NZ724014B2 - Humanized m-csf mice - Google Patents
Humanized m-csf mice Download PDFInfo
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- NZ724014B2 NZ724014B2 NZ724014A NZ72401412A NZ724014B2 NZ 724014 B2 NZ724014 B2 NZ 724014B2 NZ 724014 A NZ724014 A NZ 724014A NZ 72401412 A NZ72401412 A NZ 72401412A NZ 724014 B2 NZ724014 B2 NZ 724014B2
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- csf
- human
- cells
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
- C12N2015/8527—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic for producing animal models, e.g. for tests or diseases
- C12N2015/8536—Animal models for genetic diseases
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
- G01N2500/10—Screening for compounds of potential therapeutic value involving cells
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5082—Supracellular entities, e.g. tissue, organisms
- G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates
Abstract
Disclosed is a humanized M-CSF mouse, comprising a nucleic acid sequence incorporated into the genome of the humanized M-CSF mouse, which sequence encodes a human M-CSF protein and is operably linked to a regulatory sequence 5’ of the mouse M-CSF structural gene locus; wherein the mouse is immunocompromised for a mouse immune system as a result of one or more of the following: sub-lethal irradiation, the mouse is a NOD-SCID mouse, the mouse is a NOD-scid ?c?/? (NSG) mouse, and the mouse is a NOD/Shi-scid ?c?/? (NOG) mouse; and wherein the mouse expresses M-CSF RNA encoded by the nucleic acid sequence in bone marrow, spleen, blood, liver, brain, lung, testis and kidney Further disclosed is a method for making an activated human monocyte and/or activated human macrophage, comprising exposing a humanized M-CSF mouse to an immune stimulant under conditions sufficient for human monocytes and/or macrophages in the mouse to become activated, and isolating human monocytes and/or human macrophages from the mouse, wherein the mouse comprises a nucleic acid sequence incorporated into the genome of the mouse, which sequence encodes a human M-CSF protein and is operably linked to the endogenous promoter of the mouse M-CSF gene at the mouse M-CSF locus, wherein the mouse is immunocompromised for a mouse immune system, wherein the mouse comprises human hematopoietic cells, and wherein the mouse expresses M-CSF RNA encoded by the nucleic acid sequence in bone marrow, spleen, blood, liver, brain, lung, testis and kidney. Further disclosed is a method of isolating biologically active human M-CSF, the method comprising isolating biologically active human MCSF from a humanized M-CSF mouse, wherein the mouse comprises a nucleic acid sequence incorporated into the genome of the mouse, which sequence encodes a human M-CSF protein and is operably linked to the endogenous promoter of the mouse M-CSF gene at the mouse M-CSF locus, wherein the mouse is immunocompromised for a mouse immune system, and wherein the mouse expresses M-CSF RNA encoded by the nucleic acid sequence in bone marrow, spleen, blood, liver, brain, lung, testis and kidney. promised for a mouse immune system as a result of one or more of the following: sub-lethal irradiation, the mouse is a NOD-SCID mouse, the mouse is a NOD-scid ?c?/? (NSG) mouse, and the mouse is a NOD/Shi-scid ?c?/? (NOG) mouse; and wherein the mouse expresses M-CSF RNA encoded by the nucleic acid sequence in bone marrow, spleen, blood, liver, brain, lung, testis and kidney Further disclosed is a method for making an activated human monocyte and/or activated human macrophage, comprising exposing a humanized M-CSF mouse to an immune stimulant under conditions sufficient for human monocytes and/or macrophages in the mouse to become activated, and isolating human monocytes and/or human macrophages from the mouse, wherein the mouse comprises a nucleic acid sequence incorporated into the genome of the mouse, which sequence encodes a human M-CSF protein and is operably linked to the endogenous promoter of the mouse M-CSF gene at the mouse M-CSF locus, wherein the mouse is immunocompromised for a mouse immune system, wherein the mouse comprises human hematopoietic cells, and wherein the mouse expresses M-CSF RNA encoded by the nucleic acid sequence in bone marrow, spleen, blood, liver, brain, lung, testis and kidney. Further disclosed is a method of isolating biologically active human M-CSF, the method comprising isolating biologically active human MCSF from a humanized M-CSF mouse, wherein the mouse comprises a nucleic acid sequence incorporated into the genome of the mouse, which sequence encodes a human M-CSF protein and is operably linked to the endogenous promoter of the mouse M-CSF gene at the mouse M-CSF locus, wherein the mouse is immunocompromised for a mouse immune system, and wherein the mouse expresses M-CSF RNA encoded by the nucleic acid sequence in bone marrow, spleen, blood, liver, brain, lung, testis and kidney.
Description
Humanized M-CSF Mice
This application is a divisional application of New Zealand patent application 708040,
itself a divisional application of New Zealand patent application 613948, filed 14 February
2012, which is incorporated herein by reference.
FIELD OF INVENTION
The invention relates to genetically modified mice comprising a gene
encoding a human M-CSF protein, and mice that comprise further modifications that
support engraftment of human hematopoietic cells.
BACKGROUND
The development of animal models to study human diseases has
significantly advanced understanding of the underlying mechanisms of several diseases,
including cancer. To date, animal models, particularly mice, have proven to be excellent
candidates for the evaluation of the efficiency and efficacy of drugs and therapy options.
While the utilization of these surrogate models to study human biology and diseases can
be largely justified (due to ethical and technical constrains on the conduct of
experimental therapies in humans) studies have highlighted potential limitations of
extrapolating data from mice to humans (Mestas J, Hughes CC. (2004) Of mice and not
men: differences between mouse and human immunology. J Immunol. 172:2731-2738).
To overcome these issues, there has been a long-standing interest in
developing humanized mouse models. Intensive work by several groups have
successfully demonstrated the feasibility of studying human biology and diseases in
mice. Since having a functional and effective immune system in recipients will result in
the elimination of the transplanted tissues/cells of human origin, using genetic mutants
that lack cells of the adaptive immune system such as T, B and NK cells has significantly
contributed to the success of the humanized mouse model. Accordingly, the most
effective candidates of humanized mouse models include the NOD-SCID and the Balb/c
strains that lack genes including recombination activating genes (RAG), common
gamma chain (γC, also known as “interleukin 2 receptor, gamma”, or IL2rg), beta2
microglobin (B2M) and Perforin 1(Prf1) (Shultz LD, et al. (2007) Humanized mice in
translational biomedical research, Nat. Rev. Immunol. 7:118-130). Several studies over
the past few decades have demonstrated the feasibility of transplanting several types of
human tissues, including peripheral blood leukocytes, fetal liver cells, fetal bone, fetal
thymus, fetal lymph nodes, vascularized skin, artery segments and either mobilized or
cord blood hematopoietic stem cells (HSCs), into certain humanized mice (Macchiarini
F., et al. (2005) Humanized mice: are we there yet? J. Exp. Med. 202:1307-1311). This
approach is thought to provide better model systems since the data obtained from
human cells in these mice might reflect the physiology of the human system. A major
avenue of investigation in the field is to generate mice with a complete hematopoietic
system and a functional immune system of the human origin. While significant progress
has been made in generating immunocompromised mice with human T lymphocytes, B
lymphocytes, NK cells and dendritic cells (DCs), there are still several challenges in the
field, one of which is poor myeloid differentiation in the humanized mice.
Interestingly, there has been much progress in generating human T cells,
B cells, NK cells and dendritic cells (DCs) from hematopoietic stem cells (HSCs) in
humanized mice. In addition to the individual hematopoietic compartment, injection of
human HSCs in these mice resulted in the reconstitution of lymphoid organs such as
thymus and spleen. Nevertheless, the frequencies of myeloid cells, particularly
granulocytes, macrophages, erythrocytes and megakaryocytes, are very low—a result
that is probably due to inefficient myelopoiesis from human HSCs in these mice (Shultz
et al. (2007); Macchiarini et al. (2005)). In view of the fact that the cells of myeloid origin
(such as erythrocytes and megakaryocytes) are vital for the normal functioning of the
blood system, and granulocytes and macrophages are critical for the development of the
adaptive immune system, generating humanized mice with an efficient human
myelopoiesis is of paramount importance.
Accordingly, there is a need in the art for genetically modified mice that
are capable of improved human myelopoiesis upon engraftment with human HSCs
(Manz MG. Human-hemato-lymphoid-system mice: opportunities and challenges.
Immunity. 2007 May;26(5):537-41).
SUMMARY
[0006a] In one aspect, the invention provides a humanized M-CSF mouse,
comprising:
a nucleic acid sequence incorporated into the genome of the humanized M-CSF
mouse, which sequence encodes a human M-CSF protein and is operably linked to a
regulatory sequence 5’ of the mouse M-CSF structural gene locus; wherein the mouse is
immunocompromised for a mouse immune system as a result of one or more of the
following: sub-lethal irradiation, the mouse is a NOD-SCID mouse, the mouse is a NOD-
scid γc−/− (NSG) mouse, and the mouse is a NOD/Shi-scid γc−/− (NOG) mouse; and
wherein the mouse expresses M-CSF RNA encoded by the nucleic acid
sequence in bone marrow, spleen, blood, liver, brain, lung, testis and kidney.
[0006b] In another aspect, the invention provides a method for making an
activated human monocyte and/or activated human macrophage, comprising exposing a
humanized M-CSF mouse to an immune stimulant under conditions sufficient for human
monocytes and/or macrophages in the mouse to become activated, and isolating human
monocytes and/or human macrophages from the mouse, wherein the mouse comprises a
nucleic acid sequence incorporated into the genome of the mouse, which sequence
encodes a human M-CSF protein and is operably linked to the endogenous promoter of
the mouse M-CSF gene at the mouse M-CSF locus, wherein the mouse is
immunocompromised for a mouse immune system, wherein the mouse comprises human
hematopoietic cells, and wherein the mouse expresses M-CSF RNA encoded by the
nucleic acid sequence in bone marrow, spleen, blood, liver, brain, lung, testis and kidney.
[0006c] In another aspect, the invention provides a human immune cell isolated
from a humanized M-CSF mouse, wherein the mouse comprises a nucleic acid sequence
incorporated into the genome of the mouse, which sequence encodes a human M-CSF
protein and is operably linked to the endogenous promoter of the mouse M-CSF gene at
the mouse M-CSF locus, wherein the mouse is immunocompromised for a mouse
immune system, wherein the mouse comprises human hematopoietic cells, and wherein
the mouse expresses M-CSF RNA encoded by the nucleic acid sequence in bone
marrow, spleen, blood, liver, brain, lung, testis and kidney.
[0006d] Certain statements that appear below are broader than what appears in
the statements of the invention above. These statements are provided in the interests of
providing the reader with a better understanding of the invention and its practice. The
reader is directed to the accompanying claim set which defines the scope of the
invention.
Genetically modified mice comprising a nucleic acid sequence encoding a
human M-CSF protein are described. Also described are genetically modified mice
comprising a nucleic acid sequence encoding a human M-CSF protein that have been
engrafted with human cells such as human hematopoietic cells, and methods for making
such engrafted mice. These mice find use in a number of applications, such as in
modeling human immune disease and pathogen infection; in in vivo screens for agents
that modulate hematopoietic cell development and/or activity, e.g. in a healthy or a
diseased state; in in vivo screens for agents that are toxic to hematopoietic cells; in in
vivo screens for agents that prevent against, mitigate, or reverse the toxic effects of toxic
agents on hematopoietic cells; in in vivo screens of human hematopoietic cells from an
individual to predict the responsiveness of an individual to a disease therapy, etc.
