetically modified, irradiated, and transplanted mice that can be infected with human pathogens that do not infect wild-type mice are provided. Mice that initiate an immune response with characteristics (e.g., formation of well-defined granulomas or granulomas containing human immune cells) not observed in wild-type mice in response to exposure to human pathogens (e.g., Mycobacterium tuberculosis) are provided.

[0009] Genetically modified, irradiated, and transplanted mice, as well as compositions and methods for using them, are provided for identifying drug-resistant strains of human pathogens, testing human vaccines, and developing and testing anti-pathogen drugs.

[0010] Genetically modified mice that can receive and expand human immune cells are provided, including mice that can harbor human hematopoietic malignancies.

[0011] In one aspect, genetically modified mice are provided that include (a) a mouse RAG gene knockout; (b) a mouse Il2rgIl2rg gene knockout; and (c) humanization of one or more genes selected from (i) the mouse IL-3 (mIL-3) gene, (ii) the mouse GM-CSF (mGM-CSF) gene, and (iii) the mouse thrombopoietin (mTPO) gene.

[0012] In one embodiment, the RAG gene knockout is a RAG2 gene knockout.

[0013] In one aspect, humanization involves the replacement of the mTPO gene with the hTPO gene. In certain aspects, humanization consists essentially of the humanization of the mTPO gene with the hTPO gene.

[0014] In one aspect, humanization involves the replacement of the mIL-3 gene with the human IL-3 (hIL-3) gene and the replacement of the mGM-CSF gene with the human GM-CSF (hGM-CSF) gene. In another aspect, the mouse further involves the replacement of the mTPO gene with the human TPO (hTPO) gene. In certain aspects, humanization consists essentially of the humanization of the mIL-3 gene with the hIL-3 gene and the humanization of the mGM-CSF gene with the hGM-CSF gene.

[0015] In one aspect, humanization involves the replacement of the mGM-CSF gene with the human GM-CSF gene, and human GM-CSF is not predominantly expressed in the liver and circulation in the mouse. In one aspect, human GM-CSF is predominantly expressed in the mouse lung. In one aspect, the expression of human GM-CSF is tissue-specific and reflects the tissue-specific expression in humans.

[0016] In one aspect, the genetically modified mouse is treated to eliminate endogenous hematopoietic cells that may be present in the mouse. In one aspect, the treatment includes irradiating the genetically modified mouse. In certain aspects, the neonates of the genetically modified mouse are irradiated sub-lethally. In certain aspects, the neonates are irradiated with 2 × 200 cGy at 4-hour intervals.

[0017] In one aspect, the genetically modified and treated mouse is transplanted with human hematopoietic cells or human hematopoietic stem cells (HPSCs) to form a genetically modified and transplanted mouse. In one aspect, the hematopoietic cells are selected from human umbilical cord blood cells and human fetal liver cells. In one aspect, about 1 - 2 × 10 5 human CD34+ cells are used for transplantation.

[0018] In one aspect, the genetically modified and transplanted mouse gives rise to human cells selected from: CD34+ cells, hematopoietic stem cells, hematopoietic cells, bone marrow progenitor cells, bone marrow cells, dendritic cells, monocytes, granulocytes, neutrophils, mast cells, thymocytes, T cells, B cells, platelets, and combinations thereof. In one aspect, the human cells are present at 4, 5, 6, 7, 8, 9, 10, 11, or 12 months post-transplantation.

[0019] In one aspect, the genetically modified and transplanted mouse gives rise to a human blood-lymphatic system comprising human hematopoietic stem cell progenitors, human bone marrow progenitor cells, human bone marrow cells, human dendritic cells, human monocytes, human granulocytes, human neutrophils, human mast cells, human thymocytes, human T cells, human B cells, and human platelets. In one aspect, the human blood-lymphatic system is present at 4, 5, 6, 7, 8, 9, 10, 11, or 12 months post-transplantation.

[0020] In one aspect, the genetically modified and transplanted mouse exhibits an inflammatory response mediated by human cells. In certain aspects, the human cells are macrophages. In certain aspects, the macrophage-mediated inflammatory response is mediated by alveolar macrophages. In certain aspects, the inflammatory response mediated by alveolar macrophages includes the formation of granulomas. In certain aspects, the granulomas contain human immune cells. In certain aspects, the granulomas are well-organized granulomas. In certain aspects, the granulomas are formed following exposure to mycobacteria, such as Mycobacterium tuberculosis. In one aspect, the mouse exhibits an immune response comprising two or more granulomas. In one aspect, the genetically modified and transplanted mouse is a model for human Mycobacterium tuberculosis infection.

[0021] In one aspect, the genetically modified and transplanted mice comprise a Mycobacterium tuberculosis infection that is at least partially characterized by the formation of granulomas containing human immune cells. In certain aspects, the granulomas are well-organized granulomas. In certain aspects, the Mycobacterium tuberculosis is a drug-resistant or multi-drug resistant strain of Mycobacterium tuberculosis that infects the human population. In one aspect, the Mycobacterium tuberculosis-infected mice have granulomas in the lungs. In certain aspects, the granulomas are well-developed granulomas. In certain aspects, the lung granulomas contain human immune cells. In certain aspects, the human immune cells of the granulomas are selected from activated human macrophages, activated human T cells, and combinations thereof.

[0022] In one aspect, the genetically modified and transplanted mice exhibit enhanced mucosal immunity compared to transplanted mice lacking humanization of one or more of the IL-3, GM-CSF, and TPO genes. In certain aspects, the enhanced mucosal immunity includes a high level of expression of interferon β (IFNβ) after influenza A infection.

[0023] In one aspect, the genetically modified and transplanted mice comprise an infection selected from Mycobacterium tuberculosis infection and Salmonella typhi infection. In one aspect, the mice are colonized with Salmonella typhi or Mycobacterium tuberculosis. In one aspect, the mice initiate an anti-mycobacterial immune response against human pathogenic mycobacteria, the immune response being mediated by human immune cells and including the formation of granulomas containing human immune cells. In certain aspects, the granulomas are well-developed granulomas.

[0024] In one aspect, the genetically modified and transplanted mouse includes humanization of the mTPO gene and forms a mouse transplanted with hTPO. In one aspect, the mouse transplanted with hTPO shows an increase in human bone marrow cells in the bone marrow compared to a transplanted mouse that contains the mTPO gene but does not contain the hTPO gene. In certain aspects, the human bone marrow cells increase 1.5-fold, 2-fold, 2.5-fold, or 3-fold relative to a transplanted mouse lacking the hTPO gene. In certain aspects, the increase in granulocytes is about 1.5-fold, 2-fold, 2.5-fold, or 3-fold. In another aspect, an increase in peripheral blood monocytes is observed compared to a transplanted mouse lacking the hTPO gene, and in this case the increase in peripheral blood monocytes is about 1.5-fold, 2-fold, 2.5-fold, or 3-fold. In one aspect, the genetically modified and transplanted mouse includes humanization consisting essentially of an hTPO gene that replaces the mTPO gene, and the mouse expresses human TPO without expressing mouse TPO.

[0025] In one aspect, there is provided a genetically modified and transplanted mouse that includes knockout of the Rag gene, knockout of Il2rgIl2rg, and humanization of TPO, the mouse being transplanted with human hematopoietic stem cells or human immune cells and including a human hematopoietic organ malignancy that develops from early human hematopoietic cells. In certain aspects, the malignancy is selected from myeloid leukemia and myeloproliferative tumors.

[0026] In one aspect, the mouse further includes the human IL-3 gene and the human GM-CSF gene, as well as knockout of the endogenous mouse IL-3 gene and knockout of the endogenous mouse GM-CSF gene.

[0027] In one aspect, there is provided a mouse comprising a gene modification including RAG gene knockout, Il2rg gene knockout, and a gene modification that provides human bone marrow cells that are competitively dominant over mouse bone marrow cells. In one embodiment, the gene modification is a replacement of a mouse gene required for the development and / or maintenance of mouse bone marrow cells with the corresponding human gene. In one embodiment, the gene modification is selected from the replacement of the mouse IL-3 gene with the human IL-3 gene, the replacement of the mouse GM-CSF gene with the human GM-CSF gene, and combinations thereof. In one embodiment, the mouse lacks or substantially lacks endogenous mouse hematopoietic cells and contains human hematopoietic cells.

[0028] In one aspect, there is provided a method of producing a mouse capable of being infected with a human pathogen, the method comprising the steps of genetically modifying a mouse as described herein and transplanting the mouse, exposing the genetically modified and transplanted mouse to a human pathogen, and maintaining the mouse under conditions sufficient for the human pathogen to infect the mouse. In one embodiment, the human pathogen is selected from Mycobacterium tuberculosis and Salmonella typhi. In one embodiment, the human pathogen is a human pathogen that is not pathogenic to a mouse lacking the gene modification. In one embodiment, the human pathogen is a human pathogen that does not infect a mouse lacking the gene modification.

[0029] In one aspect, there is provided a method of determining the effect of a drug against a human pathogen, the method comprising the steps of exposing a genetically modified and transplanted mouse to a human pathogen as described herein, and infecting the mouse with the pathogen and measuring the parameters of infection over time in the presence and absence of the drug. In one embodiment, the human pathogen is a pathogen that does not infect a mouse lacking the gene modification. In one embodiment, the human pathogen is selected from Mycobacterium tuberculosis and Salmonella typhi. In one embodiment, the mouse is exposed to a known number of infectious units of the human pathogen, and the parameter of infection is the number of infectious units of the human pathogen in the body fluid or tissue of the mouse.

[0030] In one aspect, the parameter of infection is the titer in the body fluid of the mouse. In one aspect, the infection is selected from Mycobacterium tuberculosis infection and Salmonella typhi infection. In a particular aspect, the infection is Mycobacterium tuberculosis infection and the parameter is the formation of granulomas. In a particular aspect, the granulomas are pulmonary granulomas. In another particular aspect, the granulomas are well-defined granulomas.

[0031] In one aspect, there is provided a genetically modified mouse comprising: (a) a mouse RAG gene knockout; (b) a mouse Il2rg gene knockout; and (c) humanization of (i) the mouse IL-3 (mIL-3) gene and (ii) the mouse GM-CSF (mGM-CSF) gene; the mouse maintains its human hematopoietic stem cells and generates a human immune cell population comprising differentiated functional human immune cells from the human hematopoietic stem cells after being irradiated to remove endogenous mouse hematopoietic cells and after transplantation of human hematopoietic stem cells. The differentiated human immune cells include human bone marrow progenitor cells, human bone marrow cells, human dendritic cells, human monocytes, human granulocytes, human neutrophils, human mast cells, human thymocytes, human T cells, human B cells, and human platelets. In another aspect, the mouse further comprises humanization of (iii) the mouse thrombopoietin (mTPO) gene.

[0032] In one aspect, the mouse maintains a human immune cell population that is as diverse in cell types as the population of human immune cells. In one aspect, the human immune cells are maintained for at least 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after transplantation.

[0033] In one aspect, a mouse initiates a cellular and / or humoral immune response that serves as a model of human infection when exposed to a human pathogen or an antigen of a human pathogen. In one aspect, the human pathogen is a pathogen that does not infect wild-type mice. In another aspect, the human pathogen is a pathogen that infects wild-type mice, and the wild-type mice after infection do not model the immune response that a human initiates in response to that pathogen. In one aspect, the pathogen is a virus, mycobacterium, fungus, or bacterium. In certain aspects, the pathogen is a human or porcine or avian influenza virus, Salmonella typhi, or Mycobacterium tuberculosis.

[0034] Further applications and aspects of the invention will be apparent to those skilled in the art upon reading this disclosure. [Invention 1001] (a) Mouse RAG gene knockout; (b) Mouse Il2rg gene knockout; (c) Replacement of the mouse IL-3 gene with the human IL-3 gene, wherein the human IL-3 gene is present at the mouse IL-3 locus; and (d) Replacement of the mouse GM-CSF gene with the human GM-CSF gene, wherein the human GM-CSF gene is present at the mouse GM-CSF locus A genetically modified mouse comprising the above. [Invention 1002] Replacement of the mouse thrombopoietin gene with the human thrombopoietin gene, wherein the human thrombopoietin gene is present at the mouse thrombopoietin locus The mouse of Invention 1001 further comprising the above. [Invention 1003] The mouse of Invention 1001, wherein the RAG gene knockout is selected from a RAG1 gene knockout, a RAG2 gene knockout, and combinations thereof. [Invention 1004] The mouse of Invention 1001 further comprising transplantation of human hematopoietic cells. [Invention 1005] The mouse of the present invention 1004, wherein the human hematopoietic cells are selected from the group consisting of cord blood cells and fetal liver cells. [The present invention 1006] The mouse of the present invention 1005, wherein the human hematopoietic cells are human cord blood cells that are CD34+ cells. [The present invention 1007] The mouse of the present invention 1003, comprising human cells that are CD34-positive cells, hematopoietic stem cells, hematopoietic cells, bone marrow progenitor cells, bone marrow cells, dendritic cells, monocytes, granulocytes, neutrophils, mast cells, or combinations thereof. [The present invention 1008] The mouse of the present invention 1003, comprising human hematopoietic stem and progenitor cells, human bone marrow progenitor cells, human bone marrow cells, human dendritic cells, human monocytes, human granulocytes, human neutrophils, human mast cells, human thymocytes, human T cells, human B cells, and human platelets. [The present invention 1009] The mouse of the present invention 1004, comprising infection with Salmonella typhi. [The present invention 1010] The mouse of the present invention 1009, wherein the Salmonella typhi infection is a systemic Salmonella typhi infection. [The present invention 1011] The mouse of the present invention 1004, comprising infection with Mycobacterium tuberculosis. [The present invention 1012] The mouse of the present invention 1011, comprising a pulmonary granuloma containing human immune cells. [The present invention 1013] (a) Administering an agent to a mouse that has been genetically modified, transplanted, and infected with a human pathogen, wherein the mouse is (i) a RAG1 gene knockout or a RAG2 gene knockout, or a combination thereof, (ii) an Il2rg gene knockout, (iii) substitution of the human IL-3 gene for the mouse IL-3 gene, wherein the human IL-3 gene is present at the mouse IL-3 locus, and (iv) Substitution of the mouse GM-CSF gene with the human GM-CSF gene, where the human GM-CSF gene is present at the mouse GM-CSF locus comprising the step of transplanting the mouse with CD34-positive human umbilical cord blood cells or CD34-positive human fetal liver cells; and (b) determining whether the agent reduces the amount of the human pathogen in the mouse infected with the human pathogen A method for identifying an agent that suppresses human pathogen infection, comprising [Invention 1014] The method of Invention 1013, wherein the mouse further comprises substitution of the mouse thrombopoietin gene with the human thrombopoietin gene. [Invention 1015] The method of Invention 1013, wherein the human pathogen is Salmonella typhi. [Invention 1016] The method of Invention 1013, wherein the human pathogen is mycobacteria, and the mycobacteria cause granulomas containing human immune cells in the mouse. [Invention 1017] The method of Invention 1016, wherein the mycobacteria are Mycobacterium tuberculosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0035]

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BEST MODE FOR CARRYING OUT THE INVENTION

[0036] DETAILED DESCRIPTION The present invention is not limited to the specific embodiments described, but is defined by the allowed claims.

[0037] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Any methods and materials similar to or equivalent to those described can be used in practicing or using the present invention, but specific embodiments, methods, and materials are described herein. All publications mentioned are incorporated herein by reference. In case of conflict, the present disclosure prevails over any disclosure of the incorporated publications.

[0038] The singular forms "a," "an," and "the" include their corresponding plural forms unless the context clearly indicates otherwise. Thus, for example, a reference to "a gene" includes a reference to more than one such gene, and a reference to "the gene knockout" includes a reference to one or more knockouts and their equivalents.

[0039] Modified mouse that supports human immune cells: hIL-3 / GM-CSF mouse Mice containing components of the human immune system (HIS mice) are quite promising for in vivo studies of the human immune system, testing of human vaccines, and testing and development of drugs for treating human diseases and disorders. HIS mice are generated by transplanting human hematopoietic stem cell progenitor cells into severely immunodeficient mouse strains (e.g., recombinant activation gene 2 (Rag2) knockout (KO) interleukin 2 receptor gamma (Il2rg) KO mice). Compared to non-human primates, HIS mice have the advantages of small animal models, namely, they allow for more versatile experiments, are more accessible to research groups, and are more ethically acceptable than conducting experiments using human subjects. Most importantly, the experimental results obtained from HIS mice are highly relevant to humans and may be applicable to humans because infections with human-specific pathogens, as well as studies of human-specific immune responses and immunopathology, are now becoming possible.

[0040] Although many advances have been made in recent years, current HIS mouse models have several major limitations, such as poor development, maintenance, and function of human bone marrow cells and T cells. As a result, strong human T cell responses, such as human inflammation and immune responses at mucosal surfaces or delayed-type hypersensitivity (DTH), are rarely observed. Therefore, current HIS mice are not suitable for studying infections and pathologies caused by Mycobacterium tuberculosis, a serious human pathogen. Indeed, granulomas, particularly those containing human cells, which are a prominent feature of the human immune response to mycobacteria, have not been reported in HIS mice to date (see, for example, Manz et al. (2009) Renaissance for mouse models of human hematopoiesis and immunobiology, Nat. Immunol. 10:1039-1042).

[0041] Current HIS mouse hosts are not suitable for modeling some infectious diseases because, at least in part, the current HIS mouse hosts do not present a physiological environment suitable for human cells. Several mouse cytokines, such as IL-3 and GM-CSF, do not act on human cognate receptors. Furthermore, Rag2 KO Il2rg KO mice have an intact mouse bone marrow compartment, and human bone marrow cells may be competitively disadvantaged relative to host cells. To overcome these limitations, the present disclosure describes the generation of human cytokine knock-in mice in which genes encoding mouse cytokines are replaced by their human counterparts. The criteria for cytokine replacement are as follows. (1) The mouse cytokine does not act or acts weakly on human cells. (2) The human cytokine does not act or acts weakly on mouse cells to confer a competitive advantage to human cells. (3) The human cytokine is not exclusively produced by hematopoietic (transplanted) cells. (4) The lack of the mouse cytokine is not lethal to the mouse host, or the human KI cytokine is sufficiently cross-reactive to rescue the mouse KO phenotype. The KI strategy should enable faithful expression at physiological concentrations in the appropriate organ. Importantly, in homozygous KI mice, cells expressing the human cognate receptor need to acquire a competitive advantage over their respective mouse cells.

[0042] IL-3 and GM-CSF are two cytokines essential for the development and function of bone marrow cells. Neither cytokine has cross-reactivity between humans and mice. IL-3 stimulates early hematopoietic progenitor cells in vitro, but is dispensable for steady-state hematopoiesis in vivo. However, together with GM-CSF, it is required for an effective DTH response in vivo. IL-3 also specifically stimulates the proliferation of alveolar macrophages (AMs) in vitro. GM-CSF is highly expressed in the lung and is important for lung homeostasis in vivo. This is demonstrated by the fact that GM-CSF KO mice develop alveolar proteinosis (PAP) characterized by the accumulation of protein in the lung due to defects in surfactant clearance. Alveolar macrophages from GM-CSF KO mice have defects in terminal differentiation, which leads to a decrease in innate immunity against lung pathogens. GM-CSF also stimulates the proliferation of human AMs in vitro. Similar to IL-3, GM-CSF is mostly unnecessary for steady-state hematopoiesis, and the same applies to mice lacking both cytokines. In contrast, GM-CSF is required for inflammatory responses such as the production of inflammatory cytokines by macrophages and the mobilization and replenishment of monocytes. GM-CSF is also essential for protective immunity against a wide range of pathogens including Mycobacterium tuberculosis. In particular, GM-CSF KO mice infected with Mycobacterium tuberculosis do not develop granulomas, a prominent feature of tuberculosis.

[0043] The present disclosure is based, at least in part, on the recognition that generating hIL-3 / GM-CSF KI mice would be valuable in supporting human bone marrow cell reconstitution and function as well as the human innate immune response to pathogens in mice. The results described in the present disclosure obtained using such KI mice demonstrate that this strategy provides significant improvements compared to current models of HIS mice from the perspective of human bone marrow cell development, human lung mucosal immunity, and further granuloma formation after mycobacterial infection. These and other beneficial properties of such mice are described elsewhere in the present disclosure.

[0044] The ability to study human tissues in an in vivo setting in mice has opened up a wide range of possible research tools. Major limitations have hindered the application of this approach, and one of the most important of these deficiencies is the inability of mouse factors to support human cells. Indeed, in the immune system, many of the essential factors required for the development and function of human immune cells are species-specific and cannot be effectively provided by mice. Therefore, it was decided to follow a strategy of replacing mouse genes with their human counterparts, enabling the proper development and function of human cells and potentially disabling that of the corresponding mouse cells. By applying this concept to human cytokine KI mice, proof of the concept that replacement of mouse host immune genes with human genes improves HIS mice is provided herein. Specifically, the present disclosure supports the view that inappropriate cytokine cross-reactivity between mice and humans, and the need to compete with mouse cells, actually limit the engraftment and function of human bone marrow cells in current HIS mice.

[0045] Human cytokines can be administered to HIS mice by intravenous injection, and for example, by injection of the IL-15 / IL-15Rα complex and IL-7, the reconstitution of human NK cells and T cells can be enhanced respectively. Another approach is the hydrodynamic injection of plasmid DNA expressing human cytokines that leads to transient expression in the liver. This approach was recently used to improve the reconstitution of human DCs by hydrodynamic delivery of GM-CSF and IL-4 (see Chen et al. (2009) Expression of human cytokines dramatically improves reconstitution of specific human-blood lineage cells in humanized mice, Proc Natl Acad Sci USA, 106(51):21783-21788). In contrast to the present disclosure, functional responses of bone marrow cells or in vivo responses to pathogens have not been reported at all in these mice. Finally, human cytokines can also be overexpressed as transgenes in HIS mice. This approach was adopted to generate human IL-3 / GM-CSF / stem cell factor (SCF) transgenic (tg) mice (see Nicolini et al. (2004) NOD / SCID mice engineered to express human IL-3, GM-CSF and Steel factor constitutively mobilize engrafted human progenitors and compromise human stem cell regeneration, Leukemia 18:341-347). In these mice, the expression of human cytokines is driven by the cytomegalovirus promoter that results in ubiquitous expression. However, hIL-3 / GM-CSF / SCF tg HIS mice are hampered by a decrease in the maintenance of human hematopoietic stem cells in the bone marrow and an increase in terminal bone marrow hematopoiesis. Again, in contrast to the present disclosure, improved bone marrow cell function or in vivo response has not been described.In contrast, in the systems described herein, the physiological expression of target genes in steady state and inflammation enables proper development and function of only the appropriate cell types. Importantly, the approach described in this disclosure is to generate mouse strains that can be maintained and propagated under highly reproducible conditions and are available worldwide for research.

[0046] The hIL-3 / GM-CSF KI mice described in this disclosure show significant improvements compared to previous HIS mice and the alternative approaches described above. First, delivery of human IL-3 and GM-CSF by the KI strategy described herein leads to long-term cytokine expression, which avoids the need for repeated injections of expensive cytokines. Second, faithful expression is achieved in the organs where IL-3 and GM-CSF are normally expressed. Under physiological conditions, GM-CSF is mainly expressed in the lung (Figure 5a). In contrast, hydrodynamic delivery results in predominant expression in the liver and circulation. In neither organ is GM-CSF expressed under steady state conditions. Third, in contrast to delivery by hydrodynamic injection or ubiquitous overexpression in hIL-3 / GM-CSF / SCF tg mice, physiological amounts of IL-3 and GM-CSF are expressed in the KI mice. Physiological levels of GM-CSF have been demonstrated to be important for the protective immune response against Mycobacterium tuberculosis. Thus, transgenic mice with local overexpression of GM-CSF in the lung show impaired granuloma formation and increased susceptibility to Mycobacterium tuberculosis. Similarly, intravenous administration of GM-CSF also induces a decrease in the protection against Mycobacterium tuberculosis infection in mice. Fourth, homozygous hIL-3 / GM-CSF KI mice allow for simultaneous ablation of the mouse bone marrow compartment because mouse IL-3 and GM-CSF are not expressed in homozygous mice. This provides a competitive advantage for human bone marrow cells, as shown in this disclosure.

[0047] In tuberculosis caused by Mycobacterium tuberculosis infection, 1.7 million people die annually. Therefore, new effective preventive and treatment measures are urgently needed. Mice can be infected with Mycobacterium tuberculosis, but they do not represent an ideal model of human tuberculosis. This is due to species-specific differences in the immune response to Mycobacterium tuberculosis. For example, infected mice do not develop clearly organized granulomas. Granulomas are a prominent feature of the immune response in humans with tuberculosis and contain activated macrophages and activated T cells that fuse to form epithelioid and multinucleated giant cells. Granulomas play an important role in restricting bacterial replication and further controlling the spread of mycobacteria. GM-CSF promotes the differentiation of AMs into multinucleated giant cells in vitro. Studies with transgenic mice have also revealed the role of GM-CSF in macrophage fusion to form multinucleated giant cells in vivo. Furthermore, GM-CSF is essential for granuloma formation after mycobacterial infection. The absence of granulomas in GM-CSF KO mice infected with Mycobacterium tuberculosis is associated with increased bacterial replication and decreased survival. Finally, humans with PAP due to defects in GM-CSF signaling show increased susceptibility to mycobacterial infection.

[0048] The human anti-mycobacterial immune response, particularly the formation of granulomas by human cells, has not been previously reported in HIS mice. This is probably due to the weak responses of human macrophages and T cells. In the present disclosure, antigen-specific T cell responses against mycobacteria were detected in a subset of mice transplanted with human cells. Furthermore, considering the prominent role of GM-CSF in the biology of granulomas, it is hypothesized that transplanted hIL-3 / GM-CSF KI mice are excellent hosts for supporting granuloma formation. This was indeed the case for at least a subset of BCG-infected mice. Importantly, although the lung granulomas of these mice had a loose configuration characteristic of mouse granulomas, the granulomas contained human T cells and human macrophages. Future efforts should aim to further enhance the responses of human T cells and macrophages in HIS mice. This should lead to the development of a small animal model that enables the study of the human immune response to Mycobacterium tuberculosis in vivo. hIL-3 / GM-CSF KI mice may also be useful in other settings for studying the human immune response in vivo. These include infections by pulmonary pathogens, autoimmunity, and human cancers. In summary, the hIL-3 / GM-CSF KI mice provided in the present disclosure correspond to a considerably improved HIS mouse model that can serve as a versatile tool for future research.

[0049] Modified mice that support human immune cells: hTPO Hematopoietic stem cells (HSCs) are characterized by two main properties: lifelong self-renewal ability and the ability to differentiate into all mature hematopoietic lineage cells. To ensure the homeostasis of the HSC pool, during cell division, HSCs give rise to one functional HSC, while the other progeny cells undergo a highly organized program of differentiation and cell proliferation, during which progenitor cells directed towards multiple lineages, and finally terminally differentiated cells, are generated.

[0050] Mouse hematopoiesis has been extensively studied over the past few decades, leading to the identification and functional analysis of immunophenotypically defined cell populations that are very rich in stem cells and progenitor cells in vivo. However, prospective experimental in vivo studies of human hematopoiesis are limited by obvious practical and ethical constraints.

[0051] To circumvent this limitation, several xenograft models for in vivo studies of human hematopoiesis have been developed. Among these, transplantation of human hematopoietic cells into immunodeficient mice has been widely established in experimental hematology laboratories. The most commonly used models today rely on mice of the BALB / c Rag2 - / - γ c - / - or NOD-SCIDγ c - / - strains. Both strains lack B cells, T cells, and NK cells and are highly immunodeficient, and their genetic backgrounds are tolerant to human hematopoietic transplantation and differentiation. Transplantation of human CD34 + hematopoietic stem cell progenitor cells allows the development of most human hematopoietic populations (including B cells, T cells, monocytes, dendritic cells, erythrocytes, and platelets), which are detectable in these models. However, in such chimeric animals, there is generally a bias towards the development of lymphocytes with initially high B cell numbers corresponding to up to 80% of human cells at most, and the development of bone marrow monocytes is minimal. Moreover, the engraftment level usually begins to decline 4 - 6 months after transplantation. Furthermore, xenotransplantation of human cells into mice requires transplantation of a large number of cells compared to the optimal number for transplantation of mouse hematopoietic stem cell progenitor cells into mice or human cells into humans, respectively. In addition, in contrast to mouse HSCs transplanted into mouse recipients, human HSCs do not expand and are not maintained in the xenogeneic mouse environment. Therefore, the mouse background does not provide an optimal environment for studying the physiology of human HSCs. This may be due to the absence or limited cross-reactivity of growth factors necessary to support the function and maintenance of HSCs.

[0052] Thrombopoietin (TPO) was first identified as a growth factor that promotes the development of megakaryocytes and platelets. TPO is constitutively produced in the liver and kidneys and released into the bloodstream. Its receptor, c-Mpl, is expressed by hematopoietic stem cell progenitor cells in the bone marrow. c-Mpl is also expressed on circulating platelets. However, the binding of TPO to platelets does not activate any signaling pathways. Thus, platelets act as a sink or scavenger for TPO and contribute to the negative regulation of platelet production through this mechanism. Subsequently, the important function of TPO in supporting the proliferation and self-renewal of HSCs has been recognized. TPO deficiency leads to a decrease in the number of HSCs in adult mice, and the presence of TPO is necessary for the maintenance of quiescent adult HSCs. Furthermore, TPO is required to support the post-transplantation proliferation of HSCs, which is necessary to replenish the hematopoietic compartment of irradiated hosts. Interestingly, osteoblasts, which are involved in the formation of HSC niches in the bone marrow, have been demonstrated to produce TPO, which is important for HSC function and maintenance.

[0053] Mouse and human TPO are both cross-reactive with their cognate receptors when used at supra-physiological doses in vitro, but their affinity and biological activity may differ when the cytokine acts at physiological, restricted doses in the in vivo environment. Thus, mouse TPO may not provide an appropriate stimulus to the human c-Mpl receptor in vivo and could therefore be a major cause of impaired human HSC properties in the mouse environment. To correct this potential defect, the gene encoding mouse TPO was replaced with its human counterpart in Rag2 - / - γ c - / - mice.