In some embodiments, a humanized M-CSF mouse is described, where
the humanized M-CSF comprises a nucleic acid sequence that encodes a human M-
CSF protein and is operably linked to regulatory sequence 5’ of the mouse M-CSF
structural gene locus, e.g. the mouse M-CSF promoter, 5’UTR, etc. In some
embodiments the mouse comprises two copies of the nucleic acid sequence. In some
embodiments, the nucleic acid sequence is located in the mouse genome within the
mouse M-CSF locus. In some embodiments, the nucleic acid sequence is operably
linked to the endogenous mouse M-CSF promoter at the mouse M-CSF locus, i.e. the
mouse is a M-CSF mouse. In some embodiments, the mouse comprises two alleles
in which the nucleic acid sequence is located in the mouse genome within the mouse M-
CSF locus. In some embodiments, the nucleic acid sequence of both alleles is operably
linked to the endogenous mouse M-CSF promoter at the mouse M-CSF locus, i.e. the
mouse is a M-CSF mouse. In some embodiments, the humanized M-CSF mouse
comprises a null mutation in at least one mouse M-CSF allele. In some embodiments,
the humanized M-CSF mouse comprises a null mutation in both mouse M-CSF alleles.
In some such embodiments, the null mutation is a deletion of mouse M-CSF exons 2-9.
In some embodiments, the mouse expresses human M-CSF in bone
marrow, spleen, blood, liver, brain, lung, testis, and kidney. In some embodiments, the
amount of human M-CSF expressed is substantially the same as the amount of mouse
M-CSF expressed in a wild-type mouse. In some embodiments, bone marrow
mesenchymal stromal cells of the humanized M-CSF mouse express an amount of
human M-CSF that is substantially the same as the amount of mouse M-CSF expressed
by wild-type mouse bone marrow mesenchymal stromal cells. In some embodiments,
the humanized M-CSF mouse exhibits a physiological concentration of M-CSF in blood
and/or tissue. In some embodiments, the mouse expresses both mouse M-CSF and
human M-CSF. In other embodiments, the only M-CSF expressed by the mouse is
human M-CSF.
In some embodiments, the mouse secretes sufficient human M-CSF to
differentiate engrafted human hematopoietic stem cells into human monocytes, human
macrophages, and human osteoclasts. In some embodiments, the mouse secretes an
effective amount of M-CSF to stimulate the development of human macrophages from
human monocytes that result from an engraftment of human hematopoietic stem cells
into the mouse. In some embodiments, the mouse secretes an effective amount of M-
CSF to stimulate the development of a human hematopoietic stem cell into a monoblast,
a monoblast into a human promonocyte, a human promonocyte into a human monocyte,
and a human monocyte into a human macrophage, in a mouse engrafted with human
hematopoietic stem cells. In some embodiments, the effective amount of human M-CSF
secreted in the mouse is substantially the same amount of mouse M-CSF secreted by a
wild-type mouse to achieve a corresponding result (e.g., an effective amount of mouse
M-CSF to stimulate development of a mouse macrophage from a mouse monocyte).
In some embodiments, the transcriptional and translational control of
human M-CSF in the genetically modified mouse is identical or substantially identical to
the transcriptional and translational control of mouse M-CSF in a mouse that lacks a
modification of its endogenous mouse M-CSF gene.
In some embodiments, the physiological concentration of human M-CSF
in the humanized M-CSF mouse results from secretion of human M-CSF from the same
cell types that secrete mouse M-CSF in a wild-type mouse that has a mouse M-CSF
gene and that lacks a nucleic acid encoding a human M-CSF protein. In other words,
one or more M-CSF isoforms are expressed in a normal tissue-specific and
developmental pattern.
In some embodiments, the mouse expresses a human M-CSF isoform
selected from proteoglycan M-CSF, glycoprotein M-CSF, and cell surface M-CSF, and a
combination thereof. In one embodiment, the mouse expresses at least two of the
isoforms in a normal tissue-specific and developmental pattern. In a specific
embodiment, the mouse expresses human proteoglycan CSF-1 and human glycoprotein
M-CSF and human cell surface M-CSF.
In some embodiments, the mouse comprises human macrophages that
are not thymic T cell-derived macrophages. In some embodiments, the mouse
comprises human macrophages that exhibit M-CSF-dependent podosome formation
stimulated by human M-CSF expressed in the mouse.
In some embodiments, the mouse is homozygous null for Rag2. In some
embodiments, the mouse is homozygous null for IL2rg. In some embodiments, the
mouse is homozygous null for Rag2 and for IL2rg. In some embodiments, the mouse
comprises human cells. In some embodiments, the human cells are hematopoietic cells.
In some embodiments, a mouse model of the human immune system is
described, the mouse model comprising 2 null alleles for Rag2, 2 null alleles for IL2rg, a
nucleic acid sequence that encodes a human M-CSF protein operably linked to the
promoter of the mouse M-CSF gene, and human hematopoietic cells. In other words,
-/- -/-
the mouse is an engrafted Rag2 IL2rg hM-CSF mouse, where hM-CSF denotes that
the mouse comprises at least one nucleic acid encoding a human M-CSF gene. In
-/- -/-
some embodiments, the engrafted Rag2 IL2rg hM-CSF mouse is a BALB/c strain
mouse comprising these genetic modifications. In some embodiments, the mouse
comprises other genetic modifications as well.
-/- -/-
In some embodiments, the engrafted Rag2 IL2rg hM-CSF mouse at
about 12 weeks of age exhibits an increased frequency of human CD14 CD33
(hCD14 CD33 ) cells in bone marrow, spleen, and peripheral blood as compared with a
mouse comprising human hematopoietic cells that expresses mouse M-CSF but not
human M-CSF. In a specific embodiment, the increase in hCD14 CD33 cells of bone
marrow over a mouse expressing only mouse M-CSF is about 5 to about 15 fold, in one
embodiment about 12- to about 14-fold. In a specific embodiment, the increase in
hCD14 CD33 cells of spleen over a mouse comprising human hematopoietic cells that
expresses only mouse M-CSF is about 2- to about 6-fold, in one embodiment about 5- to
about 6-fold. In a specific embodiment, the increase in hCD14 CD33 cells of peripheral
blood over a mouse comprising human hematopoietic cells that expresses only mouse
M-CSF is about 2- to about 8-fold, in one embodiment about 5- to about 7-fold.
-/- -/-
In some embodiments, the engrafted Rag2 IL2rg hM-CSF mouse at
about 12 weeks of age exhibits a level of hCD14 CD33 monocyte/macrophage lineage
cells in blood of about 15 to about 40%, in one embodiment about 30%. In one
embodiment, the genetically modified engrafted mouse at about 16 weeks of age
exhibits a level of hCD14 CD33 monocyte/macrophage lineage cells in blood of about
to about 30%, in one embodiment about 22%. In one embodiment, the genetically
modified engrafted mouse at about 20 weeks of age exhibits a level of hCD14 CD33
monocyte/macrophage lineage cells in blood of about 5 to about 15%, in one
embodiment about 10%. In one embodiment, the genetically modified engrafted mouse
at about 20 weeks of age exhibits a level of hCD14 CD33 monocyte/macrophage
lineage cells in blood that is about 4- to 8-fold higher than the level in an engrafted
mouse that expresses mouse M-CSF but not human M-CSF, in one embodiment about
6-fold higher.
-/- -/-
In some embodiments, the engrafted Rag2 IL2rg hM-CSF mouse at
+ + +
about 12 weeks of age exhibits a level of hCD14 CD33 CD45 cells in liver that is about
1.5- to about 6-fold higher than an engrafted mouse that expresses mouse M-CSF but
not human M-CSF. In one embodiment, the genetically modified engrafted mouse at
+ + +
about 12 weeks of age exhibits a level of hCD14 CD33 CD45 cells in lung that is about
1.5- to about 10-fold higher than an engrafted mouse that expresses mouse M-CSF but
not human M-CSF. In one embodiment, the genetically modified engrafted mouse at
+ + +
about 12 weeks of age exhibits a level of human hCD14 CD33 CD45 cells in
peritoneum or in skin that is about 2- to about 3-fold higher than an engrafted mouse that
expresses mouse M-CSF but not human M-CSF.
-/- -/-
In some embodiments, the engrafted Rag2 IL2rg hM-CSF mouse
exhibits a response to LPS injection that is about 1.5- to about 6-fold greater with
respect to percentage of hCD14 CD33 cells in liver than mice that lack a human M-
CSF, in one embodiment about 2- to about 4-fold; in lung the LPS response with respect
to hCD14 CD33 cells is about 1.5- to 10-fold, in one embodiment about 2- to 3-fold; in
skin the LPS response with respect to hCD14 CD33 is about 2- to about 5-fold, in one
embodiment about 3- to about 4-fold; in peritoneum the LPS response with respect to
hCD14 CD33 is about 2- to about 5-fold, in one embodiment about 3- to about 4-fold.
-/- -/-
In some embodiments, the engrafted Rag2 IL2rg hM-CSF mouse
exhibits in response to LPS stimulation an enhanced pro-inflammatory cytokine
response, wherein the enhancement over a genetically modified and engrafted mouse
that lacks a hM-CSF gene is about 2- to at least about 5-fold with respect to the level of
activation and/or differentiation of a cell type that is responsive to the pro-inflammatory
cytokine.
-/- -/-
In some embodiments, the engrafted Rag2 IL2rg hM-CSF mouse
+ + +
exhibits an enhanced production of hCD14 CD33 hCD45 cells in spleen about 48 hours
following LPS injection, wherein the enhancement is about 2- to about 5-fold, in one
embodiment 4- to about 5-fold, over an engrafted mouse that expresses mouse M-CSF
but not human M-CSF.
-/- -/-
In some embodiments, the engrafted Rag2 IL2rg hM-CSF mouse
exhibits an enhanced production of serum human IL-6 in response to LPS, wherein the
level of hIL-6 about 6 hours after LPS injection is enhanced about 2- to about 5-fold over
an engrafted mouse that expresses mouse M-CSF but not human M-CSF, in one
embodiment about 3- to about 4-fold.
-/- -/-
In some embodiments, the engrafted Rag2 IL2rg hM-CSF mouse
exhibits en enhanced production of serum human TNFα in response to LPS, wherein the
level of hTNFα about 6 hours after LPS injection is enhanced about 2- to about 4-fold
over an engrafted mouse that expresses mouse M-CSF but not human M-CSF, in one
embodiment about 2- to about 3-fold.
In some embodiments, a monocyte and/or macrophage isolated from the
-/- -/-
engrafted Rag2 IL2rg hM-CSF mouse exhibits in vitro secretion upon LPS stimulation
that is about 2- to 3-fold higher with respect to hTNFα than an engrafted mouse that
expresses mouse M-CSF but not human M-CSF.
In some embodiments, a monocyte and/or macrophage isolated from the
-/- -/-
engrafted Rag2 IL2rg hM-CSF mouse exhibits in vitro secretion upon LPS stimulation
that is about 2- to 4-fold higher with respect to hIL-6 than an engrafted mouse that
expresses mouse M-CSF but not human M-CSF.
In some embodiments, a monocyte and/or macrophage isolated from the
-/- -/-
engrafted Rag2 IL2rg hM-CSF mouse exhibits in vitro secretion upon poly I:C
stimulation that is about 3- to 6-fold higher with respect to hIFNα than an engrafted
mouse that expresses mouse M-CSF but not human M-CSF.
In some embodiments, a monocyte and/or macrophage isolated from the
-/- -/-
engrafted Rag2 IL2rg hM-CSF mouse exhibits in vitro secretion upon poly I:C
stimulation that is about 2- to 3-fold higher with respect to hIFNβ than an engrafted
mouse that expresses mouse M-CSF but not human M-CSF.
In some embodiments, a human monocyte and/or macrophage isolated
-/- -/-
from the engrafted Rag2 IL2rg hM-CSF mouse exhibits enhanced phagocytosis as
compared with an engrafted mouse that expresses mouse M-CSF but not human M-
CSF. In one embodiment, the enhancement is about double the rate of phagocytosis, as
measured by incorporation of labeled bacteria at 37ºC over a 60-minute time period, as
compared with human cells from an engrafted mouse that expresses mouse M-CSF but
not human M-CSF. In one embodiment, the phagocytosis rate as measured above is
two fold or more the rate of human cells from an engrafted mouse that expresses mouse
M-CSF but not human M-CSF, e.g. 2-fold, 3-fold, or 4-fold or more.
In some embodiments, a human monocyte and/or macrophage isolated
-/- -/-
from the engrafted Rag2 IL2rg hM-CSF mouse exhibits enhanced chemotaxis in vitro
in response to Mip3β as compared with an engrafted mouse that expresses mouse M-
CSF but not human M-CSF. In one embodiment, the enhancement is about 1.5-fold to
3-fold or more, e.g. about 1.5-fold, 2-fold, 3-fold, 4-fold or more, as measured by number
of migrated cells at 30 or 60 minutes following Mip3β exposure, as compared with a
human monocyte and/or macrophage from a engrafted mouse that expresses mouse M-
CSF but not human M-CSF.
In some embodiments, a human monocyte and/or macrophage isolated
-/- -/- h
from the engrafted Rag2 IL2rg M-CSF mouse exhibits in vitro secretion upon poly I:C
stimulation that is about 3- to 6-fold higher with respect to hIFNα than an engrafted
mouse that expresses mouse M-CSF but not human M-CSF.
In some embodiments, a human monocyte and/or macrophage isolated
-/- -/-
from the engrafted Rag2 IL2rg hM-CSF mouse exhibits upregulation in vitro of a co-
stimulatory molecule in response to LPS stimulation. In one embodiment, the co-
stimulatory molecule is selected from human CD40, human CD80, human CD86, human
HLA-DR, and a combination thereof.
In some embodiments, a genetically modified engrafted mouse is
described, wherein the mouse comprises an engraftment of human hematopoietic cells,
is Rag2 Il2rg , comprises a null allele for mouse M-CSF, and comprises a nucleic acid
sequence encoding a human M-CSF at the endogenous M-CSF locus, wherein the
mouse exhibits an enhancement, or increased number, of human myeloid cells as
compared with that expresses mouse M-CSF but not human M-CSF.
In some embodiments, the enhancement comprises at least a doubling in
the number of hCD14 CD33 cells in a portion of the mouse selected from bone marrow,
spleen, and peripheral blood. In a specific embodiment, the enhancement comprises a
CD33 cells. In another embodiment, the enhancement comprises
tripling of the hCD14
a 4- to 5-fold increase or more in the number of hCD14 CD33 cells.
In some embodiments, the enhancement comprises a 2- to 3-fold
+ + +
increase in the number of hCD14 CD33 hCD45 cells in a compartment of the mouse
selected from skin and peritoneum.
In some embodiments, the enhancement comprises a 1.5- to 10-fold
+ + +
increase in the number of hCD14 CD33 hCD45 cells in a compartment of the mouse
selected from liver and lung.
In some embodiments, the enhancement comprises a 4- to 5-fold
+ + +
increase in the number of hCD14 CD33 hCD45 spleen cells at about 48 hours post-
LPS stimulation.
In some embodiments, the enhancement comprises a 2- to 4-fold
increase in LPS-stimulated serum hIL-6 or LPS-stimulated serum hTNFα.
In some embodiments, the enhancement comprises a 2- to 3-fold
increase in human MIP3β-stimulated in vitro migration of hCD14 CD33 cells.
In some embodiments, a mouse model for a human pathogen is
described, the mouse model comprising 2 null alleles for Rag2, 2 null alleles for IL2rg, a
nucleic acid sequence that encodes a human M-CSF protein operably linked to the
promoter of the mouse M-CSF gene, human hematopoietic cells, and an infection by a
-/- -/-
human pathogen. In other words, the mouse is an engrafted Rag2 IL2rg hM-CSF
mouse that has been infected with a human pathogen. In some embodiments, the
pathogen is a virus, a fungus, or a bacterium. In some embodiments, the virus is a
human or porcine or avian influenza virus. In some embodiments, the bacterium is a
mycobacterium, e.g. Mycobacterium tuberculosis (M. tuberculosis). In some
embodiments, the bacterium is an enterobacterium, e.g. Salmonella typhi (S. typhi).
In some embodiments, a pluripotent, induced pluripotent, or totipotent
mouse cell is described, comprising a nucleic acid sequence encoding a human M-CSF
protein operably linked to the promoter of the mouse M-CSF gene. In one embodiment,
the mouse cell is a mouse ES cell.
In some embodiments, a mouse embryo is described, comprising a
nucleic acid sequence encoding a human M-CSF protein operably linked to the promoter
of the mouse M-CSF gene.
In some embodiments, a targeting construct for targeting a mouse M-CSF
gene is described, comprising (a) upstream and downstream targeting arms that are
complementary or substantially complementary to upstream and downstream nucleotide
sequences of either (i) a nucleotide sequence encoding a mouse M-CSF protein, or, (ii)
a nucleotide sequence complementary to a nucleotide sequence encoding a mouse M-
CSF protein; (b) human nucleic acid sequence encoding a human M-CSF protein or
fragment thereof, or a nucleotide sequence encoding the complement of a human M-
CSF protein or fragment thereof; and, (c) a marker and/or a selection cassette.
In some embodiments, a human immune cell from a mouse as described
herein is described. In one embodiment, the human immune cell is selected from a
human monocyte and a human macrophage. In one embodiment, the human immune
cell is selected from a human NK cell, a human B cell, and a human T cell.
In some embodiments, an antibody encoded by a human nucleotide
sequence from a mouse as described herein is described. In one embodiment, the
antibody is selected from an IgA, IgD, IgE, IgM, or IgG isotype antibody.
In some embodiments, a nucleotide sequence encoding a human
immunoglobulin sequence is described, wherein the nucleotide sequence is obtained
from an engrafted humanized M-CSF mouse described herein. In one embodiment, the
nucleotides sequence encodes a human variable region of a human immunoglobulin
gene or a fragment thereof. In one embodiment, the nucleotide sequence encodes a
human TCR variable region or fragment thereof.
In some embodiments, a method for making a humanized M-CSF mouse
expressing biologically active human M-CSF is described. In some embodiments, the
method comprises contacting a mouse pluripotent stem cell, e.g. an ES cell or an iPS
cell, with a nucleic acid sequence comprising coding sequence for a human M-CSF
protein or a ragment thereof and culturing the pluripotent stem cell under conditions that
promote the integration of the nucleic acid sequence into the mouse genome; making a
mouse from the mouse ES cell that comprises the nucleic acid sequence encoding a
human M-CSF protein; and maintaining the mouse under conditions sufficient for the
mouse to express human M-CSF from the human M-CSF gene. In some embodiments,
the nucleic acid sequence is integrated randomly into the genome. In other
embodiments, the nucleic acid sequence is integrated into a target locus. In some such
embodiments, the target locus is the endogenous mouse M-CSF locus, e.g. the nucleic
acid sequence comprising coding sequence for a human M-CSF protein is flanked by
sequences that are homologous to the endogenous mouse M-CSF locus, and the
nucleic acid sequence is integrated into the endogenous mouse M-CSF locus by
homologous recombination. In some embodiments, the mouse is homozygous null for
Rag2. In some embodiments, the mouse is homozygous null for IL2rg. In some
-/- -/-
embodiments, the mouse is homozygous null for Rag2 and IL2rg, i.e., it is Rag2 IL2rg
In some embodiments, a method for making a humanized M-CSF mouse
comprising a human hematopoietic system is described. In some embodiments, the
-/- -/-
method comprises transplanting into a humanized M-CSF mouse, e.g. a Rag2 IL2rg
hM-CSF mouse or a sublethally irradiated hM-CSF mouse, a population of cells
comprising human hematopoietic progenitor cells. In some embodiments, the human
hematopoietic progenitor cells are CD34+ cells. In some embodiments, the human
hematopoietic progenitor cells are CD133+. In some embodiments, the human
hematopoietic progenitor cells pluripotent stem cells, e.g. ES cells or iPS cells. In some
embodiments, the source of the population of cells comprising human hematopoietic
progenitor cells is fetal liver. In some embodiments, the source of the cells is bone
marrow. In some embodiments, the source of the cells is peripheral blood. In some
embodiments, the source of the cells is an in vitro population of cells.
In some embodiments, a method for making a mouse that is infected with
a human pathogen is described. In some embodiments, the method comprises exposing
a humanized M-CSF comprising human hematopoietic cells, e.g. an engrafted Rag2
IL2rg hM-CSF mouse or an engrafted sublethally irradiated mouse, to a human
pathogen, and maintaining the mouse under conditions sufficient for the human
pathogen to infect the mouse. In some embodiments, the human pathogen is a human
pathogen that does not infect a mouse that lacks one or more of the genetic
modifications described herein. In some embodiments, the human pathogen is a human
pathogen that is not pathogenic in a mouse that lacks one or more of the genetic
modifications described herein.
In some embodiments, a method for making biologically active human M-
CSF in a mouse is described, the method comprising making a humanized M-CSF
mouse expressing biologically active human M-CSF as described above and elsewhere
herein. In some embodiments, the method comprises purifying biologically active
human M-CSF from blood, e.g. serum, or tissue of the mouse. In some embodiments,
the method comprises obtaining a cell that expresses biologically active human M-CSF
from the mouse, culturing the cell under conditions sufficient for the cell to express and
secrete biologically active human M-CSF, and isolating the secreted biologically active
human M-CSF. It being noted that in this embodiment the mouse is not required to have
any other genetic modifications and that the mouse is useful in making preparations of
certain human immune cells. As such, in some embodiments, isolated biologically active
human M-CSF obtained from a transgenic mouse is described.
In some embodiments, a method for making an activated human
monocyte or activated human macrophage in a mouse is described, comprising
exposing a humanized M-CSF mouse engrafted with human hematopoietic cells to an
immune stimulant, allowing human monocytes or macrophages in the mouse to become
activated, and isolating from the mouse human monocytes or human macrophages,
wherein the fraction of activated monocytes or activated macrophages are about two-
fold to five-fold higher than obtained from an engrafted mouse that is not a humanized
M-CSF mouse, i.e. that lacks a human M-CSF gene. In some embodiments, the
immune stimulant is an endotoxin. In a specific embodiment, the endotoxin is LPS.
In some embodiments, a method of screening a candidate agent for
activity in modulating human hematopoietic cell function is described. In some
embodiments, the method comprises contacting a humanized M-CSF mouse engrafted
-/- -/-
with human hematopoietic cells, e.g. an engrafted Rag2 IL2rg hM-CSF mouse or an
engrafted sublethally irradiated hM-CSF mouse, with a candidate agent; and comparing
the function of the hematopoietic cells in the mouse model contacted with the candidate
agent to the function of the hematopoietic cells in the mouse model that was not
contacted with the candidate agent; wherein a modulation in the function of the
hematopoietic cells in the mouse contacted with the candidate agent indicates that the
candidate agent modulates hematopoietic cell function.
In some embodiments, a method for determining the effect of an agent on
a human pathogen is described, comprising exposing an engrafted humanized M-CSF
-/- -/-
mouse, e.g. an engrafted Rag2 IL2rg hM-CSF mouse or an engrafted sublethally
irradiated hM-CSF mouse, to an effective amount of a human pathogen, the effective
amount of a pathogen being the amount of pathogen required to produce an infection in
the mouse; allowing the pathogen to infect the mouse; measuring a parameter of the
infection over time in the presence of the agent; and comparing that measurement to the
measurement from an engrafted humanized M-CSF mouse not exposed to the agent. In
some embodiments, the agent is provided prior to exposing the mouse to the human
pathogen, e.g. to determine the protective effect. In some embodiments, the agent is
provided concurrently with exposing the mouse to the human pathogen, e.g. to
determine the protective or therapeutic effect. In some embodiments, the agent is
provided after exposing the mouse to the human pathogen, e.g. to determine the
therapeutic effect. In some embodiments, the mouse upon exposure to a human
pathogen mounts a cellular and/or humoral immune response that models infection of a
human exposed to the pathogen. In some embodiments, the human pathogen is a
pathogen that does not infect a mouse that lacks one or more of the genetic
modifications described herein. In some embodiments the human pathogen is a
pathogen that infects a wild-type mouse, wherein the wild-type mouse following infection
does not model an immune response that a human mounts in response to the pathogen.