[0054] The present disclosure relates, at least in part, to RAG2 - / - γ c - / -The generation of hTPO KI mice in the background is based on the recognition that it is valuable in supporting human bone marrow cell reconstitution and function as well as the human innate immune response to pathogens in mice in the mice themselves and the progeny of such mice mated with hIL-3 / GM-CSF mice. RAG2 - / - γ c - / - The results obtained from hTPO KI mice in the background are described in this disclosure. Homozygous hTPO KI mice had significantly increased human engraftment levels in the bone marrow, and the multi-lineage differentiation of hematopoietic cells was improved compared to mTPO mice, and hTPO KI mice showed an increase in the ratio of the bone marrow monocyte lineage to the lymphocyte lineage. The number and self-renewal ability of human stem cell progenitor cells were both improved as demonstrated by serial transplantation. Thus, among several applicable examples, hTPO KI mice are particularly useful for the expansion of human cells by serial transplantation.

[0055] Mating of hIL-3 / GM-CSF mice and hTPO mice The progeny of hIL-3 / GM-CSF and hTPO mice described herein are expected to have at least the same relevant characteristics and exhibit at least the same advantages as the parental strains (i.e., hIL-3 / GM-CSF mice and hTPO mice). For example, it is possible to isolate a human cell population from either one of the parental strains, or both, or their progeny and serially transplant it into hTPO mice or the progeny of hIL-3 / GM-CSF and hTPO mice. Thus, in one aspect, there is provided a genetically modified mouse that is the progeny of hIL-3 / GM-CSF mice and hTPO mice described herein (including progeny mated homozygously for each relevant gene), and this genetically modified mouse exhibits both the advantages and characteristics of both hIL-3 / GM-CSF mice and hTPO mice. In one aspect, there is provided such a progeny mouse that is suitable for transplantation and / or serial transplantation from any transplanted mouse (e.g., the transplanted mice described herein) including a removed immune system (e.g., irradiated mice).

[0056] Transplantation of genetically modified mice The genetically modified mice according to the present invention are used as recipients of human hematopoietic cells that can develop human immune cells from transplanted human hematopoietic cells. In one aspect, human hematopoietic cells or human hematopoietic stem cell progenitor cells (HSPCs) are placed (transplanted) into mice that have been genetically modified and irradiated according to the present invention. The human hematopoietic cells or human hematopoietic stem cells give rise to cells selected from the following in the genetically modified mice: human CD34-positive cells, human hematopoietic stem cells, human hematopoietic cells, bone marrow progenitor cells, bone marrow cells, dendritic cells, monocytes, neutrophils, mast cells, and the human blood-lymphatic system (including human hematopoietic stem cell progenitor cells, human bone marrow progenitor cells, human bone marrow cells, human dendritic cells, human monocytes, human granulocytes, human neutrophils, human mast cells, human thymocytes, human T cells, human B cells, human platelets), and combinations thereof.

[0057] The genetically modified mice can be irradiated to eliminate any endogenous hematopoietic cells that may be present, and the mice can be transplanted with human hematopoietic cells from any suitable source. One suitable source known in the art of hematopoietic cells is human umbilical cord blood cells, particularly CD34-positive cells. Another source of hematopoietic cells is the human fetal liver.

[0058] In one aspect, transplantation using human hematopoietic cells of the mice according to the present invention results in mice that show an increased number of human hematopoietic cells compared to immunodeficient mice lacking humanization of the TPO gene, lacking humanization of the IL-3 and GM-CSF genes, or lacking humanization of the TPO gene and the IL-3 and GM-CSF genes.

[0059] In one aspect, transplantation using human hematopoietic cells of the invention results in mice that exhibit an increased number of human blood cells (e.g., mature hematopoietic cells) compared to immunodeficient mice lacking humanization. In certain aspects, the human hematopoietic cells are selected from: human CD34-positive cells, hematopoietic stem cells, hematopoietic cells, bone marrow progenitor cells, bone marrow cells, dendritic cells, monocytes, granulocytes, neutrophils, mast cells, and the human blood-lymphatic system (including human hematopoietic stem cell progenitors, human bone marrow progenitor cells, human bone marrow cells, human dendritic cells, human monocytes, human granulocytes, human neutrophils, human mast cells, human thymocytes, human T cells, human B cells, human platelets), and combinations thereof.

[0060] Non-limiting applications of genetically modified transplanted mice Genetically modified mice transplanted with human hematopoietic cells are useful animals for studying pathogens that do not normally infect mice. One such example is Salmonella typhi, the causative pathogen of typhoid fever.

[0061] Typhoid fever afflicts over 21 million people worldwide, mainly in developing countries, including approximately 400 cases / year in the United States. Typhoid fever is treated with the following drugs: amoxicillin, ampicillin, cefotaxime, ceftriaxone, ceftazidime, chloramphenicol, ciprofloxacin, cotrimoxazole, ertapenem, imipenem, fluoroquinolones (e.g., ciprofloxacin, gatifloxacin, ofloxacin), streptomycin, sulfadiazine, sulfamethoxazole, tetracycline, and combinations thereof. Recurrent infections are common, limiting disease management by antibiotic therapy. Furthermore, multidrug resistance is also frequently seen in Salmonella typhi infections.

[0062] There is a need for novel therapeutic agents, novel vaccines, and novel methods for testing the effectiveness of therapeutic agents and vaccines. Mice that can be infected by Salmonella typhi, for example, would be useful for identifying novel therapeutic agents and novel vaccines. Novel therapeutic agents and novel vaccines can be tested in such mice, for example, by measuring the amount of Salmonella typhi in the mice (in blood or a given tissue) that respond to treatment with a putative anti-Salmonella typhi agent, or by inoculating the mice with a putative vaccine, subsequently exposing them to an infectious dose of Salmonella typhi, and observing any change in infectivity due to the inoculation of the putative vaccine by comparing it to a control that is not inoculated with the vaccine but is infected with Salmonella typhi.

[0063] The genetically modified and transplanted mice according to the present invention are useful for creating mice that can be infected by human pathogens that do not infect mice. For example, mice are useful as non-human animals that can be infected by Salmonella typhi. In one aspect, the genetically modified and transplanted mice show enhanced engraftment of human cells compared to transplanted mice lacking the genetic modification, and the enhancement is sufficient to maintain Salmonella typhi infection. In certain aspects, the maintenance of Salmonella typhi infection includes the ability of Salmonella typhi to multiply in the mice. In certain aspects, Salmonella typhi infection includes the ability of the infected mice to propagate Salmonella typhi. In certain aspects, the mice can propagate Salmonella typhi for at least 1 week, 10 days, 2 weeks, 3 weeks, or 4 weeks after the initial introduction or infectious exposure to Salmonella typhi.

[0064] A method for identifying an anti-Salmonella typhi agent is also provided, which method uses the mice described herein that can be infected by Salmonella typhi. Wild-type mice and other known immunodeficient mice (e.g., RAG1 / RAG2 gene knockout mice) cannot be infected by Salmonella typhi.

[0065] Genetically modified mice are provided that include Il2rg gene knockout and RAG gene knockout (e.g., RAG2 gene knockout) (the first type), and further include replacement of the endogenous mouse IL-3 gene with the human IL-3 gene and replacement of the endogenous mouse GM-CSF gene with the human GM-CSF gene (the second type). The genetically modified mice are capable of being infected by Salmonella typhi when transplanted with human hematopoietic cells.

[0066] The data shown in FIG. 1 are representative of both the first and second types of mice. The genetic modification of the mice in FIG. 1 includes (a) mouse RAG gene knockout; and (b) mouse Il2rg gene knockout. The mice in FIG. 1 also include transplantation of human hematopoietic cells. This mouse can be further modified by two additional modifications to generate the second type of mouse, which are (c) replacement of the endogenous mouse IL-3 gene with the human IL-3 gene; and (d) replacement of the mouse GM-CSF gene with the human GM-CSF gene.

[0067] FIGS. 2, 3, and 4 are obtained using only the first type of modified mice (including the modifications of (a) mouse RAG gene knockout and (b) mouse Il2rg gene knockout, as well as transplantation of human hematopoietic cells).

[0068] In various aspects, Salmonella typhi-infected, genetically modified mice include a proliferative infection of Salmonella typhi. In one aspect, the mouse can harbor and propagate Salmonella typhi in one or more of its cells. In one aspect, the mouse can maintain the titer or level of Salmonella typhi in its blood or at least one tissue for at least 1 week, 10 days, 2 weeks, 3 weeks, or 4 weeks after infectious exposure to Salmonella typhi.

[0069] In one aspect, the method comprises administering an agent to a genetically modified mouse according to the present invention, wherein the genetically modified mouse is infected with Salmonella typhi; detecting the level of Salmonella typhi in the blood or tissue of the mouse after administration of the agent; and optionally determining whether the administration of the agent reduces the level of Salmonella typhi in the blood or tissue of the mouse. In one aspect, the agent is a vaccine. In another aspect, the agent is an antibiotic or an agent expected to have the properties of an antibiotic. In one aspect, the agent is an antigen-binding protein, and in a particular aspect, an antibody. In one aspect, the agent is an approved pharmaceutical for human use.

[0070] In one aspect, the method comprises infecting a genetically modified and transplanted mouse according to the present invention with a known amount of Salmonella typhi, administering an agent to the infected mouse, and measuring the amount of Salmonella typhi in the genetically modified and transplanted mouse after administration of the agent. In one aspect, if the agent reduces the amount of Salmonella typhi in the blood or tissue of the mouse by at least half over a predetermined period after one administration of the agent or two or more administrations, the agent is determined to be an anti-Salmonella typhi agent.

[0071] In one aspect, a method is provided for determining whether an isolate or strain of Salmonella typhi of interest is drug resistant or multidrug resistant, the method comprising administering to a genetically modified and transplanted mouse according to the invention a drug or combination of drugs used in the treatment of Salmonella typhi, wherein the mouse is infected with an isolate or strain of Salmonella typhi of interest. The method includes the steps of measuring (a) the titer of the isolate or strain of Salmonella typhi of interest in the blood or tissue of the mouse at a time point after administration of the drug or combination of drugs, (b) the ability of the isolate or strain of Salmonella typhi of interest to maintain infection in the mouse or the level of Salmonella typhi in the tissues of the mouse after one or more administrations of the drug or combination of drugs, or (c) the effect, if any, of the drug or combination of drugs on the ability of the isolate or strain of Salmonella typhi of interest to replicate in the mouse at a time point after administration of the drug or combination of drugs. In certain embodiments, the drug is selected from the group consisting of amoxicillin, ampicillin, ceftaxime, ceftriaxone, cefotaxime, chloramphenicol, ciprofloxacin, cotrimoxazole, ertapenem, imipenem, fluoroquinolones (e.g., ciprofloxacin, gatifloxacin, ofloxacin), streptomycin, sulfadiazine, sulfamethoxazole, tetracycline, and combinations thereof. In certain embodiments, administration of the drug or combination of drugs is at least 1 week, 10 days, 2 weeks, 3 weeks, or 4 weeks after exposure that results in infection with Salmonella typhi.

[0072] In various aspects and embodiments, the level of Salmonella typhi in the blood or tissue is measured by determining the number of colony forming units per unit of blood or tissue (e.g., weight or volume).

[0073] In one embodiment, a mouse genetically modified according to the invention and transplanted with human hematopoietic cells has a Salmonella typhi level, measured in colony forming units (cfu), that is at least 100-fold, 1,000-fold, or 10,000-fold higher than that of a mouse not transplanted with human hematopoietic cells.

[0074] Provided are methods and compositions useful for confirming the effectiveness of an anti- typhoid vaccine. In one aspect, a method for confirming the effectiveness of an anti-typhoid vaccine is provided, the method comprising exposing a genetically modified and transplanted mouse according to the present invention to an anti-typhoid vaccine, then exposing the genetically modified and transplanted mouse to typhoid bacteria, and determining whether or not the genetically modified and transplanted mouse can be infected by typhoid bacteria, or to what extent it can be infected.

[0075] In one embodiment, the anti-typhoid vaccine comprises a cell surface protein of typhoid bacteria or an immunogenic fragment thereof. In one embodiment, the vaccine comprises a membrane fraction of a typhoid strain. In one embodiment, the vaccine comprises a recombinant typhoid protein or an immunogenic fragment thereof. In one embodiment, the vaccine comprises an expression vector encoding a typhoid protein or an immunogenic fragment thereof. In one embodiment, the vaccine comprises an inactivated typhoid strain or a mixture of inactivated typhoid strains.

[0076] The genetically modified and transplanted mice described in the present disclosure are also useful for more precisely modeling human pathogen infections than existing mice. For example, infection by Mycobacterium tuberculosis. The genetically modified and transplanted mice described herein are useful for modeling human infections by mycobacteria, for example, by providing a Mycobacterium tuberculosis mouse model that develops granulomas containing human immune cells and granulomas with well-defined boundaries. The methods for testing drugs and vaccines described in connection with the typhoid bacteria infection of the genetically modified and transplanted mice described are also applicable to applications for Mycobacterium tuberculosis, such as identification of drug-resistant strains, testing the effectiveness of Mycobacterium tuberculosis vaccines, testing anti- Mycobacterium tuberculosis agents, measuring cfu in response to anti- Mycobacterium tuberculosis agents, and the like.

[0077] The genetically modified and transplanted mice described in the present disclosure are also useful for modeling human hematopoietic malignancies that develop from early human hematopoietic cells, for example human hematopoietic or progenitor cells. Further applications of the genetically modified and transplanted mice described in the present disclosure will be apparent to those skilled in the art upon reading this disclosure.

[0078] Thrombopoietin and transplantation Thrombopoietin (TPO) was first identified as a growth factor that promotes the development of megakaryocytes and platelets (Wendling, F. et al. (1994) cMpl ligand is a humoral regulator of megakaryocytopoiesis, Nature 369:571-574; Kaushansky, K. et al. (1994) Promotion of megakaryocyte progenitor expansion and differentiation by the c-Mpl ligand thrombopoietin, Nature 369:568-571; Lok, S. et al. (1994) Cloning and expression of murine thrombopoietin cDNA and stimulation of platelet production in vivo, Nature 369:565-568; de Sauvage, F.J. et al. (1994) Stimulation of megakaryocytopoiesis and thrombopoiesis by the c-Mpl ligand, Nature 369:533-538; Bartley, T.D. et al. (1994) Identification and cloning of a megakaryocyte growth and development factor that is a ligand for the cytokine receptor Mpl, Cell 77:1117-1124; Kaushansky, K. (1998) Thrombopoietin, N Engl J Med 339:746-754; Kaushansky, K. (2005) The molecular mechanisms that control thrombopoiesis, J Clin Invest 115:3339-3347; Kaushansky, K.(2008) Historical review: megakaryopoiesis and thrombopoiesis, Blood 111:981-986).