In some embodiments, the virus is a human or porcine or avian influenza virus. In some
embodiments, the bacterium is a mycobacterium, e.g. Mycobacterium tuberculosis (M.
tuberculosis). In some embodiments, the bacterium is an enterobacterium, e.g.
Salmonella typhi (S. typhi). In some embodiments, 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 a fluid or tissue of the mouse. In
some embodiments, the parameter of the infection is a titer in a body fluid of the mouse.
In some embodiments, the parameter of the infection is the formulation of a granuloma.
In some such embodiments, the granuloma is a lung granuloma. In some such
embodiments, the granuloma is a well-defined granuloma.
In some embodiments, a method for determining the effect of an agent on
a human pathogen is described, comprising exposing an engrafted humanized M-CSF
-/- -/-
mouse, e.g. an engrafted Rag2 IL2rg hM-CSF mouse or an engrafted sublethally
irradiated hM-CSF mouse, to an effective amount of an antigen of a human pathogen,
the effective amount of antigen being the amount of antigen required to promote a
cellular and/or humoral response in the mouse; allowing a cellular and/or humoral
response to develop; measuring a parameter of the cellular and/or humoral response
over time in the presence of the agent; and comparing that measurement to the
measurement from an engrafted humanized M-CSF mouse not exposed to the agent. In
some embodiments, the agent is provided before exposing the mouse to the antigen
from the human pathogen, e.g. to determine the protective effect of the agent. In some
embodiments, the agent is provided concurrently with exposing the mouse to the antigen
from the human pathogen, e.g. to determine the protective or therapeutic effect of the
agent. In some embodiments, the agent is provided after exposing the mouse to antigen
from the human pathogen, e.g. to determine the therapeutic effect of the agent. In some
embodiments, the mouse upon exposure to a human pathogen mounts a cellular and/or
humoral immune response that models infection of a human exposed to the pathogen.
In some embodiments, the antigen is from a human pathogen that does
not infect a mouse that lacks one or more of the genetic modifications described herein.
In other embodiments the antigen is from a human pathogen that infects a wild-type
mouse, wherein the wild-type mouse following infection does not model an immune
response that a human mounts in response to the pathogen. In some embodiments, the
pathogen is a virus, a fungus, or a bacterium. In some embodiments, the virus is a
human or porcine or avian influenza virus. In some embodiments, the bacterium is a
mycobacterium, e.g. Mycobacterium tuberculosis (M. tuberculosis). In some
embodiments, the bacterium is an enterobacterium, e.g. Salmonella typhi (S. typhi).
In some embodiments, a method of screening a candidate agent for
toxicity to human hematopoietic cells is described. In some embodiments, the method
comprises contacting a humanized M-CSF mouse engrafted with human hematopoietic
-/- -/-
cells, e.g. an engrafted Rag2 IL2rg hM-CSF mouse, with a candidate agent; and
comparing the viability and/or function of the hematopoietic cells in the mouse contacted
with the candidate agent to the viability and/or function of the hematopoietic cells in a
humanized M-CSF mouse engrafted with human hematopoietic cells that was not
contacted with the candidate agent; wherein a decrease in the viability and/or function of
the hematopoietic cells in the mouse contacted with the candidate agent indicates that
the candidate agent is toxic to the hematopoietic cells.
In some embodiments, a method of screening a candidate agent for the
ability to protect human hematopoietic cells from a toxic agent, mitigate the effects of a
toxic agent on human hematopoietic cells, or reverse the effects of a toxic agent on
human hematopoietic cells is described. In some embodiments, the method comprises
contacting a humanized M-CSF mouse engrafted with human hematopoietic cells, e.g.
-/- -/-
an engrafted Rag2 IL2rg hM-CSF mouse or an engrafted sublethally irradiated hM-
CSF mouse, with a toxic agent; contacting the mouse with a candidate agent; and
comparing the viability and/or function of the hematopoietic cells in the mouse contacted
with the candidate agent to the viability and/or function of hematopoietic cells in a
humanized M-CSF mouse engrafted with human hematopoietic cells that were not
contacted with the candidate agent; wherein an enhancement in viability and/or function
of hematopoietic cells in the mouse model contacted with the candidate agent indicates
that the candidate agent protects hematopoietic cells from the toxic agent.
In some embodiments, a method for predicting responsiveness of an
individual to treatment with a therapeutic agent is described. In some embodiments, the
method comprises contacting a humanized M-CSF mouse engrafted with human
-/- -/-
hematopoietic cells from the individual, e.g. an engrafted Rag2 IL2rg hM-CSF mouse
or an engrafted sublethally irradiated hM-CSF mouse, with a therapeutic agent; and
comparing the viability and/or function of the hematopoietic cells in the mouse model
contacted with the candidate agent to the viability and/or function of the hematopoietic
cells in a humanized M-CSF mouse engrafted with human hematopoietic cells that was
not contacted with the candidate agent; wherein a modulation in the viability and/or
function of the hematopoietic cells in the mouse contacted with the candidate agent
indicates that the individual will have a response to treatment with the therapeutic agent.
BRIEF DESCRIPTION OF THE FIGURES
illustrates, for bone marrow mesenchymal stromal cells, (A)
m/m h/h
expression of M-CSF; indicated organs from M-CSF and M-CSF were isolated,
RNA was extracted and reverse transcription (RT)-PCR analysis was performed either
using mouse M-CSF (top) or human M-CSF (middle) specific primers; HPRT level
(bottom) was used as control for the input cDNA; data are representative of 2
independent experiments. (B) Indicated organs from M-CSF were isolated, RNA was
extracted and RT-PCR analysis was performed either using mouse M-CSF (top) or
human M-CSF (bottom) specific primers. RNA extracted either from mouse liver or
human fetal liver served as positive controls for mouse and human primer pairs,
respectively, no RT, and no template PCR reactions served as negative controls. Data
are representative of 2 independent experiments. (C) Bone associated stromal cells from
m/m m/h h/h
M-CSF , M-CSF and M-CSF mice were isolated and cultured in vitro for 10 days,
cells were lysed, and RNA was extracted and real time PCR analysis was performed
either using mouse M-CSF (white) or human M-CSF (black) specific primers; mean
values of duplicate samples are shown; error bars indicate ± SEM; input cDNA quantity
was normalized according to HPRT (hypoxanthine guanine phosphoribosyl transferase)
expression levels; data are representative of 2 independent experiments; and, (D) bone
m/m m/h h/h
associated stromal cells from M-CSF , M-CSF and M-CSF mice were isolated and
cultured in vitro for 10 days; cell culture supernatants were collected and the secreted
levels of mouse (white) and human (black) M-CSF were quantified using species-
specific M-CSF ELISA kits; mean values of triplicate samples are shown; error bars
indicate ± SEM; data are representative of 2 independent experiments. (E) M-CSF ,
h/m h/h
M-CSF , and M-CSF mice were bled and the serum levels of human and mouse M-
CSF were quantified through ELISA. Shown are the mean values of triplicate samples.
Error bars indicate ± SEM.
illustrates absolute numbers of bone marrow (BM) cells of M-
m/m m/h h/h
CSF , M-CSF and M-CSF mice, as average per animal (two tibia and fibula); each
group contains n=5 mice, age 4 weeks; error bars indicate ± SEM; data are
representative of 3 independent experiments.
illustrates flow cytometry analysis of a stained single cell
suspension of BM (top), Spleen (middle) and Peripheral Blood (PB) from M-CSF , M-
m/h h/h
CSF and M-CSF mice; stained with Gr1 and CD11b antibodies.
illustrates flow cytometry analysis of a stained single cell
m/m m/h h/h
suspension of BM (top) and Spleen (middle) from M-CSF , M-CSF and M-CSF
mice; stained with F4/80 and CD11b antibodies.
illustrates flow cytometry analysis of BM cells that were isolated
and cultured either in the presence of recombinant mouse M-CSF (left) or human M-CSF
(right) for 7 days; cells were stained with F4/80 and CD11b antibodies.
illustrates flow cytometry analysis of BM cells that were isolated
and cultured either in the presence of recombinant mouse M-CSF (filled) or human M-
CSF (open) for 7 days; cells were stained with indicated surface markers.
illustrates flow cytometry of single cell suspensions of BM (top),
Spleen (middle) and Peripheral Blood (PB) from human CD34 cells engrafted in M-
m/m m/h h/h
CSF , M-CSF and M-CSF mice; staining is with CD45, CD14 and CD33 human
antibodies; cells that are human CD45 were pre-gated and discriminated based on
CD14 and CD33 expression.
illustrates relative frequencies of human CD45+ CD14+CD33+
cells of BM (top), spleen (middle) and peripheral blood (PB); absolute numbers of BM
cells were determined as average per animal (two tibia and fibula) and of peripheral
blood were determined per mL volume of blood; each group contains n=20 mice; each
symbol represents an individual mouse, horizontal bars indicate the mean values; data
are representative of 5 independent experiments
illustrates absolute frequencies of human CD45+ CD14+CD33+
cells of BM (top), spleen (middle) and peripheral blood (PB); absolute numbers of BM
cells were determined as average per animal (two tibia and fibula) and of peripheral
blood were determined per mL volume of blood; each group contains n=20 mice; each
symbol represents an individual mouse, horizontal bars indicate the mean values; data
are representative of 5 independent experiments.
illustrates flow cytometry analysis of stained cells from human
+ m/m m/h h/h
CD34 cells engrafted M-CSF , M-CSF and M-CSF mice bled after 12, 16 and 20
weeks of transplantation; cells were stained with CD45, CD14 and CD33 human
antibodies; cells that are human CD45 were pre-gated and discriminated based on
CD14 and CD33 expression.
+ + +
illustrates relative frequencies of human CD45 CD14 CD33
cells; each group contains n=10 mice; each symbol represents an individual mouse,
horizontal bars indicate the mean values; data are representative of 3 independent
experiments.
illustrates analysis of flow cytometry results from M-CSF , M-
m/h h/h +
CSF and M-CSF mice engrafted with human CD34 cells and 12 weeks after
transplantation, when mice were sacrificed and perfused with PBS; Liver (A), Lungs (B)
and Skin (C) were harvested and single cell suspensions were prepared; peritoneal
cavity cells (D) were collected by aspirating with PBS; cells were stained with human
CD45, CD14 and CD33 antibodies, and analyzed by flow cytometry; each symbol
represents an individual mouse, horizontal bars indicate the mean values; data are
representative of 3 independent experiments.
m/m m/h
illustrates results of LPS stimulation. (A) M-CSF and M-CSF
mice were engrafted with human CD34 cells and 12 weeks after transplantation, LPS
was injected i.p. and 48 hours later mice were sacrificed and the frequencies of human
+ + +
CD45 CD14 CD33 cells in the spleen were determined; PBS-injected mice served as
controls; each symbol represents an individual mouse, horizontal bars indicate mean
m/m m/h +
values. (B), (C) M-CSF and M-CSF mice were engrafted with human CD34 cells
and 12 weeks after transplantation, LPS was injected i.p. Six hours later, mice were bled
and the serum levels of human (right) and mouse (left) IL-6 and TNFα were quantified by
ELISA; PBS-injected mice served as controls; mean values of triplicate samples are
shown; error bars indicate ± SEM.
(for hTNFα) and 7B (for hIL-6) illustrate the ability of
monocytes/macrophages to secrete pro-inflammatory cytokines in vitro following LPS
+ + + +
stimulation. Human CD45 CD14 CD33 cells from the spleens of human CD34 cells-
m/m h/h
engrafted M-CSF and M-CSF mice were isolated after 12 weeks of transplantation;
+ + +
human CD45 CD14 CD33 cells obtained from the fetal liver served as controls; cells
were stimulated in vitro with LPS either for 24 or 48 hours, cell culture supernatants were
collected, and levels of human TNFα (A) and IL-6 (B) were quantified through ELISA;
mean values of triplicate samples are shown; error bars indicate ± SEM.
illustrates levels of interferon-α and –β mRNA in response to poly
+ + +
I:C stimulation. Human CD45 CD14 CD33 cells were stimulated poly I:C for either 6 or
12 hours and IFNα (left) and IFNβ (right) mRNA levels were quantified by real time PCR;
mean values of duplicate samples are shown; error bars indicate ± SEM
illustrates phagocytosis, migration, and activation properties of
+ + +
cells from engrafted mice. Human CD45 CD14 CD33 cells were isolated from
humanized mice and incubated with FITC-labeled bacteria at 37°C either for 30 or 60
minutes and measured by flow cytometry; cells incubated with FITC-labeled bacteria on
mice, dotted
ice served as controls. Open histograms represent cells from M-CSF
histograms represent cells from M-CSF mice, and filled histograms represent cells
from human fetal liver.
illustrates chemotaxis of cells in response to MIP3β. Human
+ + + m/m h/h
CD45 CD14 CD33 cells isolated from M-CSF mice, M-CSF , mice and human fetal
liver were kept in upper wells and medium containing MIP3β was added in to lower
wells; cells were incubated for either 30 or 60 minutes and the number of cells that
migrated from upper wells to lower wells was calculated and plotted; mean values of
duplicate samples are shown; error bars indicate ± SEM
illustrates enhanced activation of human
monocytes/macrophages from engrafted mice based on up-regulation of hCD40,
hCD80, hCD86, and hHLA-DR following in vitro LPS stimulation. Human
+ + + m/m h/h
CD45 CD14 CD33 cells isolated from M-CSF mice, M-CSF mice, and human fetal
liver were cultured either in the presence or in the absence of LPS; after 24 hours of
stimulation, cells were stained with indicated surface markers and measured by flow
cytometry. Open histograms represent cells from M-CSF mice, dotted histograms
mice, and filled histograms represent cells from human
represent cells from M-CSF
fetal liver.
provides a schematic representation of the mouse M-CSF locus
indicating the relative location of the exons 1-9, and final targeted allele with human M-
CSF gene.
,B illustrates the frequencies of the HSC compartment and
m/m h/m h/h
myeloid progenitor compartment in M-CSF , M-CSF , and M-CSF mice. BM cells
m/m m/h h/h
from M-CSF , M-CSF and M-CSF mice were stained with lineage, c-Kit, Sca1,
CD150, CD48, CD16/32, and CD34 antibodies, and analyzed by flow cytometry. (A)
Lineage cells (top) were gated and discriminated based on Sca1 and c-Kit expression
- + +
(middle). Lineage Sca1 c-Kit (LSK) cells were gated and further discriminated based on
CD150 and CD48 expression (bottom). (B) Lineage cells were pre-gated and
- + -
discriminated based on Sca1 and c-Kit expression (top). Lineage c-Kit Sca1 cells were
gated and further discriminated based on CD16/32 and CD34 expression (bottom).
DETAILED DESCRIPTION
Before the present methods and compositions are described, it is to be
understood that this invention is not limited to particular method or composition
described, as such may, of course, vary. It is also to be understood that the terminology
used herein is for the purpose of describing particular embodiments only, and is not
intended to be limiting, since the scope of the present invention will be limited only by the
appended claims.
Unless defined otherwise, 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. Although any methods and materials similar or equivalent
to those described herein can be used in the practice or testing of the present invention,
particular methods and materials are now described. All publications mentioned herein
are incorporated herein by reference to disclose and describe the methods and/or
materials in connection with which the publications are cited. It is understood that the
present disclosure supersedes any disclosure of an incorporated publication to the extent
there is a contradiction.
As will be apparent to those of skill in the art upon reading this disclosure,
each of the individual embodiments described and illustrated herein has discrete
components and features which may be readily separated from or combined with the
features of any of the other several embodiments without departing from the scope or
spirit of the present invention. Any recited method can be carried out in the order of
events recited or in any other order which is logically possible.
It must be noted that as used herein and in the appended claims, the
singular forms "a", "an", and "the" include plural referents unless the context clearly
dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such
cells and reference to "the peptide" includes reference to one or more peptides and
equivalents thereof, e.g. polypeptides, known to those skilled in the art, and so forth. The
term ‘comprising’ as used in this specification and claims means ‘consisting at least in
part of’. When interpreting statements in this specification and claims which includes the
‘comprising’, other features besides the features prefaced by this term in each statement
can also be present. Related terms such as ‘comprise’ and ‘comprised’ are to be
interpreted in similar manner.
The publications discussed herein are provided solely for their disclosure
prior to the filing date of the present application. Nothing herein is to be construed as an
admission that the present invention is not entitled to antedate such publication by virtue
of prior invention.
Genetically modified mice comprising a nucleic acid sequence encoding a
human M-CSF protein are described. Also described are genetically modified mice
comprising a nucleic acid sequence encoding a human M-CSF protein that have been
engrafted with human cells such as human hematopoietic cells, and methods for making
such engrafted mice. These mice find use in a number of applications, such as in
modeling human immune disease and pathogen infection; in in vivo screens for agents
that modulate hematopoietic cell development and/or activity, e.g. in a healthy or a
diseased state; in in vivo screens for agents that are toxic to hematopoietic cells; in in
vivo screens for agents that prevent against, mitigate, or reverse the toxic effects of toxic
agents on hematopoietic cells; in in vivo screens of human hematopoietic cells from an
individual to predict the responsiveness of an individual to a disease therapy, etc.
HUMANIZED M-CSF MICE
In some embodiments described herein, a humanized M-CSF mouse is
described. By a humanized M-CSF mouse, or “hM-CSF mouse”, it is meant a mouse
comprising a nucleic acid sequence that encodes a human M-CSF protein. By a human
M-CSF protein, it is a meant a protein that is human M-CSF or is substantially identical
to human M-CSF, e.g., it is 80% or more identical, 85% or more identical, 90% or more
identical, or 95% or more identical to human M-CSF, for example, 97%, 98%, or 99%
identical to human M-CSF. A nucleic acid sequence that encodes a human M-CSF
protein is, therefore, a polynucleotide that comprises coding sequence for a human M-
CSF protein, i.e. human M-CSF or a protein that is substantially identical to human M-
CSF. M-CSF (also known as CSF-1, for “colony stimulating factor 1”) is a cytokine that
controls the production, differentiation, and function of macrophages. Polypeptide
sequence for human M-CSF and the nucleic acid sequence that encodes for human M-
CSF may be found at Genbank Accession Nos. NM_000757.5 (variant 1), NM_172210.2
(variant 2), and NM_172212.2 (variant 4). The genomic locus encoding the human M-
CSF protein may be found in the human genome at Chromosome 1; NC_000001.10
(110453233-110472355). Protein sequence is encoded by exons 1 through 8 at this
locus, while exon 9 comprises untranslated sequence. As such, a nucleic acid
sequence comprising coding sequence for human M-CSF comprises one or more of
exons 1-8 of the human M-CSF gene. In some instances, the nucleic acid sequence also
comprises aspects of the genomic locus of the human M-CSF, e.g. introns, 3’ and/or 5’
untranslated sequence (UTRs). In some instances, the nucleic acid sequence
comprises whole regions of the human M-CSF genomic locus. In some instances, the
nucleic acid sequence comprises exon 2 of the human M-CSF genomic locus to 633 nt
downstream of noncoding exon 9.
In the humanized M-CSF mice of the subject application, the nucleic acid
sequence that encodes a human M-CSF protein is operably linked to one or more
regulatory sequences of the mouse M-CSF gene. Mouse M-CSF regulatory sequences
are those sequences of the mouse M-CSF genomic locus that regulate mouse M-CSF
expression, for example, 5’ regulatory sequences, e.g. the M-CSF promoter, M-CSF 5’
untranslated region (UTR), etc.; 3’ regulatory sequences, e.g. the 3’UTR; and
enhancers, etc. Mouse M-CSF is located on chromosome 3 at about positions
107,543,966-107,563,387, and the mouse M-CSF coding sequence may be found at
Genbank Accession Nos. NM_007778.4 (isoform 1), NM_001113529.1 (isoform 2), and
NM_001113530.1 (isoform 3). The regulatory sequences of mouse M-CSF are well
defined in the art, and may be readily identified using in silico methods, e.g. by referring
to the above Genbank Accession Nos. on the UCSC Genome Browser, on the world
wide web at genome.ucsc.edu, or by experimental methods as described below and in
the art, e.g., Abboud et al. (2003) Analysis of the Mouse CSF-1 Gene Promoter in a
Transgenic Mouse Model. J. Histochemistry and Cytochemistry 51(7):941-949, the
disclosure of which is incorporated herein by reference. In some instances, e.g. when
the nucleic acid sequence that encodes a human M-CSF protein is located at the mouse
M-CSF genomic locus, the regulatory sequences operably linked to the human CSF
coding sequence are endogenous, or native, to the mouse genome, i.e. they were
present in the mouse genome prior to integration of human nucleic acid sequences.
In some instances, the humanized M-CSF mouse is generated by the
random integration, or insertion, of human nucleic acid sequence encoding human M-
CSF protein or a fragment thereof, i.e. “human M-CSF nucleic acid sequence”, or
“human M-CSF sequence”, into the genome. Typically, in such embodiments, the
location of the nucleic acid sequence encoding a human M-CSF protein in the genome is
unknown. In other instances, the humanized M-CSF mouse is generated by the
targeted integration, or insertion, of human M-CSF nucleic acid sequence into the
genome, by, for example, homologous recombination. In homologous recombination, a
polynucleotide is inserted into the host genome at a target locus while simultaneously
removing host genomic material, e.g. 50 base pairs (bp) or more, 100 bp or more, 200
bp or more, 500 bp or more, 1 kB or more, 2 kB or more, 5 kB or more, 10 kB or more,
kB or more, 20 kB or more, or 50 kB or more of genomic material, from the target
locus. So, for example, in a humanized M-CSF mouse comprising nucleic acid sequence
that encodes a human M-CSF protein created by targeting human M-CSF nucleic acid
sequence to the mouse M-CSF locus, human M-CSF nucleic acid sequence may
replace some or all of the mouse sequence, e.g. exons and/or introns, at the M-CSF
locus. In some such instances, human M-CSF nucleic acid sequence is integrated into
the mouse M-CSF locus such that expression of the human M-CSF sequence is
regulated by the native, or endogenous, regulatory sequences at the mouse M-CSF
locus. In other words, the regulatory sequence(s) to which the nucleic acid sequence
encoding a human M-CSF protein is operably linked are the native M-CSF regulatory
sequences at the mouse M-CSF locus.
In some instances, the integration of human M-CSF sequence does not
affect the transcription of the gene into which the human M-CSF sequence has
integrated. For example, if the human M-CSF sequence integrates into coding
sequence as an intein, or the human M-CSF sequence comprises a 2A peptide, the
human M-CSF sequence will be transcribed and translated simultaneously with the gene
into which the human M-CSF sequence has integrated. In other instances, the
integration of the human M-CSF sequence interrupts the transcription of the gene into
which the human M-CSF sequence has integrated. For example, upon integration of the
human M-CSF sequence by homologous recombination, some or all of the coding
sequence at the integration locus may be removed, such that the human M-CSF
sequence is transcribed instead. In some such instances, the integration of human M-
CSF sequence creates a null mutation, and hence, a null allele. A null allele is a mutant
copy of a gene that completely lacks that gene's normal function. This can be the result
of the complete absence of the gene product (protein, RNA) at the molecular level, or
the expression of a non-functional gene product. At the phenotypic level, a null allele is
indistinguishable from a deletion of the entire locus.