[0079] TPO is constitutively produced in the liver and kidneys and released into the blood circulation. Its receptor, c-Mpl, is expressed by hematopoietic stem cell progenitor cells in the bone marrow. c-Mpl is also expressed on circulating platelets. However, binding of TPO to platelets does not activate any signaling pathway. Thus, platelets act as a sink or scavenger for TPO and contribute to the negative regulation of platelet production via this mechanism (Kuter, D.J. and Rosenberg, R.D. (1995) The reciprocal relationship of thrombopoietin (c-Mpl ligand) to changes in the platelet mass during busulfan-induced thrombocytopenia in the rabbit, Blood 85:2720-2730). Subsequently, the important function of TPO in supporting the proliferation and self-renewal of HSCs has been recognized (Fox, N., et al. (2002) Thrombopoietin expands hematopoietic stem cells after transplantation, J Clin Invest 110, 389-3894; Kirito, K. et al. (2003) Thrombopoietin stimulates Hoxb4 expression: an explanation for the favorable effects of TPO on hematopoietic stem cells, Blood 102:3172-3178).

[0080] TPO deficiency leads to a decrease in the number of HSCs in adult mice, and the presence of TPO is necessary for the maintenance of quiescent adult HSCs (Yoshihara, H. et al. (2007) Thrombopoietin / MPL signaling regulates hematopoietic stem cell quiescence and interaction with the osteoblastic niche, Cell Stem Cell 1, 685-697; Qian, H. et al. (2007) Critical role of thrombopoietin in maintaining adult quiescent hematopoietic stem cells, Cell Stem Cell 1:671-684). Furthermore, TPO is required to support the post-transplantation expansion of HSCs, which is necessary to replenish the hematopoietic compartment of irradiated hosts. Interestingly, osteoblasts, which are involved in the formation of the HSC "niche" in the bone marrow, have been demonstrated to produce TPO, which is important for the function and maintenance of HSCs.

[0081] Mouse and human TPOs are both cross-reactive with their cognate receptors when used at supra-physiological doses in vitro, but their affinity and biological activity may differ when the cytokine acts at physiological limiting doses in the in vivo environment. The inventors hypothesized that mouse TPO may not provide an appropriate stimulus to the human c-Mpl receptor in vivo and could therefore be a major cause of impaired human HSC properties in the mouse environment. To correct this potential defect, the inventors replaced the gene encoding mouse TPO with its human counterpart in Rag2 - / - γ c - / - mice. Such mice were hypothesized to have an improved ability to maintain the differentiation and function of the human blood-lymphoid system.

[0082] In the development of mice that maintain the differentiation and function of the human blood-lymphocyte generation system, significant progress has been achieved since the publication of the first model more than 20 years ago. However, several limitations remain, including the following (i)-(iii): (i) transient human cell engraftment that does not persist throughout the lifespan of the recipient mouse, (ii) non-physiological bias towards the lymphoid lineage and insufficient differentiation of myeloid cells, and (iii) variability in engraftment levels between different animals, even when groups of mice are transplanted with cells from a single human donor. These limitations may be due to the non-physiological location of human cells, residual xenoreactivity of immunodeficient hosts, different compositions of blood-lymphoid cells in the mouse and human species, and / or lack or deficiency of cross-reactivity of hematopoietic support factors between mouse and human, which lead to preferential support of mouse cells. Therefore, providing physiological levels of human growth factors and deleting the respective mouse homologs in the host may more favorably affect the development and survival of human cell populations. Herein, the inventors describe a novel recipient mouse strain in which the gene encoding thrombopoietin, a cytokine important for the maintenance and self-renewal of hematopoietic stem cells, has been humanized.

[0083] When human CD34+ hematopoietic stem cell progenitor cells are transplanted into these humanized thrombopoietin mice, significant improvements are seen for all three of the limitations listed above, compared to previously available models. Namely, higher bone marrow chimerism was achieved and maintained for at least 6 months. Multilineage, particularly myeloid, differentiation was enhanced. And variability in engraftment levels was reduced.

[0084] The main difference between the mouse and human immune systems is the proportion of granulocytes present in the blood. In mice, lymphocytes are dominant, while human blood is rich in granulocytes, and the species difference has an unclear significance. Interestingly, the presence of human TPO improved the differentiation of human granulocytes (Figure 14). Therefore, the presence of human TPO in recipient mice likely acts favorably on the balance of granulocytes and lymphocytes that better reflects the human physiological state, perhaps due to the improved maintenance and / or differentiation of human bone marrow progenitor cells.

[0085] More importantly, these results indicate that humanized TPO acts favorably on the maintenance of secondary recipients that repopulate human hematopoietic stem cell progenitor cells in a mouse environment (Figure 15). Therefore, Rag2 - / - γ c - / - TPO h / h Mice represent a novel model for studying various aspects of the function of human stem cell progenitor cells in vivo.

[0086] Nevertheless, despite the observation of a good balance between myeloid and lymphoid lineages in the blood, no significant effect of humanized TPO on the overall engraftment level in peripheral lymphoid tissues (including spleen, blood, and thymus) was observed (Figure 13(g) - (i)). This can be explained by various factors. First, recipient mice are sub-lethally irradiated before transplantation, but a large population of mouse bone marrow cells still exists. Among these cells, macrophages can function as phagocytes of human cells and limit the overall engraftment level in the periphery. Therefore, genetic depletion of mouse macrophages, or their functional inactivation, may allow for higher peripheral engraftment levels. Second, human cells may require additional human growth factors to favorably promote their terminal differentiation, egress from the bone marrow, and / or their survival in the periphery. A diverse panel of cytokines can be considered for each lineage. Finally, although secondary lymphoid organs are formed in humanized mice, their structure is not optimal compared to human tissues. Their partially defective structure may represent a limitation to the number of human cells that can survive in these organs.

[0087] To further improve the mouse recipient, additional gene replacements can be employed. To achieve this, the approach used in this study, based on knock-in replacement of mouse genes with their human homologs, offers two main advantages compared to classical transgenic approaches. First, since it maintains most of the mouse-derived regulatory sequences, it ensures that the humanized gene is faithfully expressed within the mouse host. Second, since the knock-in strategy replaces mouse cytokines with their human homologs, it can affect populations of mouse-derived cells that are dependent on mouse cytokines if the human cytokine does not show sufficient cross-reactivity with the mouse receptor. This can provide additional competitive advantage to the human cell population after transplantation. Indeed, this is thought to be the case for human TPO. Because homozygous replacement of TPO results in decreased mouse platelets and HSCs in non-transplanted animals (Figures 13(a), 15(a) and 15(b)).

[0088] In the case of human TPO knock-in mice, an improved model that can generally be useful for studying human hematopoiesis in vivo physiology, particularly human hematopoietic stem cell progenitor cells, is provided. Furthermore, these mice maintain in vivo human hematopoietic malignancies that arise from early hematopoietic cells, such as myeloid leukemia and myeloproliferative tumors, for example.

Example

[0089] The following examples are not intended to limit the scope of what the inventors regard as their invention. Unless otherwise specified, parts are by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric pressure.

[0090] Example 1 Generation of Human IL-3 / GM-CSF and Human TPO Mice hIL-3 / GM-CSF Targeting A targeting construct for replacing the mouse IL-3 gene with the human IL-3 gene and the mouse GM-CSF gene with the human GM-CSF gene in a single targeting step was constructed using the VELOCIGENE® technology that utilizes gap repair cloning (see, for example, U.S. Patent No. 6,586,251 and Valenzuela et al. (2003) "High-throughput engineering of the mouse genome coupled with high-resolution expression analysis," Nat Biot 21(6):652-659, which are incorporated herein by reference).

[0091] The mouse sequence was obtained from bacterial artificial chromosome (BAC) RPCI-23, clone 5E15. The human sequence was obtained from the Caltech D library (CTD), BAC clone 2333J5.

[0092] The gap repair donor vector containing the p15 origin of replication was constructed by cloning a 5' mouse homology arm immediately upstream of the mIL-3 ATG, a human 5' IL-3 homology arm spanning the hIL-3 gene from about 274 nt from the hIL-3 ATG, a polylinker, a 3' hGM-CSF (about 233 bases) starting about 2.9 kb downstream of the polyA sequence of the hGM-CSF gene, and a drug selection cassette, followed by a mouse 3' homology arm having a sequence downstream (about 2.9 kb downstream) of the mGM-CSF polyA sequence. This gap repair vector was linearized and inserted into Escherichia coli (E. coli) DH10B strain containing the human CTD BAC clone 2333J5 and the recombinase vector as described by Valenzuela et al.

[0093] Cells were grown in drug selection medium. Individual clones were grown, the gap repair donor vector DNA was extracted, and portions of the vector were sequenced for appropriate mouse-human junctions. Pulse field gel electrophoresis was used to confirm the size of the insert and the expected restriction fragment lengths.

[0094] A captured donor containing the mouse upstream and downstream homology boxes adjacent to the hIL-3 gene, the hGM-CSF gene, and a loxed drug selection cassette was obtained from a repair donor vector. The captured donor was linearized, and the linearized captured donor was introduced into Escherichia coli DH10B containing RPCI23 clone 5E15 and the pABG vector. The cells were grown in a drug selection medium. Individual clones containing the captured donor DNA in the RPCI23 clone 5E15 DNA (forming the targeting vector) were isolated, the targeting vector DNA was extracted, and the portion of the vector was sequenced for the appropriate mouse-human junction. Pulse field gel electrophoresis was used to confirm the size of the insert and the expected restriction fragment lengths.

[0095] Electroporation The targeting vector was linearized and used to electroporate mouse ES cells as described by Valenzuela et al. Mouse ES cells containing the targeting vector that had been electroporated were further electroporated using a transient Cre expression vector to remove the loxed drug selection cassette. The targeting vector was introduced into Rag2 HET Il2rg y / - ES cells by electroporation. The parental ES cell line with knockouts of the RAG2 gene and the Il2rg gene was the commercially available V17 ES cells (BALB / c×129 heterozygote). The ES cells targeted with the hIL-3 and hGM-CSF genes were used for introduction into mouse embryos.

[0096] hIL-3 / GM-CSF mouse Targeted donor ES cells are introduced into 8-cell stage mouse embryos by the VELOCIMOUSE® method (see, for example, U.S. Patent No. 7,294,754 and Poueymirou et al. (2007) "F0 generation mice that are essentially fully derived from the donor gene-targeted ES cells allowing immediate phenotypic analyses," Nat Biot 25(1):91-99, which are incorporated herein by reference). VELOCIMICE® (F0 mice that are completely derived from donor ES cells) carrying humanized IL-3 and GM-CSF constructs are identified by genotyping for loss of the mouse allele and acquisition of the human allele using a modified allele assay method (see, for example, Valenzuela et al.). These mice are first mated to BALB / cAnNCR and then mice that are heterozygous for Rag2 and Il2rg and contain the human IL-3 / GM-CSF KI are mated to Rag2 / Il2rg double KO mice for transplantation studies.

[0097] Phenotypic Classification of hIL-3 / GM-CSF Mice Humanized mice were tested for human GM-CSF production by RT-PCR using hGM-CSF-specific primers. The expression pattern of human GM-CSF in the tissues tested was consistent with that of mouse GM-CSF (expressed mainly in the lung). ELISA of splenocytes from humanized mice stimulated with ConA and IL-2 for 48 hours was performed to detect the presence of hIL-3 and hGM-CSF. Splenocytes were positive for the expression of both hIL-3 and hGM-CSF.

[0098] hTPO Targeting The targeting construct (Figure 11(d)) for replacing the mouse Tpo (mTpo) gene with the human TPO (hTPO) gene in a single targeting step was constructed using the VELOCIGENE® technology that utilizes gap repair cloning (Valenzuela et al.). This vector replaces the sequence containing the open reading frame of Tpo but is designed to maintain the mouse-derived promoter and 5'UTR. The mouse sequence was obtained from bacterial artificial chromosome (BAC) RPCI-23, clone 98H7. The human sequence was obtained from BAC RPCI-11, clone 63m3. The gap repair donor vector containing the p15 origin of replication was constructed by cloning a 5'mouse homology arm immediately upstream of mTpo ATG, a human 5'TPO homology arm spanning the hTPO gene from hTPO ATG to approximately 275 nt, a polylinker, a 3'hTPO homology arm starting approximately 1.5 kb downstream of the polyA sequence of the hTPO gene, and a loxP drug selection cassette, followed by a mouse 3'homology arm with the sequence downstream (approximately 3.5 kb downstream) of the mTpo polyA sequence. This gap repair vector was linearized and inserted into the Escherichia coli DH10B strain containing the human BAC clone RPCI-11, 63m3 and the recombination enzyme vector. The cells were grown in drug selection medium. Individual clones were grown, and the gap repair donor vector DNA was extracted and the portion of the vector was sequenced for the appropriate mouse-human junction. Pulse field gel electrophoresis was used to confirm the size of the insert and the expected restriction fragment lengths. A capture donor containing the mouse upstream and downstream homology boxes adjacent to the hTPO gene and the loxP drug selection cassette was obtained from the repair donor vector, the capture donor was linearized, and the linearized capture donor was introduced into Escherichia coli DH10B containing RPCI-23 clone 98H7 and the pABG vector. The cells were grown in drug selection medium. Individual clones containing the capture donor DNA in the RPCI-23 clone 98H7 DNA (forming the targeting vector) were isolated, the targeting vector DNA was extracted, and the portion of the vector was sequenced for the appropriate mouse-human junction.Pulse field gel electrophoresis was used to confirm the size of the insert and the expected restriction fragment lengths. The targeting vector was linearized and used to electroporate mouse embryonic stem (ES) cells. The targeting vector was RAG2+ / -γ. c Y / -ES cells were introduced by electroporation. The parental RAG2 + / - γ c Y / - ES cell lines were generated from the commercially available V17 ES cell line (BALB / c×129 heterozygote). Correctly targeted ES cells were further electroporated with a transient Cre expression vector to remove the loxP drug selection cassette. ES cells targeted with hTPO and lacking the selection cassette were introduced into 8-cell stage mouse embryos by the VELOCIMOUSE® method (Poueymirou et al). Wild-type Tpo (TPO m / m ), heterozygous (TPO h / m ) or homozygous (TPO h / h ) TPO gene replacement Rag2 - / - γ c - / - mice were obtained.

[0099] Example 2 hIL-3 / GM-CSF mice: transplantation Isolation of human hematopoietic stem cells Human umbilical cord blood and fetal liver samples were obtained from Yale - New Haven Hospital and Albert Einstein Medical College New York, respectively, under the approval of the Human Investigation Committee of Yale University. CD34+ cells were isolated from human umbilical cord blood or fetal liver by density gradient centrifugation and immunomagnetic selection using CD34 microbeads (Miltenyi Biotec). The purity of the isolated CD34 - positive cells was confirmed by flow cytometry. The purified human CD34 - positive cells were cryopreserved and stored in liquid nitrogen before use.

[0100] Transplantation of Mice with Human Hematopoietic Stem Cells Transplantation was performed as previously described (Traggiai et al. (2004) Development of a human adaptive immune system in cord blood cell - transplanted mice, Science 304:104 - 107). Briefly, on the day of birth, neonatal mice from the background of RAG2 gene knockout / Il2rg gene knockout (with or without hIL - 3 / hGM - CSF) were sub - lethally irradiated (2×200 cGy at 4 - hour intervals). After irradiation, the neonates received 1 - 2×10 5 individual human CD34+ cells (resuspended in 25 μL of PBS) by intra - hepatic injection using a 30 - gauge needle. As a control, only PBS was injected. The mice were weaned at 3 - 4 weeks of age and maintained under specific pathogen - free conditions. To prevent opportunistic infections, prophylactic antibiotics (Sulfatrim) in the drinking water were administered to the mice. All animal experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Yale University and were conducted in accordance with the IACUC regulations.

[0101] Analysis of transplanted mice Engraftment of human hematopoietic cells was determined 8 - 12 weeks after transplantation. Blood samples were collected from the retro-orbital sinus and erythrocytes were lysed using ACK lysis buffer (Lonza). Samples were then stained with fluorescently labeled monoclonal antibodies against mouse CD45, human CD45, human CD3, and human CD14 (all from BD Biosciences) and analyzed by flow cytometry using a FACScalibur™ (BD Biosciences). Mice used in the infection experiments had a blood engraftment level of >4% hCD45+ cells unless otherwise specified. Matched mice, i.e., mice transplanted with the same batch of CD34+ cells, were used in the experiments. Unless otherwise specified, experiments were performed using mice transplanted with CD34+ cells from FL.

[0102] Flow cytometry For the hIL-3 / GM-CSF study, cell suspensions were prepared from the lungs, BAL, bone marrow, thymus, spleen, and blood of mice 10 - 14 weeks after transplantation. RBC lysis was performed using ACK lysis buffer (Lonza). Samples were then stained with fluorescent dye-labeled monoclonal antibodies (mAbs) against mouse and human cell surface antigens. The following mAbs were used: (1) anti-human: CD3 (UCHT1), CD4 (RPA-T4), CD8 (HIT8a), CD11c (B-ly6), CD14 (MoP9), CD19 (HIB19), CD33 (WM53), CD45 (HI30 and 2D1), CD56 (NCAM 16.2), CD66 (B1.1), CD116 (4H1), CD123 (9F5). (2) anti-mouse: CD45 (30-F11), F4 / 80 (BM8). CD116, CD45 (30-F11), and F4 / 80 mAbs were purchased from eBioscience. All other mAbs were purchased from BD Biosciences. Samples were analyzed using a FACScalibur™ or LSRII™ flow cytometer (BD Biosciences).

[0103] Methylcellulose CFU assay method For hIL-3 / GM-CSF studies, human CD34+ bone marrow cells from transplanted mice were purified by cell sorting. The sorted cells (1 - 1.5×10 5 cells) were cultured in Iscove's Modified Dulbecco's Medium (IMDM, GIBCO)-based methylcellulose medium (Methocult™ H4100, StemCell Technologies). The medium was supplemented with 20% FBS, 1% BSA, 2 mM L-glutamine, 55 μM 2-mercaptoethanol, and the following human cytokines: stem cell factor (10 ng / ml), FLT3 ligand (10 ng / ml), thrombopoietin (50 ng / ml), IL-3 (20 ng / ml), IL-6 (10 ng / ml), IL-11 (10 ng / ml), GM-CSF (50 ng / ml), and erythropoietin (4 U / ml) (all from R&D Systems). The cells were incubated in 60 mm Petri dishes at 37 °C / 5% CO2. After 12 - 14 days, the number of colonies was counted microscopically.

[0104] Inflammatory response to LPS Mice were given two intraperitoneal injections of Ultrapure LPS E. coli 0111:B4 (Invivogen) at 48-hour intervals (35 and 17.5 μg). Serum was collected 2 - 3 hours after each injection. The serum concentration of human IL-6 was measured by ELISA (R&D Systems). Mice were sacrificed 72 hours after the first LPS injection, and blood was collected by cardiac puncture for use in flow cytometry.

[0105] Intracellular cytokine staining For the hIL-3 / GM-CSF study, overlapping peptides covering the entire TB10.4 protein (Skjot et al. (2002) Epitope mapping of the immunodominant antigen TB10.4 and the two homologous proteins TB10.3 and TB12.9, which constitute a subfamily of the esat-6 gene family, Infect. Immun. 70:5446-5453) were synthesized at the W.M. Keck Facility of Yale University. Splenocytes (2×10 6 cells / well) from BCG-infected mice were incubated at 37°C / 5% CO2 for 5 hours at a total volume of 200 μl / well in 96-well U-bottom microtiter plates (Becton Dickinson) with the mixed peptides (5 μg / ml of each peptide). RPMI 1640 medium (Invitrogen) supplemented with 10% FCS, 1% penicillin-streptomycin, 1% L-glutamine, and 55 μM 2-mercaptoethanol was used for cell culture. Intracellular cytokine staining was performed using the Cytofix / Cytoperm™ kit (BD Biosciences) according to the manufacturer's instructions. The following mAbs were used for intracellular staining (all from BD Biosciences). Anti-human IFNγ (B27), anti-mouse IFNγ (XMG1.2). Isotype-matched mAbs were used as controls.

[0106] Histology and immunohistochemistry For hIL-3 / GM-CSF studies, organs were removed and fixed in 10% neutral buffered formalin or Zinc Fixative (BD Biosciences) for histological analysis. Paraffin-embedded tissue sections were prepared and stained with H&E or PAS, or processed for immunohistochemistry at Yale Pathology Tissue Services. The following anti-human antibodies were used for immunohistochemistry (all from Dako): CD45 (2B11+PD7 / 26), CD3 (F7.2.38), CD68 (PG-M1). Evaluation of tissue sections for the presence of granulomas was performed blindly.

[0107] Statistical Analysis For hIL-3 / GM-CSF studies, the non-parametric Mann-Whitney U test was used to determine statistical significance (α = 0.05) between two groups. For multiple group comparisons, we employed post hoc testing using Tukey's multiple comparison test (α = 0.05) together with one-way ANOVA. Only statistically significant P values (P < 0.05) are shown.

[0108] Example 3 Mice transplanted with hIL-3 / GM-CSF: Infection Salmonella typhimurium ISP2825 (Galan J.E. and Curtiss, R. (1991) Distribution of the invA, -B, -C, and -D genes of S. thyphimurium among other S. serovars: invA mutants of S. typhi are deficient for entry into mammalian cells, Infect. Immun. 59(9):2901-2908; incorporated herein by reference) is a clinical isolate from a patient with enteric typhoid, and this S. typhimurium was grown overnight in LB broth. The next day, 40 μL of the bacterial cell culture was transferred to 2 mL of fresh LB broth containing 0.3 M NaCl, and the culture was adjusted to an OD of approximately 0.9 600It was grown at 37°C for about 3 hours until it reached [a certain state]. The bacterial culture was spin - down and resuspended in buffered saline for use in infection. Humanized mice and control mice at 9 - 12 weeks of age were intraperitoneally inoculated with 1×10 3 or 1×10 4 or 1×10 5 Salmonella typhi bacteria on day 0. The infected mice were carefully monitored and sacrificed 4 weeks after infection. The spleen, liver, and gallbladder were aseptically removed and mechanically homogenized in 3 - 5 mL of sterile PBS containing 0.05% sodium deoxycholate. The tissue homogenates were serially diluted, plated on LB agar plates, and incubated overnight at 37°C for colony counting. The number of colonies was counted and the total number of colony - forming units recovered was calculated. Mouse data are provided in Figures 1 - 4. In Figure 1, "control" mice are gene - modified mice (RAG KO, Il2rg KO / hIL - 3, hGM - CSF) that were not transplanted. In Figures 2 - 4, "control" mice are non - transplanted RAG KO / Il2rg KO mice (i.e., they lack humanization of IL - 3 and GM - CSF but instead have endogenous mouse IL - 3 and endogenous mouse GM - CSF). "Control" mice were injected with PBS instead of human CD34+ cells.

[0109] As shown in Figure 1, Salmonella typhi infection in the spleen was detected in 2 gene - modified mice (RAG KO, Il2rg KO / hIL - 3, hGM - CSF) 10 days after infection.

[0110] As shown in Figure 2, 1 week after infection with 1×10 3 Salmonella typhi bacteria, gene - modified mice (RAG KO, Il2rg KO) with engraftment rates of 3.8 and 3% showed spleen infections about 1000 - fold higher than control mice (p < 0.01). The p - value for the difference between control mice and humanized mice was p < 0.01.

[0111] As shown in Figure 3, transplanted with CD34 - positive cells derived from fetal liver and 1×10 4Genetically modified mice (RAG KO, Il2rg KO) infected with individual S. typhi bacteria showed S. typhi infection in both the spleen (on average about 1,000 - about 10,000 times) and the liver (on average about 1,000 times) 4 weeks after infection. Each mouse in the cohort tested for S. typhi in the spleen had engraftment rates of human cells of 23.5, 40.1, 16.5, 50, 26, and 51.7 (from top to bottom in the "humanized" cohort in the left panel of Figure 3). Each mouse in the cohort tested for S. typhi in the liver had engraftment rates of human cells of 16.5, 40.1, 23.5, 26, 50, and 51.7 (from top to bottom in the "humanized" cohort in the right panel of Figure 3). The p-value for the difference between control mice and humanized mice in the spleen was p < 0.01; in the liver, p < 0.03.

[0112] As shown in Figure 4, genetically modified mice (RAG KO, Il2rg KO) transplanted with CD34-positive cells derived from fetal liver and infected with 1 × 10 4 individual S. typhi bacteria showed S. typhi infection in the gallbladder 4 weeks after infection, and the S. typhi cfu was on average 1 million times higher than that of control mice. Each mouse in the cohort tested for S. typhi in the gallbladder had engraftment rates of human cells of 23.5, 16.5, 40.1, 50, 26, and 51.7 (from top to bottom in the "humanized" cohort in Figure 3). The p-value for the difference between control mice and humanized mice was p < 0.03.

[0113] These results demonstrate that genetically modified mice (RAG KO, Il2rg KO / hIL-3, hGM-CSF) can be colonized by S. typhi bacteria after systemic infection.

[0114] Example 4 Mice transplanted with hIL-3 / GM-CSF: Validation of a mouse model for the human inflammatory response to pulmonary pathogens The genes encoding GM-CSF (Csf2) and IL-3 are closely linked (<10 kb) on chromosomes 5 and 11 in humans and mice, respectively. This enabled the replacement of the mouse loci of both genes with the human locus to generate hIL-3 / GM-CSF KI mice (Figure 5(e)). The human IL-3 KI allele is under the control of mouse regulatory elements, while the human Csf2 KI allele remains under the control of its human regulatory elements. The expression of mouse and human GM-CSF mRNAs was analyzed by RT-PCR in hIL-3 / GM-CSF KI mice (referred to as IL-3 / GM-CSF “human / mouse” (h / m) mice) that express one allele of each mouse gene and one allele of each human gene. Wild-type mice that carry only the mouse alleles of IL-3 and GM-CSF are referred to as IL-3 / GM-CSF “mouse / mouse” (m / m) mice.

[0115] RT-PCR and ELISA analyses of hIL-3 / GM-CSF KI mice Total RNA was extracted from homogenized tissues using TRIzol™ reagent (Invitrogen) according to the manufacturer's instructions. Equal amounts of DNase-treated RNA were used for cDNA synthesis by the SuperScript™ First-Strand Synthesis System (Invitrogen). Conventional RT-PCR was performed using the following primers. Quantitative RT-PCR was performed using a 7500 Fast real-time PCR system with a primer-probe set purchased from ABI. Expression levels were calculated using the comparative threshold cycle method and normalized to mouse or human HPRT. Mouse and human IL-3 and GM-CSF proteins were detected using species-specific ELISA kits from R&D Systems according to the manufacturer's instructions. Splenocytes were activated with 5 μg / ml concanavalin A (ConA) and 100 U / ml IL-2, and the supernatant was collected for ELISA after 48 hours of stimulation.

[0116] Expression in hIL-3 / GM-CSF KI mice Human GM-CSF mRNA showed the highest expression in the lung and was expressed in a pattern similar to its mouse counterpart (Figure 5(a)). IL-3 is mainly expressed by activated T cells, which also produce GM-CSF. Therefore, ELISA was performed on the supernatant from activated splenocytes isolated from h / m mice. Both human IL-3 and GM-CSF proteins could be detected (Figure 6(f), 6(g)). To confer competitive superiority to human hematopoietic cells, homozygous KI mice called IL-3 / GM-CSF "human / human" (h / h) mice that express two alleles of human IL-3 and GM-CSF were generated. Conventional RT-PCR analysis and quantitative RT-PCR analysis of lung tissue showed that h / h mice expressed only human GM-CSF mRNA and not mouse GM-CSF mRNA (Figure 5(b), 5(c)). Human GM-CSF protein could be detected by ELISA in the bronchoalveolar lavage (BAL) fluid of h / h mice (Figure 5(d)). These results indicate that hIL-3 / GM-CSF KI mice faithfully express human GM-CSF (and IL-3).

[0117] Figures 5(a)-(d) show the verification of human GM-CSF expression in non-transplanted hIL-3 / GM-CSF KI mice. Figure 5(a) shows a representative RT-PCR analysis of GM-CSF mRNA expression in various tissues from KI mice (h / m) having one allele of human Csf2 and one allele of mouse Csf2. Li, liver; Br, brain; Lu, lung; Mu, muscle; Sp, spleen; Th, thymus; LN, lymph node; BM, bone marrow. Lower: The specificity of the primers for detecting human GM-CSF was verified by RT-PCR analysis of tissues from control mice (m / m). Ribosomal protein L13 (Rpl13) was used as an endogenous control. Figure 5(b) shows an RT-PCR analysis of GM-CSF mRNA expression in the lungs from m / m mice or homozygous KI mice (h / h) expressing two alleles of human Csf2 (n = 5 each). Rpl13 was used as an endogenous control; NTC, no-template control. Figure 5(c) shows a quantitative RT-PCR analysis of the same GM-CSF mRNA expression. GM-CSF expression was normalized to mouse Hprt (n = 5 each). Figure 5(d) shows an ELISA of human GM-CSF protein in BAL fluid recovered from m / m or h / h KI mice (n = 6 each). The results represent two independent experiments. Each dot represents one mouse. The horizontal bar indicates the mean value.

[0118] Figure 5(e) shows the strategy for generating hIL-3 / GM-CSF KI mice. The genomic organization of the IL3 and Csf2 loci in mouse (top) and human (bottom) is shown on chromosomes 11 and 5, respectively. The mouse locus was replaced with the human locus as described in this disclosure.

[0119] Figures 6(f) and (g) show the expression of human IL-3 and GM-CSF in non-transplanted hIL-3 / GM-CSF KI mice. ELISA results of human IL-3 (f) and GM-CSF (g) production by activated splenocytes are presented. Splenocytes from m / m mice (white bars) or h / m KI mice (black bars) were stimulated with ConA and IL-2 for 48 hours, and the supernatants were collected (n = 1 for each). Human IL-3 and GM-CSF were not detected (ND) in m / m mice.

[0120] Example 5 Mice transplanted with hIL-3 / GM-CSF: Enhanced human inflammatory response hIL-3 / GM-CSF KI mice were generated from embryonic stem (ES) cells in which one allele of both Rag2 and Il2rg had already been deleted. Subsequently, by mating into the Rag2 KO Il2rg KO background, it became possible to transplant human CD34+ hematopoietic cells. The overall chimerism of human CD45+ hematopoietic cells, as well as the distribution of T cells, B cells, and natural killer (NK) cells in the bone marrow, thymus, spleen, and blood, did not increase significantly in hIL-3 / GM-CSF KI mice (data not shown). Furthermore, the frequencies of total human CD33+ bone marrow cells, CD66+ granulocytes, CD14+ monocytes / macrophages, CD14loCD16+ non-classical monocytes, CD11c+ dendritic cells (DCs), and CD123+CD11c- plasmacytoid DCs did not increase significantly in hIL-3 / GM-CSF KI mice (data not shown). This was true for both h / m and h / h mice under steady-state conditions. Finally, human bone marrow cells from transplanted hIL-3 / GM-CSF KI mice had a similar ability to form bone marrow colonies in methylcellulose in vitro (data not shown). These findings are consistent with the results from KO mouse studies indicating that both IL-3 and GM-CSF are largely dispensable for steady-state bone marrow hematopoiesis in the organs analyzed herein.