In some instances, the humanized M-CSF mouse comprises one copy of
the nucleic acid sequence encoding a human M-CSF protein. For example, the mouse
may be heterozygous for the nucleic acid sequence. In other words, one allele at a
locus will comprise the nucleic acid sequence, while the other will be the endogenous
allele. For example, as discussed above, in some instances, human M-CSF nucleic acid
sequence is integrated into the mouse M-CSF locus such that it creates a null allele for
mouse M-CSF. In some such embodiments, the humanized M-CSF mouse may be
heterozygous for the nucleic acid sequence encoding, i.e. the humanized M-CSF mouse
comprises one null allele for mouse M-CSF (the allele comprising the nucleic acid
sequence) and one endogenous M-CSF allele (wild type or otherwise). In other words,
mouse, where “h” represents the allele comprising the human
the mouse is a M-CSF
sequence and “m” represents the endogenous allele. In other instances, the humanized
M-CSF comprises two copies of the nucleic acid sequence encoding a human M-CSF
protein. For example, the mouse may be homozygous for the nucleic acid sequence,
i.e. both alleles for a locus in the diploid genome will comprise the nucleic acid
sequence, ,i.e. the humanized M-CSF mouse comprises two null alleles for the mouse
M-CSF (the allele comprising the nucleic acid sequence). In other words, the mouse is a
M-CSF mouse.
Strikingly, humanized M-CSF mice, e.g. such as those described above,
h/h h/m
e.g. M-CSF and M-CSF mice, exhibit normal, or wild type, development and function
of macrophages and monocytes and tissues that develop from cells of the macrophage
lineage, e.g., bone. For example, humanized mice normal teeth and bone properties as
well as normal bone marrow content, myeloid cell frequencies in the bone marrow,
spleen and peripheral blood, and macrophage frequencies in the bone marrow and
spleen.
In some instances, the humanized M-CSF mouse comprises other
genetic modifications. For example, the humanized M-CSF mouse may comprise at
least one null allele for the Rag2 gene (“recombination activating gene 2”, the coding
sequence for which may be found at Genbank Accession No. 1.NM_009020.3). In some
embodiments, the humanized M-CSF mouse comprises two null alleles for Rag2. In
other words, the humanized M-CSF mouse is homozygous null for Rag2. As another
example, the humanized M-CSF mouse comprises at least one null allele for the IL2rg
gene (“interleukin 2 receptor, gamma”, also known as the common gamma chain, or γC,
the coding sequence for which may be found at Genbank Accession No.
1.NM_013563.3). In some embodiments, the humanized M-CSF mouse comprises two
null alleles for IL2rg. In other words, the humanized M-CSF mouse is homozygous null
for IL2rg. In some embodiments, the mouse comprises a null allele for both Rag2 and
-/- -/-
IL2rg, i.e. it is Rag2 IL2RG . Other genetic modifications are also contemplated. For
example, the humanized M-CSF mouse may comprise modifications in other genes
associated with the development and/or function of hematopoietic cells and the immune
system, e.g. the replacement of one or other mouse genes with nucleic acid sequence
encoding the human ortholog. Additionally or alternatively, the humanized M-CSF
mouse may comprise modifications in genes associated with the development and/or
function of other cells and tissues, e.g. genes associated with human disorders or
disease, or genes that, when modified in mice, provide for mouse models of human
disorders and disease.
-/- -
In some embodiments, the humanized M-CSF mouse, e.g. a Rag2 IL2rg
hM-CSF mouse or a sublethally irradiated hM-CSF mouse, is engrafted, or
transplanted, with cells. Cells may be mitotic cells or post-mitotic cells, and include such
cells of interest as pluripotent stem cells, e.g. ES cells, iPS cells, and embryonic germ
cells; and somatic cells, e.g. fibroblasts, hematopoietic cells, neurons, muscle cells,
bone cells, vascular endothelial cells, gut cells, and the like, and their lineage-restricted
progenitors and precursors. Cell populations of particular interest include those that
comprise hematopoietic stem or progenitor cells, which will contribute to or reconstitute
the hematopoietic system of the humanized M-CSF mouse, for example, peripheral
blood leukocytes, fetal liver cells, fetal bone, fetal thymus, fetal lymph nodes,
vascularized skin, artery segments, and purified hematopoietic stem cells, e.g.
mobilized HSCs or cord blood HSCs. Cells may be from any mammalian species, e.g.
murine, rodent, canine, feline, equine, bovine, ovine, primate, human, etc. Cells may be
from established cell lines or they may be primary cells, where “primary cells”, “primary
cell lines”, and “primary cultures” are used interchangeably herein to refer to cells and
cells cultures that have been derived from a subject and allowed to grow in vitro for a
limited number of passages, i.e. splittings, of the culture. For example, primary cultures
are cultures that may have been passaged 0 times, 1 time, 2 times, 4 times, 5 times, 10
times, or 15 times, but not enough times go through the crisis stage. Typically, the
primary cell lines described herein are maintained for fewer than 10 passages in vitro.
If the cells are primary cells, they may be harvest from an individual by
any convenient method. For example, cells, e.g. blood cells, e.g. leukocytes, may be
harvested by apheresis, leukocytapheresis, density gradient separation, etc. As another
example, cells, e.g. skin, muscle, bone marrow, spleen, liver, pancreas, lung, intestine,
stomach tissue, etc. may be harvested by biopsy. An appropriate solution may be used
for dispersion or suspension of the harvested cells. Such solution will generally be a
balanced salt solution, e.g. normal saline, PBS, Hank’s balanced salt solution, etc.,
conveniently supplemented with fetal calf serum or other naturally occurring factors, in
conjunction with an acceptable buffer at low concentration, generally from 5-25 mM.
Convenient buffers include HEPES, phosphate buffers, lactate buffers, etc.
In some instances, a heterogeneous population of cells will be
transplanted into the humanized mouse. In other instances, a population of cells that is
enriched for a particular type of cell, e.g. a progenitor cell, e.g. a hematopoietic
progenitor cell, will be engrafted into the humanized mouse. Enrichment of a cell
population of interest may be by any convenient separation technique. For example, the
cells of interest may be enriched by culturing methods. In such culturing methods,
particular growth factors and nutrients are typically added to a culture that promote the
survival and/or proliferation of one cell population over others. Other culture conditions
that affect survival and/or proliferation include growth on adherent or non-adherent
substrates, culturing for particular lengths of time, etc. Such culture conditions are well
known in the art. As another example, cells of interest may be enriched for by
separation the cells of interest from the initial population by affinity separation
techniques. Techniques for affinity separation may include magnetic separation using
magnetic beads coated with an affinity reagent, affinity chromatography, "panning" with
an affinity reagent attached to a solid matrix, e.g. plate, cytotoxic agents joined to an
affinity reagent or used in conjunction with an affinity reagent, e.g. complement and
cytotoxins, or other convenient technique. Techniques providing accurate separation
include fluorescence activated cell sorters, which can have varying degrees of
sophistication, such as multiple color channels, low angle and obtuse light scattering
detecting channels, impedance channels, etc. The cells may be selected against dead
cells by employing dyes associated with dead cells (e.g. propidium iodide). Any
technique may be employed which is not unduly detrimental to the viability of the cells of
interest.
For example, using affinity separation techniques, cells that are not the
cells of interest for transplantation may be depleted from the population by contacting
the population with affinity reagents that specifically recognize and selectively bind
markers that are not expressed on the cells of interest. For example, to enrich for a
population of hematopoietic progenitor cells, one might deplete cells expressing mature
hematopoietic cell markers. Additionally or alternatively, positive selection and
separation may be performed using by contacting the population with affinity reagents
that specifically recognize and selectively bind markers associated with hematopoietic
progenitor cells, e.g. CD34, CD133, etc. By "selectively bind" is meant that the molecule
binds preferentially to the target of interest or binds with greater affinity to the target than
to other molecules. For example, an antibody will bind to a molecule comprising an
epitope for which it is specific and not to unrelated epitopes. In some embodiments, the
affinity reagent may be an antibody, i.e. an antibody that is specific for CD34, CD133,
etc. In some embodiments, the affinity reagent may be a specific receptor or ligand for
CD34, CD133, etc., e.g. a peptide ligand and receptor; effector and receptor molecules,
a T-cell receptor specific for CD34, CD133, etc., and the like. In some embodiments,
multiple affinity reagents specific for the marker of interest may be used.
Antibodies and T cell receptors that find use as affinity reagents may be
monoclonal or polyclonal, and may be produced by transgenic animals, immunized
animals, immortalized human or animal B-cells, cells transfected with DNA vectors
encoding the antibody or T cell receptor, etc. The details of the preparation of antibodies
and their suitability for use as specific binding members are well-known to those skilled
in the art. Of particular interest is the use of labeled antibodies as affinity reagents.
Conveniently, these antibodies are conjugated with a label for use in separation. Labels
include magnetic beads, which allow for direct separation; biotin, which can be removed
with avidin or streptavidin bound to a support; fluorochromes, which can be used with a
fluorescence activated cell sorter; or the like, to allow for ease of separation of the
particular cell type. Fluorochromes that find use include phycobiliproteins, e.g.
phycoerythrin and allophycocyanins, fluorescein and Texas red. Frequently each
antibody is labeled with a different fluorochrome, to permit independent sorting for each
marker.
The initial population of cells are contacted with the affinity reagent(s) and
incubated for a period of time sufficient to bind the available cell surface antigens. The
incubation will usually be at least about 5 minutes and usually less than about 60
minutes. It is desirable to have a sufficient concentration of antibodies in the reaction
mixture, such that the efficiency of the separation is not limited by lack of antibody. The
appropriate concentration is determined by titration, but will typically be a dilution of
antibody into the volume of the cell suspension that is about 1:50 (i.e., 1 part antibody to
50 parts reaction volume), about 1:100, about 1:150, about 1:200, about 1:250, about
1:500, about 1:1000, about 1:2000, or about 1:5000. The medium in which the cells are
suspended will be any medium that maintains the viability of the cells. A preferred
medium is phosphate buffered saline containing from 0.1 to 0.5% BSA or 1-4% goat
serum. Various media are commercially available and may be used according to the
nature of the cells, including Dulbecco’s Modified Eagle Medium (dMEM), Hank's Basic
Salt Solution (HBSS), Dulbecco’s phosphate buffered saline (dPBS), RPMI, Iscove’s
medium, PBS with 5 mM EDTA, etc., frequently supplemented with fetal calf serum,
BSA, HSA, goat serum etc.
The cells in the contacted population that become labeled by the affinity
reagent are selected for by any convenient affinity separation technique, e.g. as
described above or as known in the art. Following separation, the separated cells may
be collected in any appropriate medium that maintains the viability of the cells, usually
having a cushion of serum at the bottom of the collection tube. Various media are
commercially available and may be used according to the nature of the cells, including
dMEM, HBSS, dPBS, RPMI, Iscove’s medium, etc., frequently supplemented with fetal
calf serum.
Compositions highly enriched for a cell type of interest, e.g. hematopoietic
cells, are achieved in this manner. The cells will be about 70%, about 75%, about 80%,
about 85% about 90% or more of the cell composition, about 95% or more of the
enriched cell composition, and will preferably be about 95% or more of the enriched cell
composition. In other words, the composition will be a substantially pure composition of
cells of interest.
The cells to be transplanted into the humanized M-CSF mouse, be they a
heterogeneous population of cells or an enriched population of cells, may be
transplanted immediately. Alternatively, the cells may be frozen at liquid nitrogen
temperatures and stored for long periods of time, being thawed and capable of being
reused. In such cases, the cells will usually be frozen in 10% DMSO, 50% serum, 40%
buffered medium, or some other such solution as is commonly used in the art to
preserve cells at such freezing temperatures, and thawed in a manner as commonly
known in the art for thawing frozen cultured cells. Additionally or alternatively, the cells
may be cultured in vitro under various culture conditions. Culture medium may be liquid
or semi-solid, e.g. containing agar, methylcellulose, etc. The cell population may be
conveniently suspended in an appropriate nutrient medium, such as Iscove's modified
DMEM or RPMI-1640, normally supplemented with fetal calf serum (about 5-10%),
L-glutamine, a thiol, particularly 2-mercaptoethanol, and antibiotics, e.g. penicillin and
streptomycin. The culture may contain growth factors to which the cells are responsive.