[0121] In contrast, GM-CSF plays an important role in mediating the inflammatory response. The expression of GM-CSF is induced by inflammatory stimuli, which leads to the production of inflammatory cytokines (such as IL-6 and TNFα) by monocytes / macrophages and their recruitment to the inflammatory site. Human CD14+ monocytes derived from transplanted hIL-3 / GM-CSF KI mice showed the highest expression of the GM-CSF receptor α chain (CD116) (Figure 8(d)). Therefore, the analysis of transplanted hIL-3 / GM-CSF KI mice focused on human monocytes / macrophages. First, the inflammatory response of human monocytes in transplanted hIL-3 / GM-CSF KI mice was analyzed. Systemic inflammation was induced by intraperitoneal (i.p.) injection of lipopolysaccharide (LPS). After LPS injection, the frequency of circulating human CD14+ monocytes was significantly increased in h / m mice compared to control m / m mice (Figures 8(e), (f)). The increase in human monocyte recruitment in h / m mice was associated with an increase in the serum concentration of human IL-6 after one and two LPS injections (Figure 8(g)). The LPS-induced production of human TNFα was also increased in h / m mice, but this result did not reach statistical significance. These data indicate that hIL-3 / GM-CSF KI mice transplanted with human hematopoietic cells have an enhanced human inflammatory response mediated by human bone marrow monocyte cells.

[0122] Figures 8(d)-(g) show enhanced human inflammatory responses in transplanted hIL-3 / GM-CSF KI mice. Figure 8(d) shows flow cytometric analysis of human bone marrow cells from steady-state transplanted hIL-3 / GM-CSF h / m KI mice. The dot plot (left) is gated on hCD45+mCD45- cells. The histogram (right) shows GM-CSF receptor α (CD116) expression on CD14- cells (population 1), CD14mid / SSChi granulocytes (population 2), and CD14hi monocytes (population 3). A representative example from a total of 12 analyzed mice is shown. Figure 8(e) includes representative flow cytometric analysis of human blood cells from CB-transplanted m / m or h / m KI mice 72 hours after two intraperitoneal injections of LPS. The plots are gated on hCD45+mCD45- cells. The numbers next to the boxed regions indicate the percentage of human CD14+ cells. Figure 8(f) shows the frequency of human CD14+ blood cells in transplanted m / m mice (n = 4) or h / m KI mice (n = 8) 72 hours after LPS injection. Figure 8(g) shows ELISA results for human IL-6 in sera from transplanted m / m mice (n = 4-5) or h / m KI mice (n = 8) 2-3 hours after the first (top) and second (bottom) LPS injections. One m / m mouse died after the first LPS injection. Each dot represents one mouse. The horizontal bars indicate the mean values. The results represent two independent experiments.

[0123] Example 6 Mice transplanted with hIL-3 / GM-CSF: Enhanced human macrophage engraftment in the lung BAL analysis Bronchoalveolar analysis for hIL-3 / GM-CSF studies was performed as follows. The lungs were inflated with 1 ml of PBS from a catheter inserted into the trachea. This was repeated twice, and the collected washings were pooled. After centrifugation, the cell-free supernatant was stored for measurement of GM-CSF protein concentration by ELISA or for measurement of total protein content using a BCA Protein Assay Kit (Pierce) according to the manufacturer's instructions. Red blood cells (RBCs) were lysed with ACK lysis buffer (Lonza), and the number of cell pellets was counted and used for flow cytometry or for preparation of cytospin specimens. The cells were spun onto slides and stained with a Diff-Quik™ Stain Set (Dade Behring) according to the manufacturer's instructions.

[0124] Enhanced macrophage engraftment The absence of murine GM-CSF leads to impaired function of murine alveolar macrophages (AMs), which should favorably act on the reconstitution by human macrophages in homozygous hIL-3 / GM-CSF KI mice. As evidence, human GM-CSF is highly expressed in the lungs and BAL of h / h mice, while murine GM-CSF is deficient.

[0125] AMs from non-transplanted h / h mice were enlarged and exhibited a typical "foamy" appearance (Figure 9(f)). This foamy appearance has been described for AMs from GM-CSF KO mice. GM-CSF KO mice develop PAP due to a defect in surfactant clearance by AMs with impaired terminal differentiation. Similar to what has been reported for GM-CSF KO mice, non-transplanted h / h mice exhibited features of PAP, such as subpleural accumulation of AMs filled with periodic acid-Schiff (PAS)-positive material (Figure 9(g)). Therefore, it was concluded that non-transplanted h / h mice exhibit impaired differentiation of murine AMs and develop PAP, and are thus functionally equivalent to GM-CSF KO mice.

[0126] Figures 9(f) and (g) show the development of PAP in non-transplanted homozygous hIL-3 / GM-CSF KI mice. Figure 9(f) shows Diff-Quick™ staining of BAL cells from non-transplanted m / m or h / h KI mice (magnification 400x); a representative example of a total of 6 mice analyzed per group is shown. Figure 9(g) shows PAS staining of lung tissue sections from non-transplanted m / m or h / h KI mice (magnification 400x); a representative example of a total of 12 mice analyzed per group is shown.

[0127] Next, the lung compartments of h / h mice after transplantation of human hematopoietic cells were examined. FACS analysis showed that h / h mice had a significant number of human CD45+ cells in the BAL (Figures 6(a) and (b)). Quantitative RT-PCR of lung tissue revealed that this increase in human cells consisted mainly of cells expressing the mRNA of human bone marrow markers CD33, CD11b, CD11c, and CD14 (Figure 6(c)). Furthermore, the mRNA expression of human CD68, a mature macrophage marker mainly expressed intracellularly, was significantly increased in transplanted h / h mice (Figure 6(d)). This increase in h / h mice was associated with higher expression of two transcription factors expressed by AMs, namely PU.1 (Spi1) and peroxisome proliferator-activated receptor γ (PPARγ) (Figure 6(d)). PU.1 is highly expressed in terminally differentiated AMs in a GM-CSF-dependent manner. Importantly, transduction of GM-CSF KO AMs with PU.1 in vitro improves their dysfunction. PPARγ is also highly expressed in AMs, and similar to GM-CSF KO mice, PPARγ KO mice develop PAP. Immunohistological staining of lung sections revealed the presence of numerous hCD68+ cells at typical alveolar locations, consistent with human AMs, in transplanted h / h mice (Figure 6(e)). In contrast, only very few human AMs could be detected in transplanted m / m control mice. In summary, the lungs of h / h mice transplanted with CD34+ hematopoietic cells show markedly improved human macrophage engraftment.

[0128] Figures 6(a)-(e) show that homozygous hIL-3 / GM-CSF KI mice have good human macrophage engraftment in the lung. Figure 6(a) shows representative flow cytometry analysis of BAL cells from transplanted m / m and h / h KI mice. The numbers adjacent to the areas circled in the figure indicate the percentages of hCD45+ and mCD45+ hematopoietic cells. mCD45+hCD45+ cells have high autofluorescence and constitute F4 / 80+ mouse AM. Figure 6(b) provides the numbers of human hematopoietic (hCD45+) cells in BAL from transplanted m / m and h / h KI mice (results are combined from three independent experiments (total n = 15 per group)). Figure 6(c) shows the results of quantitative RT-PCR analysis of human lymphocyte and myeloid gene expression in lung tissues from transplanted m / m and h / h KI mice (n = 4 each). Expression was normalized to mouse HPRT ( * , P<0.05). Figure 6(d) shows quantitative RT-PCR analysis of human macrophage gene expression in lung tissues from transplanted m / m and h / h KI mice (n = 4 each). Expression was normalized to mouse HPRT ( * , P<0.05). Each dot represents one mouse. Horizontal bars indicate mean values. Figure 6(e) shows immunohistochemistry of lung tissue sections stained for human CD68 from transplanted m / m and h / h KI mice (magnification 100x (top) and 200x (bottom)). A representative example of a total of 10 mice analyzed per group is shown.

[0129] Example 7 Mice transplanted with hIL-3 / GM-CSF: PAP reduced by human hematopoietic cells We investigated whether the increased engraftment of human macrophages in h / h mice leads to improved human immune function in the lung. First, we examined whether human macrophages can rescue the PAP syndrome seen in non-transplanted h / h mice. Both type II alveolar epithelial cells and AM can respond to GM-CSF, but PAP can be rescued by bone marrow transplantation. This indicates that hematopoietic cells, particularly AM, are the main cell type capable of improving PAP. Therefore, it was hypothesized that h / h mice transplanted with human hematopoietic cells should have a lower severity of PAP. As expected, non-transplanted h / h mice showed alveolar accumulation of PAS-positive substances (Figure 7(a)), which is a prominent feature of PAP. Consistent with this hypothesis, transplanted h / h mice had mild protein accumulation in the lung, and the lungs of some h / h mice resembled those of (non-transplanted or transplanted) m / m control mice (Figure 7(a)). Furthermore, transplanted h / h mice had a significantly lower total protein content in the BAL fluid than non-transplanted h / h mice (Figure 7(b)). These results indicate that transplanted human hematopoietic cells (presumably AM) can reduce PAP in homozygous hIL-3 / GM-CSF KI mice.

[0130] Figures 7(a)-(e) show that human hematopoietic cells reduce PAP in homozygous hIL-3 / GM-CSF KI mice. Figure 7(a) shows PAS staining of lung tissue sections from non-transplanted or transplanted m / m or h / h KI mice. Lung sections from two different transplanted h / h KI mice are shown (magnification 400x). Representative examples of a total of 10-12 mice analyzed per group are shown. Figure 7(b) shows quantification of total protein in BAL fluid from non-transplanted (non) or transplanted h / h KI mice or m / m control mice (n = 6 per group). P < 0.0001 (one-way ANOVA test). P values determined by Tukey's multiple comparison test are indicated by stars ( ** , P < 0.01; *** , P < 0.001).

[0131] Example 8 Mice transplanted with hIL-3 / GM-CSF: Strong human type I IFN response against influenza A Influenza A infection Mice (9 - 10 weeks old) were infected with 2×10 4 plaque-forming units of influenza A / PR8 (H1N1) virus via the intranasal route. Infection was carried out by intranasally administering 50 μl of the virus stock solution diluted with PBS (or an equal volume of PBS as a control) to mice that had been deeply anesthetized with Anafane™ (Ivesco). Twenty-four hours after infection, the lungs were harvested for the above RNA extraction and quantitative RT-PCR analysis.

[0132] In addition to their role in lung homeostasis, AMs are essential for host defense in the lung. Numerous studies have shown that GM-CSF KO mice are more susceptible to the effects of various pathogens in the lung. To evaluate the functional response of transplanted human AMs to lung pathogens, transplanted h / h mice were infected with influenza A / PR8 (H1N1) virus via the intranasal route. AMs are the main producers of type I interferon (IFN) after lung virus infection, and AMs are required for an effective innate response to influenza A. The expression of human hypoxanthine phosphoribosyltransferase (HPRT) mRNA was significantly higher in the lungs of transplanted h / h mice compared to that in control m / m mice (Figure 8(a)), which indicates good human immune cell chimerism. Transplanted m / m mice did not show a significant induction of human IFNβ mRNA expression after influenza A infection when compared to transplanted m / m mice administered PBS intranasally (Figure 8(b)). In contrast, transplanted h / h mice expressed significantly more human IFNβ mRNA than both non-infected h / h mice and infected m / m mice (Figure 8(b)). The increased expression of human IFNβ mRNA in h / h mice remained significant when normalized to human HPRT, i.e., the number of human cells in the lung, compared to m / m mice (Figure 8(c)). In summary, homozygous hIL-3 / GM-CSF KI mice enable good human macrophage chimerism and a function in the lung that leads to enhanced human mucosal immunity against virus infection.

[0133] Figures 8(a)-(g) show that homozygous hIL-3 / GM-CSF KI mice initiate a strong human type I IFN response against influenza A infection. Figures 8(a)-(c) show quantitative RT-PCR analysis of gene expression in lung tissues derived from m / m and h / h KI 24 hours after intranasal infection with influenza A (PR8) (n = 8 each). Intranasal administration of PBS was used as a control (PBS) (n = 4 each). Figure 8(a) shows the expression of human Hprt normalized to mouse Hprt. P < 0.0001 (one-way ANOVA test). Figure 8(b) shows the expression of human IFNγ normalized to mouse Hprt. P = 0.0171 (one-way ANOVA test). Figure 8(c) shows the expression of human IFNγ normalized to human Hprt. P = 0.0032 (one-way ANOVA test). P values determined by Tukey's multiple comparison test are indicated by asterisks (* , P < 0.05; ** , P < 0.01; *** , P < 0.001). Each dot represents one mouse. The horizontal bar indicates the mean value. The results represent two independent experiments.

[0134] Example 9 Mice transplanted with hIL-3 / GM-CSF: Granulomas containing human cells after mycobacterial infection The ability of hIL-3 / GM-CSF KI mice to support the human inflammatory response to a second pathogen with tropism for the lung, where macrophages play a central role in pathogen-specific immune responses, was examined. The formation of granulomas after mycobacterial infection was selected. Granulomas represent a specialized local inflammatory response characteristic of mycobacterial infection. It is a classical example of a DTH response, and its formation depends on the interaction between activated T cells and macrophages. Both IL-3 and GM-CSF are required for an optimal DTH response, and importantly, GM-CSF KO mice do not form granulomas when infected with mycobacteria.

[0135] Transplanted hIL-3 / GM-CSF h / m KI mice were infected with Bacillus Calmette-Guerin (BCG) by intravenous injection. BCG is an attenuated strain of M. bovis, which is used as a vaccine against human tuberculosis. The mice used in the BCG infection experiment had a blood engraftment level of >20% hCD45+ cells, and >8% of the hCD45+ cells were T cells (hCD3+). The mice were 9 - 10 weeks old at the time of infection. 1×10 5 Colony-forming units of BCG (Statens Serum Institute, Copenhagen) were injected into the mice via the tail vein in a volume of 0.1 ml.

[0136] Since T cells are essential for granuloma formation, first, the human T cell response to BCG 4 weeks after infection was examined. Flow cytometry clearly showed the presence of human T cells in the lungs of both transplanted m / m and h / m mice (Figure 9(a)). Indeed, T cells were the major human hematopoietic cell type in the BCG-infected lungs. Compared to m / m control mice, h / m mice infected with BCG had a higher mean ratio of human CD4 to CD8 T cells in the lungs, although the difference did not fully reach statistical significance (Figures 9(b), 9(c)). No difference in the ratio of splenic hCD4 / hCD8 T cells was observed between these two mouse groups.

[0137] Next, the expression of two types of T cell-derived cytokines, namely IFNγ and TNFα, was analyzed. Both of these are extremely important for the protective immune response against mycobacteria. Intracellular cytokine staining was used to examine BCG-specific IFNγ production after restimulating splenocytes from infected mice with peptides derived from the immunodominant mycobacterial antigen TB10.4. As expected, a population of mouse T cells producing IFNγ was detected among the splenocytes from BALB / c mice (Figure 9(d)). Furthermore, a human BCG-specific T cell response was seen in subsets of the transplanted h / m and m / m mice (Figure 9(d)). Finally, most of the transplanted h / m and m / m mice expressed human IFNγ and TNFα mRNA in the lungs after BCG infection (Figure 9(e)). These results indicate that subsets of the transplanted mice can initiate a pathogen-specific human T cell response against BCG, but this response was not enhanced in hIL-3 / GM-CSF KI mice. Consistent with this, there was no difference in bacterial load between h / m and m / m mice.

[0138] Next, granuloma formation was evaluated histologically in the lungs and livers 4 weeks after infection. Non-transplanted m / m mice (lacking T cells) did not develop granulomas (Table 1), which is consistent with the requirement for T cells for granuloma formation. Similarly, m / m mice transplanted with human cells showed no granulomas in either the lungs or the livers (Table 1). In contrast, most of the h / m mice had small lesions or granulomas in the lungs (Figure 10(a)) or livers or both organs (Table 1). Generally, the observed granulomas were small and had a loose organization characteristic of mouse rather than human granulomas. However, the lung granulomas in hIL-3 / GM-CSF KI mice contained human hematopoietic cells (hCD45+) as demonstrated by immunohistochemistry (Figure 10(b)). Most of these cells were human T cells (hCD3+), and a few human macrophages (hCD68+) were present in the center (Figure 10(b)). In summary, transplanted hIL-3 / GM-CSF KI mice were able to develop granulomas containing human T cells and human macrophages in response to mycobacterial infection, which has not been previously reported in HIS mice. Table 1 shows the lesions / granulomas observed in liver and lung tissue sections from BALB / c, non-transplanted m / m (non), transplanted m / m, and transplanted hIL-3 / GM-CSF h / m KI mice 4 weeks after BCG infection.

[0139] (Table 1) Granulomas in transplanted hIL-3 / GM-CSF KI infected with BCG TIFF0007710403000002.tif175153

[0140] Figures 9(a)-(g) show the human T cell responses to BCG in transplanted hIL-3 / GM-CSF KI mice. Figures 9(a)-(c) show flow cytometric analysis of lung cells from transplanted m / m and h / m KI mice 4 weeks after BCG infection. Figure 9(a) shows the frequency of human T cells (hCD45+hCD3+) in the lung. The numbers adjacent to the boxed areas indicate the percentage of cells. Figure 9(b) shows the distribution of human CD4 and CD8 T cells in the lung. The dot plots are gated on hCD45+hCD3+ cells. The numbers within the quadrants indicate the percentage of cells. Figure 9(c) shows the ratio of human CD4 to CD8 T cells in the lung (n = 6 each). Each dot represents one mouse. The horizontal bar indicates the mean value. Figure 9(d) shows flow cytometric analysis of splenocytes from BALB / c mice, transplanted m / m mice, and transplanted h / m KI mice 4 weeks after BCG infection. Splenocytes were restimulated in vitro with a pool of overlapping peptides covering the TB10.4 protein described herein. The dot plots show the frequency of mouse IFNγ+ CD4 T cells (mCD4+) or human IFNγ+ T cells (hCD3+) measured by intracellular cytokine staining. Staining with an isotype-matched antibody was used as a control. Figure 9(e) shows the results of quantitative RT-PCR analysis of human IFNγ (left) and TNFα (right) gene expression in lung tissues from BALB / c mice, non-transplanted (non) m / m mice, transplanted m / m mice, and transplanted h / m KI mice 4 weeks after BCG infection (n = 4-7 per group). Each dot represents one mouse. The horizontal bar indicates the mean value.

[0141] Figures 10(a) and (b) show that transplanted hIL-3 / GM-CSF KI mice develop granulomas containing human cells after BCG infection. Figure 10(a) shows hematoxylin and eosin (H&E) staining of lung tissue sections from transplanted h / m KI mice 4 weeks after BCG infection (magnification 100x (left) and magnification 200x (right)). Figure 10(b) shows immunohistochemistry of lung tissue sections from transplanted h / m KI mice stained for human CD45, CD3, or CD68 4 weeks after BCG infection (magnification 200x). A representative example of two mice with lung granulomas is shown.

[0142] Example 10 hTPO Mice: Engraftment and Analysis Transplantation into TPO Mice Recipient mice were transplanted with human hematopoietic progenitor cells as described by Traggiai et al. Umbilical cord blood samples were obtained from healthy full-term neonates under approval from the Human Rights Committee of Yale University (Department of Labor and Birth, Yale New Haven Hospital, New Haven, CT). Fetal liver samples were obtained from the Human Fetal Tissue Repository at Albert Einstein College of Medicine (Bronx, NY) and from Advance Biosciences Resources, Inc. (Alameda, CA).

[0143] Fetal liver samples were cut into small pieces, treated with collagenase D (100 ng / ml, Roche) at 37°C for 45 minutes, and cell suspensions were prepared. Human CD34+ cells were purified from fetal liver samples or umbilical cord blood by density gradient centrifugation (Lymphocyte Separation Medium, MP Biomedicals) followed by positive immunomagnetic selection using anti-human CD34 microbeads according to the manufacturer's instructions (Miltenyi Biotec). Cells were either frozen in 10% DMSO containing FBS or injected directly.

[0144] Newborns (within 1 day of birth) were sub-lethally irradiated (X-ray irradiation, 2×150 cGy at 4-hour intervals), and 100,000 - 200,000 CD34+ cells in 20 μl of PBS were injected into the liver using a 22-gauge needle (Hamilton, Reno, NV).

[0145] All experiments were conducted in accordance with the Yale University Human Investigation Committee protocol and the Yale University Animal Care and Use Committee protocol.

[0146] TPO expression Serum concentrations of mouse and human TPO protein were measured by species-specific ELISA (RayBiotech) according to the manufacturer's protocol. To measure the expression of mouse and human mRNA encoding TPO, tissues were isolated from adult animals and total RNA was purified using TRIzol (Invitrogen) according to the manufacturer's instructions. Contaminating genomic DNA was removed by treatment with RNase-Free DNase I (Roche), and the RNA was reverse transcribed using SuperScript II reverse transcriptase (Invitrogen) and oligo dT primers. The following primers were used for PCR amplification. TIFF0007710403000003.tif30160

[0147] To examine whether human TPO is faithfully expressed in these mice, total RNA was extracted from various organs derived from TPO h / m mice. Similar expression patterns were observed by RT-PCR for both mouse and human mRNAs encoding TPO (Figure 11(a)). Next, TPO m / m , TPO h / m and TPO h / h expression in three mouse-derived tissues or cell types known to express TPO (liver, kidney, and mesenchymal multipotent stromal cells) was compared. TPO m / m and TPO h / m expression of mouse Tpo was detected in samples from mice, while TPO h / m and TPO h / h human TPO was expressed in mice (Figure 11(b)). The concentration of TPO protein in the serum of the target mice was also measured. Mouse TPO was detected in TPO m / m and TPO h / m animals, and human TPO was detected in TPO h / m and TPO h / h (Figure 11(c)). The measured concentration of human TPO was approximately 10-fold lower than that of mouse TPO. However, this difference is consistent with the physiological concentrations reported in healthy humans and mice (Figure 11(c)) and may be due to species-specific differences in cytokine half-life.

[0148] Figure 11 shows (a) RT-PCR analysis of mouse TPO (mTpo) and human TPO (hTPO) expression in different tissues of Rag2 + / - γ c Y / - TPO h / m mice (mouse Rpl13a was used as a housekeeping gene); (b) RT-PCR analysis of mTpo and hTPO expression in the liver, kidney, and mesenchymal multipotent stromal cells (MSC) of Rag2 - / - γ c - / - TPO m / m , TPO h / m and TPO h / h mice; (c) TPO m / m , TPO h / mand TPO h / h Shows the concentrations (pg / ml, mean ± S.D., n = 7 - 9) of mouse and human TPO proteins measured by ELISA in the sera of mice. ND: Not detected. The normal range shown is from the Thrombopoietin Quantikine kit of R&D Systems.

[0149] Isolation of mesenchymal multipotent stromal cells For TPO research, the femurs and tibias of mice were harvested and the bone marrow cells were flushed out. The bones were cut into small pieces and digested at 37°C for 45 minutes using collagenases P and D (10 μg / ml). Bone-related cells were collected by repeated pipetting. Cells were cultured for 14 days in the presence of MSC medium supplemented with stimulatory supplements (Stemcell Technologies). Hematopoietic cells were removed from the culture by immunomagnetic cell sorting (MACS, Miltenyi Biotec) using CD45 and Ter119 antibodies. Non-hematopoietic cells (CD45 - Ter119 -) were cultured for another 5 days, and the MSC phenotype (CD45 - Ter119 - Sca1 + CD90 +) was confirmed by FACS (Diminici et al. (2006) Minimal criteria for defining multipotent mesenchymal stromal cells, The International Society for Cellular Therapy position statement, Cytotherapy 8:315 - 317).

[0150] Analysis of hematopoietic cell populations in TPO mice Mice were bled 8 - 12 weeks after transplantation. Red blood cells were lysed three times using ACK (Lonza), and the cells were stained with anti-mouse CD45 and anti-human CD45 antibodies. Animals in which at least 1% of the CD45 + cells were of human origin were used for further analysis. Approximately 80% of the transplanted mice reached this engraftment threshold, and no difference was observed between the TPOm / m and TPOh / h groups.

[0151] Mice were sacrificed at 3 - 4 or 6 - 7 months after transplantation. Single cell suspensions were prepared from bone marrow (flushed from two femurs and two tibias), spleen and thymus. Red blood cells were excluded by ACK lysis, and cells were stained with the following antibodies for FACS analysis. For overall hematopoietic engraftment: anti - mouse CD45 - eFluor450 (30 - F11, eBioscience) and anti - human CD45 - APC - Cy7 (2D1). For human hematopoietic stem cell progenitors and hematopoietic lineages: anti - human CD14 - PerCP (MoP9), CD19 - APC (HIB19), CD33 - APC (WM53), CD34 - PE (AC136, Miltenyi Biotec), CD38 - FITC (HIT2), CD41a - APC (HIP8) and CD66 - FITC (B1.1).

[0152] For analysis of mouse stem cell progenitors, the anti - lineage cocktail contained biotinylated antibodies against CD3ε (145 - 2C11), CD11b (M1 / 70), CD11c (HL3), CD19 (1D3), Gr1 (RB6 - 8C5) and Ly - 76 (Ter119). Cells were then stained with streptavidin - APC - Cy7, anti - cKit - APC (2B8) and anti - Sca1 - PE - Cy7 (D7).

[0153] All antibodies were obtained from BD Biosciences, unless otherwise specified. Data were acquired on a FACSCalibur™ or LSRII™ flow cytometer (BD Biosciences) and analyzed using FlowJo™ software.

[0154] Functional Characterization of Human Hematopoietic Stem Cell Progenitors in TPO Mice Bone marrow cells obtained from 3 - 7 transplanted mice were pooled, and human CD34+ cells were purified by MACS depletion of mouse CD45+ cells (Miltenyi Biotec) followed by FACS sorting of human CD45+CD34+ cells on a FACSAria™ flow cytometer (BD Biosciences).

[0155] To evaluate the colony - forming ability of human CD34+ cells, 20% FCS, 2 mM L - glutamine, 55 μM 2 - mercaptoethanol (all reagents from GIBCO) were added to IMDM, mixed with Methocult™ H4100, 1% BSA (Stemcell Technologies), and the following human cytokines were added: SCF (10 ng / ml), FLT3I (10 ng / ml), TPO (50 ng / ml), IL - 3 (20 ng / ml), IL - 6 (10 ng / ml), IL - 11 (10 ng / ml), GM - CSF (50 ng / ml), EPO (4 U / ml) (all from R&D systems). 100,000 - 150,000 sorted cells were seeded onto 60 - mm Petri dishes and incubated at 37 °C, 5% CO2 for 12 - 14 days. Colony numbers were counted at 12 - 14 days and classified into specific bone marrow lineages under a microscope.

[0156] For the secondary transplantation experiment, TPO m / m or TPO h / h 100,000 CD34+ cells purified from the primary recipient were, as described above, sub - lethally irradiated (2 × 200 cGy) Rag2 - / - γ c - / - TPO m / m and injected into secondary recipients. These mice were sacrificed after 8 weeks, and the proportion of human CD45+ cells in the bone marrow was measured by FACS.

[0157] Statistical analysis of TPO mouse data Data were compared using unpaired two-sided t-tests. When comparing more than two samples, one-way ANOVA followed by Tukey's post hoc test was performed. The percentage of mice showing engraftment in the secondary transplantation experiments was compared using Pearson's chi-squared test. Differences were considered significant when the p-value was less than 0.05.

[0158] Example 11 Mice transplanted with hTPO: TPO h / h Improved human engraftment levels in the bone marrow of recipient mice Phenotypic classification of the bone marrow of humanized and transplanted mice Analysis of cells isolated from the bone marrow of humanized mice by flow cytometry revealed a statistically significant improvement in the engraftment of total human hematopoietic cells, human hematopoietic stem cells, human bone marrow cells, and human granulocytes compared to engraftment in non-humanized mice (i.e., RAG and Il2rg knockout mice lacking humanization of the TPO gene). See Figures 12 and 14.

[0159] Wild-type Tpo (TPO m / m ), heterozygous (TPO h / m ), or homozygous (TPO h / h ) TPO gene-replaced Rag2 - / - γ c - / - mice were generated as described. Newborn Rag2 - / - γ c - / - TPO m / m and TPO h / h mice were transplanted with human CD34 + cells purified from umbilical cord blood or fetal liver and analyzed for engraftment in the bone marrow 3 - 4 months or 6 - 7 months later.