Growth factors, as defined herein, are molecules capable of promoting survival, growth
and/or differentiation of cells, either in culture or in the intact tissue, through specific
effects on a transmembrane receptor. Growth factors include polypeptides and
non-polypeptide factors.
The cells may be genetically modified prior to transplanting to the
humanized M-CSF mouse, e.g. to provide a selectable or traceable marker, to induce a
genetic defect in the cells (e.g. for disease modeling), to repair of a genetic defect or
ectopically express a gene in the cells (e.g. to determine if such modifications will impact
the course of a disease), etc. Cells may be genetically modified by transfection or
transduction with a suitable vector, homologous recombination, or other appropriate
technique, so that they express a gene of interest, or with an antisense mRNA, siRNA or
ribozymes to block expression of an undesired gene. Various techniques are known in
the art for the introduction of nucleic acids into target cells. To prove that one has
genetically modified the cells, various techniques may be employed. The genome of the
cells may be restricted and used with or without amplification. The polymerase chain
reaction; gel electrophoresis; restriction analysis; Southern, Northern, and Western blots;
sequencing; or the like, may all be employed. General methods in molecular and
cellular biochemistry for these and other purposes disclosed in this application can be
found in such standard textbooks as Molecular Cloning: A Laboratory Manual, 3rd Ed.
(Sambrook et al., Cold Spring Harbor Laboratory Press 2001); Short Protocols in
Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein
Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy
(Wagner et al. eds., Academic Press 1999); Viral Vectors (Kaplift & Loewy eds.,
Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic Press
1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle &
Griffiths, John Wiley & Sons 1998), the disclosures of which are incorporated herein by
reference. Reagents, cloning vectors, and kits for genetic manipulation referred to in this
disclosure are available from commercial vendors such as BioRad, Stratagene,
Invitrogen, Sigma-Aldrich, and ClonTech.
The cells may be transplanted in the humanized M-CSF mouse by any
convenient method, including, for example, intra-hepatic injection, tail-vein injection,
retro-orbital injection, and the like. Typically, about 0.5 x 10 - 2 x 10 pluripotent or
6 5
progenitor cells are transplanted, e.g. about 1 x 10 – 1 x 10 cells , or about 2 x 10 – 5
x 10 cells. In some instances, the mouse is sublethally irradiated prior to transplanting
the human cells. In other words, the mouse is exposed to a sublethal dose of radiation,
e.g. as described in the examples section below and as well-known in the art. The
engrafted humanized M-CSF mouse is then maintained under laboratory animal
husbandry conditions for at least 1 week, e.g. 1 week or more, or two weeks or more,
sometimes 4 weeks or more, and in some instances 6 weeks or more, to allow sufficient
reconstitution of the immune system with the engrafted cells.
As demonstrated in the examples section below, humanized M-CSF mice
demonstrate a significantly increased ability to engraft and maintain human
hematopoietic cells as compared to other mouse strains that have been developed for
this purpose and other M-CSF transgenic mice. For example, intra-hepatic transfer of
human fetal liver-derived hematopoietic stem and progenitor cells (CD34+) to newborn
mice results in more efficient differentiation and enhanced frequencies of human
monocytes/macrophages in bone marrow, spleen, peripheral blood, lungs, liver and the
peritoneal cavity. Significant proportions of human CD14+CD33+ cells are observed at
16-20 weeks. Specifically, humanized M-CSF mice engrafted with hematopoietic cells
demonstrate one or more, in some instances two or more, in some instances, three or
more, in some instances four or more, in some instances all of the following
characteristics: they express human M-CSF in bone marrow, spleen, blood, liver, brain,
lung, testis and kidney at a level comparable to expression of mouse M-CSF in a wild-
type mouse; exhibit a frequency of hCD14 CD33 cells of spleen that is 2- to 6-fold
higher than hCD14 CD33 in an engrafted mouse that does not express hM-CSF; exhibit
a frequency in hCD14 CD33 cells of peripheral blood that is 2- to 8-fold higher than
hCD14 CD33 in an engrafted mouse that does not express hM-CSF; exhibit a level of
hCD14 CD33 monocyte/macrophage lineage cells in blood of about 15 to about 40%;
exhibit a level of hCD14 CD33 monocyte/macrophage lineage cells in blood of about 5
to about 15% at about 20 weeks of age; exhibit a response to LPS injection that is about
1.5- to about 6-fold greater with respect to percentage of hCD14 CD33 cells in liver
than mice that lack a human M-CSF; exhibit an enhanced production of
+ + +
hCD14 CD33 hCD45 cells in spleen about 48 hours following LPS injection, wherein
the enhancement is about 2- to about 5-fold over an engrafted mouse that lacks hM-
CSF; exhibit an enhanced production of serum human IL-6 in response to LPS, wherein
the level of hIL-6 about 6 hours after LPS injection is enhanced about 2- to about 5-fold
over an engrafted mouse that lacks a hM-CSF; exhibit in vitro secretion by a monocyte
and/or macrophage upon LPS stimulation that is about 2- to 3-fold higher with respect to
hTNFα than an engrafted mouse that lacks a hM-CSF gene; exhibit in vitro secretion by
a monocyte and/or macrophage upon LPS stimulation that is about 2- to 4-fold higher
with respect to hIL-6 than an engrafted mouse that lacks a hM-CSF gene; exhibit in vitro
secretion by a monocyte and/or macrophage upon I:C stimulation that is about 3- to 6-
fold higher with respect to hIFNα than an engrafted mouse that lacks a hM-CSF gene;
exhibit in vitro secretion by a monocyte and/or macrophage upon I:C stimulation that is
about 2- to 3-fold higher with respect to hIFNβ than an engrafted mouse that lacks a hM-
CSF gene; exhibit enhanced phagocytosis as compared with a genetically modified and
engrafted mouse that lacks a hM-CSF gene; exhibit enhanced chemotaxis in vitro in
response to Mip3β as compared with a genetically modified engrafted mouse that lacks
a hM-CSF gene; and; exhibit upregulation in vitro of a co-stimulatory molecule in
response to LPS stimulation, wherein the co-stimulatory molecule is selected from
human CD40, human CD80, human CD86, human HLA-DR, and a combination thereof.
UTILITY
The humanized M-CSF mice and humanize M-CSF mice engrafted with
-/- -/-
human hematopoietic cells, e.g. engrafted Rag2 IL2rg hM-CSF mice, and optionally
other genetic modifications are useful in many applications. For example, these mice
provide a useful system for modeling human immune diseases and human pathogens.
For example, the subject mice are useful for modeling a human hematopoietic
malignancy that originates from an early human hematopoietic cell, e.g. from a human
hematopoietic stem or progenitor cell. As another example, the subject mice are useful
for studying human pathogens, e.g. viruses, fungi, and bacteria, that do not normally
infect mice.
One such example of a human pathogen that does not normally infect
mice is the causative agent of typhoid fever, S. typhi. Typhoid fever afflicts over 21
million people around the world—principally in the developing world—including about 400
cases/year in the United States. Typhoid fever has been treated with the drugs
amoxicillin, ampicillin, cefotaxime, ceftriaxone, ceftazidime, chloramphenicol,
ciprofloxacin, co-trimoxazole, ertapenem, imipenem, fluoroquinolones (e.g., ciprofloxacin,
gatifloxacin, ofloxacin), streptomycin, sulfadiazine, sulfamethoxazole, tetracycline, and
combinations thereof. Recurrent infections are common, which limits disease
management by antibiotic therapy. Further, multi-drug resistance is also prevalent with
S. typhi infections.
New therapeutics, new vaccines, and new ways of testing efficacy of
therapeutics and vaccines are needed. A mouse capable of being infected by S. typhi,
for example, would be useful to identify new therapeutics and new vaccines. New
therapeutics and new vaccines could be testing in such a mouse by, e.g., determining
the amount of S. typhi in the mouse (in blood or a given tissue) in response to treatment
with a putative anti-S. typhi agent, or by inoculating the mouse with a putative vaccine
followed by exposure to an infective administration of S. typhi, and observing any
change in infectivity due to inoculation by the putative vaccine as compared to a control
not inoculated with the vaccine but infected with S. typhi.
A humanized M-CSF mouse engrafted with human hematopoietic cells,
-/- -/-
e.g. a Rag2 IL2rg hM-CSF mouse, is useful for studying human pathogens, i.e.
pathogens that infect humans; the response of the human immune system to infection
by human pathogens; and the effectiveness of agents in protecting against and/or
treating infection by human pathogens. The pathogen may be a virus, a fungus, a
bacterium, etc. Non-limiting examples of viral pathogens include human or porcine or
avian influenza virus. Non-limiting examples of bacterial pathogens include
mycobacterium, e.g. Mycobacterium tuberculosis (M. tuberculosis) and enterobacterium,
e.g. Salmonella typhi (S. typhi).
For example, engrafted humanized M-CSF mice are useful as a non-
human animal model of S. typhi infection. By contrast, wild-type mice, and other known
immune-compromised mice (e.g., RAG1/RAG2 gene knockout mice), are not capable of
being infected by S. typhi. As discussed above, engrafted human M-CSF mice as
described herein display an enhanced engraftment of human cells as compared to an
engrafted mice that do not comprise a human M-CSF protein. This enhancement is
sufficient to maintain a productive S. typhi infection, that is, the S. typhi is able to
reproduce in the mouse, i.e. the infected mouse is able to harbor and reproduce S. typhi
in one or more of its cells. In a specific embodiment, the mouse is capable of
reproducing S. typhi at least a week, 10 days, two weeks, three weeks, or four weeks
following an initial introduction or infective exposure of S. typhi In other words, the
mouse is capable of maintaining a S. typhi titer or level in its blood or in at least one
tissue for at least a week, 10 days, two week, three weeks, or four weeks following an
infective exposure to S. typhi. Examples of methods for infecting mice with S. typhi and
for assessing infection may be found in, for example, US Published Application No.
2011/0200982, the disclosure of which is incorporated herein by reference.
As another example, engrafted humanized M-CSF mice, e.g. engrafted
-/- -/-
Rag2 IL2rg hM-CSF mice, are useful as a non-human animal model of infection by M.
tuberculosis. The enhanced engraftment of human hematopoietic cells in mice
comprising a nucleic acid that encodes human M-CSF protein is sufficient to maintain a
productive M. tuberculosis infection, that is, the M. tuberculosis is able to reproduce in
the mouse, i.e. the infected mouse is able to harbor and reproduce M. tuberculosis in
one or more of its cells. In some such embodiments, the mouse mounts an anti-
mycobacterial immune response to a human pathogenic mycobacterium, wherein the
response comprises formation of a granuloma mediated by human immune cells and
that comprises a human immune cell. In some such embodiments, the granuloma is a
lung granuloma. In some such embodiments, the granuloma is a well-defined
granuloma. Examples of methods for infecting mice with M. tuberculosis and for
assessing infection may be found in, for example, US Published Application No.
2011/0200982, the disclosure of which is incorporated herein by reference.
Other examples of human pathogens that do not infect a mouse
expressing human M-CSF and in some instances, one or more other genetic
modifications e.g. as described herein, or that infect wild-type mice, wherein the wild-
type mouse following infection does not model an immune response that a human
mounts in response to the pathogen, will be well-known to the ordinarily skilled artisan.
Such mouse models of pathogen infection are useful in research, e.g. to
better understand the progression of human infection. Such mouse models of infection
are also useful in drug discovery, e.g. to identify candidate agents that protect against or
treat infection.