[0160] Figure 12 shows the results of the engraftment study. Figure 12(a) shows Rag2 + γ - / - - / - TPO m / m and TPO h / h mice 3 - 4 months after transplantation of human CD34 cHuman and Mouse CD45 in Mouse Bone Marrow + Representative FACS analysis of cells is shown. Results for two representative mice are shown for each genotype. Total (mouse + human) CD45 + Mouse and human CD45 in the cell population + The percentage of cells is shown. Figure 12(b) shows the percentage of human CD45 in bone marrow 3 - 4 months (left, n = 42 - 53) or 6 - 7 months (right, n = 20 - 25) after transplantation + The percentage of cells is shown. Each symbol represents an individual mouse and the horizontal bar indicates the mean value. Figure 12(c) shows the absolute number of human CD45 in bone marrow of the same animals as in (b) + The p - value indicates statistical significance

[0161] TPO m / m Compared to recipients, in TPO h / h the percentage (Figure 12(a) and 12(b)) and absolute number (Figure 12(c)) of human hematopoietic cells (hCD45 + ) in the bone marrow showed a significant increase at both time points. Furthermore, TPO h / h recipients showed less engraftment variability and at the 3 - 4 - month time point at least 80% human chimerism was seen in 75% of the mice (Figure 12(b)). The source of CD34 + cells did not affect this result, as similar increases in chimerism in the host were observed with cord blood - derived cells and fetal liver - derived cells (Figure 12(d)). Interestingly, in TPO h / h recipients the number of human cells decreased between the early and late time points, while in TPO m / m animals they remained constant (Figure 12(c)). These results are consistent with the previously described functions of TPO in mice. First, TPO acts favorably on the amplification of HSCs after transplantation into irradiated recipient mice, resulting in an increase in engraftment levels. Second, it acts favorably on the maintenance of adult HSCs, resulting in sustained hematopoiesis throughout adulthood h / h

[0162] Example 12 ​Mice transplanted with hTPO: Effects of TPO humanization on mouse and human platelets Platelet analysis in TPO mice The platelet count in peripheral blood was measured using a Hemavet (TM) 950FS instrument (Drew Scientific). Next, blood samples were stained with anti-mouse CD61-PE (2C9.G2) and anti-human CD41a-APC (HIP8), and the percentages of mouse and human platelets were measured by flow cytometry without placing any gates on cell size (FSC) or granulosity (SSC). Absolute mouse and human platelet counts were calculated by multiplying these respective percentages by the absolute platelet count.

[0163] Since TPO is well-known for its important function in platelet production, we investigated whether humanization of TPO affects platelet development. Humanization of both alleles of the TPO gene led to a decrease of approximately two-fold in the blood platelet count of non-transplanted Rag2 - / - γ c - / - mice (Figure 13(a)). After transplantation of human cells, the mouse platelet count in TPO h / h mice further decreased to less than 25% of the normal value (Figure 13(d)). The ratio of human to mouse platelets (Figures 13(b), 13(c)), as well as the absolute number of human platelets (Figure 13(e)), tended to be higher in TPO m / m than in TPO h / h mice, but none of these differences reached statistical significance. Furthermore, the percentage of bone marrow megakaryocytes (CD41a + cells) among human cells was equivalent in both strains (Figure 13(f)). These results demonstrate that the level or bioactivity of human TPO achieved by the knock-in strategy is not sufficient to fully replace mouse TPO function, and further suggest that human TPO alone is not sufficient to support human platelet production in the mouse environment.

[0164] Figure 13(a) shows adult non-transplanted Rag2 - / - γ c- / - TPO m / m , TPO h / m and TPO h / h indicates the platelet count in the blood of mice. p < 0.0001 (one-way ANOVA, n = 7 - 17; the p-values shown were calculated by Tukey's post hoc test). Each symbol represents an individual mouse, and the horizontal bar indicates the mean value. Figure 13(b) Three to four months after transplantation, Rag2 - / - γ c - / - TPO m / m and TPO h / h representative FACS analysis of mouse (mCD61 + ) and human (hCD41a + ) platelets in the blood of mice. The numbers indicate the ratio among all events. Figure 13(c) TPO m / m and TPO h / h Chimerism of human platelets measured by FACS in mice (n = 19 - 22). Only mice with a percentage of human CD45 + cells in the blood higher than 5% were included in this analysis. (d), (e) TPO m / m and TPO h / h Platelet counts of mouse (mCD61 + , (d)) and human (hCD41a + , (e)) in the blood of the recipient; (f) Percentage of human megakaryocytes (CD41a + ) among human CD45 + cells in the bone marrow.

[0165] Figures 13(g) - (i) show the human engraftment levels in secondary lymphoid organs. Figures 13(g), (h) show the percentage of human CD45 - / - γ c - / - TPO m / m and TPO h / h in the blood (20g; n = 43 - 53) and spleen (13h; n = 35 - 36) of mice (each symbol represents an individual mouse, and the horizontal bar indicates the mean value). Figure 13(i) shows the transplanted TPO + and TPO m / m and TPO h / hThe total cellularity of the thymus of recipients (n=24-34) is shown. More than 90% of the cells found in the thymus were of human origin (hCD45 + ) was.

[0166] Example 13 Mice engrafted with hTPO: Multilineage hematopoiesis in TPO-humanized mice Phenotyping of humanized and transplanted mouse blood cells Flow cytometric analysis of cells isolated from the blood of humanized mice showed a statistically significant improvement in engraftment of human monocytes and granulocytes compared to engraftment in non-humanized mice (i.e., RAG and Il2rg knockouts lacking humanization of the TPO gene), see Figure 14.

[0167] We investigated whether human TPO could favor the multilineage differentiation of human hematopoietic stem cell progenitors in vivo. As previously reported (Traggiai et al.; Ishikawa et al. (2005) Development of functional human blood and immune systems in NOD / SCID / IL2 receptor gamma chain(null) mice, Blood 106:1565-1573), transplanted human cells expressed wild-type Rag2 - / - Gamma c - / - In the host, mainly B cells (CD19 + ) (61.51 ± 4.71% of human cells in the spleen, mean ± SEM, n = 32), with only a small amount of bone marrow cells. m / m Recipient and TPO h / h When comparing recipients, TPO h / h CD33 in mouse bone marrow + A significant increase in the frequency of myeloid cells was observed (Figures 14(a) and 14(b)). Interestingly, this increase was mainly due to granulocytes (CD33 + CD66 hi SSC hi cells) and monocytes (CD33 hi CD66lo CD14 + ) The ratio was similar in both strains (Figs. 14(a), (c), (d), (e)). The percentage of bone marrow cells (both granulocytes and monocytes) also significantly increased in the peripheral blood of the animals (Figs. 14(a), (f), and (g)). h / h It was significantly increased in the peripheral blood of the animals (Figs. 14(a), (f), and (g)).

[0168] Figs. 14(a) to (g) show the improved multilineage hematopoiesis in hTPO mice as measured by CD33 m / m and TPO h / h mice, CD66 + , CD14 + , CD14 + cells. Fig. 14(a) shows the representative FACS analysis of the human bone marrow cell population in the bone marrow and blood of Rag2 - / - γ c - / - TPO m / m and TPO h / h mice 3 to 4 months after transplantation. The numbers indicate the percentage of the gated cell population shown. Figs. 14(b) to (e) show the analysis of the human bone marrow cell population for total human CD45 - / - γ c - / - TPO m / m and TPO h / h recipient (n = 19). Fig. 14(b) provides the total bone marrow population (CD33+ cells). Fig. 14(d) provides granulocytes (CD33 + cells, and Fig. 14(c) shows the Diff-Quick™ staining of hCD45 + CD66 hi ) cells purified from the bone marrow of the TPO h / h recipient. Fig. 14(e) shows monocytes (CD33 + SSC hi CD33 + CD66 hi ) cells. Fig. 14(e) shows monocytes (CD33 + CD66 lo CD14 + ). Each symbol represents an individual mouse, and the horizontal bar indicates the mean value. Figs. 14(f) and (g) show Rag2 - / - γ c- / - TPO m / m and TPO h / h Analysis of the human bone marrow cell population for total human CD45 in the blood of recipients (n = 6 - 7); + Fig. 14(f): granulocytes (CD66 + ); Fig. 14(g): monocytes (CD14 + ).

[0169] Example 14 Mice transplanted with hTPO: Humanization effect on mouse and human hematopoietic stem cell progenitors The effects of human TPO on the numbers and functions of HSCs and progenitor cells themselves were analyzed. Gene deletion of TPO leads to a decrease in HSCs in adult mice. To examine whether TPO humanization can affect a mouse population defined immunophenotypically including mouse HSCs, non-transplanted TPO m / m , TPO h / m and TPO h / h The percentages of lineage-negative Sca1 + c-Kit + cells in the bone marrow of adult mice were compared.

[0170] Fig. 15 shows a decrease in lin - c-Kit + Sca1 + cells in the bone marrow of human TPO knock-in mice, as well as an increase in the number and self-renewal ability of human stem cell progenitors. Fig. 15(a) shows non-transplanted Rag2 m / m ) Rag2 - / - γ c - / - mice compared with WT TPO (TPO - / - γ c - / - TPO h / m and TPO h / h Representative results of FACS analysis of mouse Lin - Sca1+c-Kit + stem cell progenitors in the bone marrow of mice. The numbers are Sca1 - among the Lin + c-Kit +Indicates the percentage of cells. Figure 15(b) shows the quantitative analysis of the results shown in (a). p = 0.0006 (one-way ANOVA; the p-values shown were calculated by Tukey's post hoc test; n = 5 per genotype; the results shown represent two independent experiments). Each symbol represents an individual mouse, and the horizontal bar indicates the mean value. Figure 15(c) shows the representative FACS analysis of human CD34 - / - γ c - / - TPO m / m and TPO h / h and human CD38 + cells in the bone marrow of mice. The numbers indicate the percentage of CD38 - cells among human CD45 + CD34 + cells. Figure 15(d) shows the quantitative analysis of the percentage of CD38 - cells in human CD45 m / m and TPO h / h recipient mice (n = 43 - 53). Figure 15(e) shows the absolute number of human CD34 + CD38 + cells in the bone marrow of the same mice as in 15(d). Figures 15(f) and (g) show the methylcellulose colony formation assay using human CD45 - CD34 + CD38 - cells purified from recipients. Figure 15(f) shows CFU-GEMM, and Figure 15(g) shows BFU-E (black), CFU-G (white), CFU-M (gray), and CFU-GM (dashed line). CD34 - / - γ c - / - TPO m / m and TPO h / h cells. CD34 + cells were pooled from groups of 3 - 4 mice to obtain 4 independent pools per recipient mouse genotype. In Figure 15(h), Rag2 + CD34 + cells. CD34 - / - γ c - / - TPO m / m and TPO h / hHuman CD45 from primary recipient mice + CD34 + cells were purified and transplanted into neonatal Rag2 - / - γ c - / - mice (100,000 cells per mouse), and human CD45 chimerism in secondary recipients was measured 8 weeks later. Results were pooled from two independent experiments (n = 7 - 12 primary recipients, n = 11 - 19 secondary recipients). +

[0171] TPO m / m Compared to TPO h / m and TPO h / h a significant decrease in the percentage of these cells was observed in both mouse lines (Figure 15(b)), suggesting that human TPO is not fully cross-reactive with the mouse receptor or is not utilized in sufficient amounts by mouse cells in this knock-in setting.

[0172] Transplanted TPO m / m and TPO h / h human CD34 populations in the host bone marrow were characterized. Human HSCs with long-term repopulating ability are contained in the Lin-CD34 + CD38 + cell fraction. The percentage of CD34 - cells in the human CD45 + population was slightly increased in TPO + mice (12.39 ± 0.79% vs 10.00 ± 0.81%, mean ± standard error, n = 43 - 53, p = 0.037). A statistically significant, albeit small (1.5-fold), increase in the percentage of CD38 h / h cells in the CD34 m / m population was observed in TPO h / h recipients compared to TPO + recipients (Figure 15(c), (d)). Overall, this indicates that in TPO-humanized mice, CD34 - CD38 + CD38 -This resulted in a significant increase in absolute cell numbers (approximately 2.8-fold) (Figure 15(e)). Thus, based on cell surface immunophenotype, human TPO supports a population of cells known to be highly enriched for HSCs.

[0173] To investigate the functional properties of this cell population, human CD34 + Cells are treated with TPO m / m and TPO h / h CFU-GEMMs were purified from mouse bone marrow and evaluated in vitro by methylcellulose colony formation assay. CFU-GEMMs are multilineage myeloid colonies derived from immature cells that contain at least all erythroblast-megakaryocyte and myeloid cell differentiation potentials. The formation of CFU-GEMMs, although in small numbers, was associated with TPO h / h CD34 isolated from recipient mice + was detected in all four cell samples, whereas TPO m / m Only one sample from the TPO strain produced CFU-GEMM (Fig. 15(f)). h / h In addition, consistent with the enhanced myeloid differentiation observed in vivo (FIG. 14), the number of CFU-M also increased with TPO. m / m Compared to mice, TPO h / h Human CD34 isolated from + The results were significantly higher in cell samples (150,000 cultured CD34 + 225.0±12.25 vs. 81.25±10.80 colonies per cell, mean±sem, p=0.0001; Figure 15(g)).

[0174] Maintenance and / or self-renewal of HSCs is functionally best demonstrated by successful secondary transplantation. Continuously engrafting SCID repopulating cells (SRCs) in mice now represent a reliable surrogate experimental standard for human HSC function. Thus, human CD34 + Cells are treated with TPO m / m and TPO h / h Purified from primary recipient bone marrow and Rag2 - / - Gammac - / - A small number (100,000 CD34 cells per animal) was similarly transplanted into neonatal mice. The bone marrow of the secondary recipients was analyzed 8 weeks later (Figure 15(h)). TPO + Human CD34 cells isolated from the primary recipients m / m were detected in only 2 out of 11 secondary recipients for human CD45 + cells, so the ability to engraft continuously was very low. In contrast, human CD45 + cells were present in the bone marrow of 15 out of 19 mice transplanted with CD34 cells isolated from the primary recipients (p = 0.0012). Since the genotypes of the secondary recipient mice were the same in both groups (TPO h / h ), this result indicates that the presence of human TPO in the primary recipient favorably affects the maintenance of human cells with enhanced self-renewal ability. + + m / m ) This result indicates that the presence of human TPO in the primary recipient favorably affects the maintenance of human cells with enhanced self-renewal ability.

[0175] In summary, these results demonstrate that homozygous TPO humanized mice provide a good environment for maintaining the self-renewal ability and multi-lineage differentiation ability of human hematopoietic stem cell progenitor cells.

[0176] Those skilled in the art can devise various modifications that are not explicitly described or represented in this disclosure but are embodied in the present invention and fall within the spirit and scope of the present invention. All examples are provided to assist the reader in understanding the principles and concepts of the present invention and are used without being limited to the specific examples and embodiments described. All principles, aspects, embodiments, and examples of the present invention are intended to include their equivalents, whether currently known or to be developed in the future. The scope of the present invention is not limited to the aspects and embodiments shown or described in this disclosure.​​

Claims

**Claim 1** Mouse embryonic stem (ES) cells containing in their genome a substitution of the mouse thrombopoietin gene with the human thrombopoietin gene at both alleles of the mouse thrombopoietin (TPO) locus, wherein the human TPO gene is operably linked to the endogenous promoter of the mouse TPO gene at the mouse TPO locus. **Claim 2** The mouse ES cells according to claim 1, further comprising in their genome a substitution of the mouse interleukin-3 (IL-3) gene with the human IL-3 gene at the mouse IL-3 locus, wherein the human IL-3 gene is operably linked to the endogenous promoter of the mouse IL-3 gene at the mouse IL-3 locus. **Claim 3** The mouse ES cells according to claim 1 or 2, further comprising in their genome a substitution of the mouse granulocyte macrophage colony-stimulating factor (GM-CSF) gene with the human GM-CSF gene at the mouse GM-CSF locus, wherein the human GM-CSF gene is operably linked to the endogenous promoter of the mouse GM-CSF gene at the mouse GM-CSF locus. **Claim 4** The mouse ES cells according to any one of claims 1 to 3, further comprising a RAG2 gene knockout. **Claim 5** The mouse ES cells according to any one of claims 1 to 4, further comprising an Il2rg gene knockout. **Claim 6** A mouse embryo comprising the mouse ES cells according to any one of claims 1 to 5.

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