Humanized M-CSF mice engrafted with human hematopoietic cells provide a
useful system for screening candidate agents for other desired activities in vivo as well,
for example, for agents that are able to modulate (i.e., promote or suppress)
hematopoietic cell development and/or activity, e.g. the activity of B cells, T cells, NK
cells, macrophages, neutrophils, eosinophils, basophils, etc., e.g. in a healthy or a
diseased state, e.g. to identify novel therapeutics and/or develop a better understanding
of the molecular basis of the development and function of the immune system; for
agents that are toxic to hematopoietic cells, e.g. B cells, T cells, NK cells, macrophages,
neutrophils, eosinophils, basophils, etc., and progenitors thereof; and for agents that
prevent against, mitigate, or reverse the toxic effects of toxic agents on hematopoietic
cells, e.g. B cells, T cells, NK cells, macrophages, neutrophils, eosinophils, basophils,
etc., and progenitors thereof; etc. As yet another example, the genetically modified mice
described herein provide a useful system for predicting the responsiveness of an
individual to a disease therapy, e.g. by providing an in vivo platform for screening the
responsiveness of an individual’s immune system to an agent, e.g. a therapeutic agent,
to predict the responsiveness of an individual to that agent.
In screening assays for biologically active agents, humanized M-CSF
-/- -/-
mice, e.g. Rag2 IL2rg hM-CSF mice, that have been engrafted with human
hematopoietic cells and in some instances, infected with human pathogens, or cells to be
engrafted into a humanized M-CSF mouse, are contacted with a candidate agent of
interest and the effect of the candidate agent is assessed by monitoring one or more
output parameters. These output parameters may be reflective of the viability of the
cells, e.g. the total number of hematopoietic cells or the number of cells of a particular
hematopoietic cell type, or of the apoptotic state of the cells, e.g. the amount of DNA
fragmentation, the amount of cell blebbing, the amount of phosphatidylserine on the cell
surface, and the like by methods that are well known in the art. Alternatively or
additionally, the output parameters may be reflective of the differentiation capacity of the
cells, e.g. the proportions of differentiated cells and differentiated cell types. Alternatively
or additionally, the output parameters may be reflective of the function of the cells, e.g.
the cytokines and chemokines produced by the cells, the ability of the cells to home to
and extravasate to a site of challenge, the ability of the cells to modulate, i.e. promote or
suppress, the activity of other cells in vitro or in vivo, etc. Other output parameters may
be reflective of the extent of pathogen infection in the animal, e.g. the titer of pathogen in
the mouse, the presence of granuloma in the mouse, etc.
Parameters are quantifiable components of cells, particularly components
that can be accurately measured, desirably in a high throughput system. A parameter
can be any cell component or cell product including cell surface determinant, receptor,
protein or conformational or posttranslational modification thereof, lipid, carbohydrate,
organic or inorganic molecule, nucleic acid, e.g. mRNA, DNA, etc. or a portion derived
from such a cell component or combinations thereof. While most parameters will provide
a quantitative readout, in some instances a semi-quantitative or qualitative result will be
acceptable. Readouts may include a single determined value, or may include mean,
median value or the variance, etc. Characteristically a range of parameter readout values
will be obtained for each parameter from a multiplicity of the same assays. Variability is
expected and a range of values for each of the set of test parameters will be obtained
using standard statistical methods with a common statistical method used to provide
single values.
Candidate agents of interest for screening include known and unknown
compounds that encompass numerous chemical classes, primarily organic molecules,
which may include organometallic molecules, inorganic molecules, genetic sequences,
vaccines, antibiotics or other agents suspected of having antibiotic properties, peptides,
polypeptides, antibodies, agents that have been approved pharmaceutical for use in a
human, etc. An important embodiment is to evaluate candidate drugs, including toxicity
testing; and the like.
Candidate agents include organic molecules comprising functional groups
necessary for structural interactions, particularly hydrogen bonding, and typically include
at least an amine, carbonyl, hydroxyl or carboxyl group, frequently at least two of the
functional chemical groups. The candidate agents often comprise cyclical carbon or
heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or
more of the above functional groups. Candidate agents are also found among
biomolecules, including peptides, polynucleotides, saccharides, fatty acids, steroids,
purines, pyrimidines, derivatives, structural analogs or combinations thereof. Included are
pharmacologically active drugs, genetically active molecules, etc. Compounds of interest
include chemotherapeutic agents, hormones or hormone antagonists, etc. Exemplary
suitable pharmaceutical agents are those described in, "The Pharmacological Basis of
Therapeutics," Goodman and Gilman, McGraw-Hill, New York, N.Y., (1996), Ninth
edition. Also included are toxins, and biological and chemical warfare agents, for
example see Somani, S. M. (Ed.), "Chemical Warfare Agents," Academic Press, New
York, 1992).
Candidate agents of interest for screening also include nucleic acids, for
example, nucleic acids that encode siRNA, shRNA, antisense molecules, or miRNA, or
nucleic acids that encode polypeptides. Many vectors useful for transferring nucleic
acids into target cells are available. The vectors may be maintained episomally, e.g. as
plasmids, minicircle DNAs, virus-derived vectors such cytomegalovirus, adenovirus, etc.,
or they may be integrated into the target cell genome, through homologous
recombination or random integration, e.g. retrovirus derived vectors such as MMLV, HIV-
1, ALV, etc. Vectors may be provided directly to the subject cells. In other words, the
pluripotent cells are contacted with vectors comprising the nucleic acid of interest such
that the vectors are taken up by the cells.
Methods for contacting cells, e.g. cells in culture or cells in a mouse, with
nucleic acid vectors, such as electroporation, calcium chloride transfection, and
lipofection, are well known in the art. Alternatively, the nucleic acid of interest may be
provided to the cells via a virus. In other words, the cells are contacted with viral
particles comprising the nucleic acid of interest. Retroviruses, for example, lentiviruses,
are particularly suitable to the method described herein. Commonly used retroviral
vectors are “defective”, i.e. unable to produce viral proteins required for productive
infection. Rather, replication of the vector requires growth in a packaging cell line. To
generate viral particles comprising nucleic acids of interest, the retroviral nucleic acids
comprising the nucleic acid are packaged into viral capsids by a packaging cell line.
Different packaging cell lines provide a different envelope protein to be incorporated into
the capsid, this envelope protein determining the specificity of the viral particle for the
cells. Envelope proteins are of at least three types, ecotropic, amphotropic and
xenotropic. Retroviruses packaged with ecotropic envelope protein, e.g. MMLV, are
capable of infecting most murine and rat cell types, and are generated by using ecotropic
packaging cell lines such as BOSC23 (Pear et al. (1993) P.N.A.S. 90:8392-8396).
Retroviruses bearing amphotropic envelope protein, e.g. 4070A (Danos et al, supra.), are
capable of infecting most mammalian cell types, including human, dog and mouse, and
are generated by using amphotropic packaging cell lines such as PA12 (Miller et al.
(1985) Mol. Cell. Biol. 5:431-437); PA317 (Miller et al. (1986) Mol. Cell. Biol. 6:2895-
2902); GRIP (Danos et al. (1988) PNAS 85:6460-6464). Retroviruses packaged with
xenotropic envelope protein, e.g. AKR env, are capable of infecting most mammalian cell
types, except murine cells. The appropriate packaging cell line may be used to ensure
that the cells of interest—in some instance, the engrafted cells, in some instance, the
cells of the host, i.e. the humanized M-CSF--are targeted by the packaged viral particles.
Vectors used for providing nucleic acid of interest to the subject cells will
typically comprise suitable promoters for driving the expression, that is, transcriptional
activation, of the nucleic acid of interest. This may include ubiquitously acting promoters,
for example, the CMV-b-actin promoter, or inducible promoters, such as promoters that
are active in particular cell populations or that respond to the presence of drugs such as
tetracycline. By transcriptional activation, it is intended that transcription will be
increased above basal levels in the target cell by at least about 10 fold, by at least about
100 fold, more usually by at least about 1000 fold. In addition, vectors used for providing
reprogramming factors to the subject cells may include genes that must later be
removed, e.g. using a recombinase system such as Cre/Lox, or the cells that express
them destroyed, e.g. by including genes that allow selective toxicity such as herpesvirus
TK, bcl-xs, etc
Candidate agents of interest for screening also include polypeptides. Such
polypeptides may optionally be fused to a polypeptide domain that increases solubility of
the product. The domain may be linked to the polypeptide through a defined protease
cleavage site, e.g. a TEV sequence, which is cleaved by TEV protease. The linker may
also include one or more flexible sequences, e.g. from 1 to 10 glycine residues. In some
embodiments, the cleavage of the fusion protein is performed in a buffer that maintains
solubility of the product, e.g. in the presence of from 0.5 to 2 M urea, in the presence of
polypeptides and/or polynucleotides that increase solubility, and the like. Domains of
interest include endosomolytic domains, e.g. influenza HA domain; and other
polypeptides that aid in production, e.g. IF2 domain, GST domain, GRPE domain, and
the like. Additionally or alternatively, such polypeptides may be formulated for improved
stability. For example, the peptides may be PEGylated, where the polyethyleneoxy
group provides for enhanced lifetime in the blood stream. The polypeptide may be fused
to another polypeptide to provide for added functionality, e.g. to increase the in vivo
stability. Generally such fusion partners are a stable plasma protein, which may, for
example, extend the in vivo plasma half-life of the polypeptide when present as a fusion,
in particular wherein such a stable plasma protein is an immunoglobulin constant domain.
In most cases where the stable plasma protein is normally found in a multimeric form,
e.g., immunoglobulins or lipoproteins, in which the same or different polypeptide chains
are normally disulfide and/or noncovalently bound to form an assembled multichain
polypeptide, the fusions herein containing the polypeptide also will be produced and
employed as a multimer having substantially the same structure as the stable plasma
protein precursor. These multimers will be homogeneous with respect to the polypeptide
agent they comprise, or they may contain more than one polypeptide agent.
The candidate polypeptide agent may be produced from eukaryotic cells,
or may be produced by prokaryotic cells. It may be further processed by unfolding, e.g.
heat denaturation, DTT reduction, etc. and may be further refolded, using methods
known in the art. Modifications of interest that do not alter primary sequence include
chemical derivatization of polypeptides, e.g., acylation, acetylation, carboxylation,
amidation, etc. Also included are modifications of glycosylation, e.g. those made by
modifying the glycosylation patterns of a polypeptide during its synthesis and processing
or in further processing steps; e.g. by exposing the polypeptide to enzymes which affect
glycosylation, such as mammalian glycosylating or deglycosylating enzymes. Also
embraced are sequences that have phosphorylated amino acid residues, e.g.
phosphotyrosine, phosphoserine, or phosphothreonine. The polypeptides may have
been modified using ordinary molecular biological techniques and synthetic chemistry so
as to improve their resistance to proteolytic degradation or to optimize solubility
properties or to render them more suitable as a therapeutic agent. Analogs of such
polypeptides include those containing residues other than naturally occurring L-amino
acids, e.g. D-amino acids or non-naturally occurring synthetic amino acids. D-amino
acids may be substituted for some or all of the amino acid residues.
The candidate polypeptide agent may be prepared by in vitro synthesis,
using conventional methods as known in the art. Various commercial synthetic
apparatuses are available, for example, automated synthesizers by Applied Biosystems,
Inc., Beckman, etc. By using synthesizers, naturally occurring amino acids may be
substituted with unnatural amino acids. The particular sequence and the manner of
preparation will be determined by convenience, economics, purity required, and the like.
Alternatively, the candidate polypeptide agent may be isolated and purified in accordance
with conventional methods of recombinant synthesis. A lysate may be prepared of the
expression host and the lysate purified using HPLC, exclusion chromatography, gel
electrophoresis, affinity chromatography, or other purification technique. For the most
part, the compositions which are used will comprise at least 20% by weight of the desired
product, more usually at least about 75% by weight, preferably at least about 95% by
weight, and for therapeutic purposes, usually at least about 99.5% by weight, in relation
to contaminants related to the method of preparation of the product and its purification.
Usually, the percentages will be based upon total protein.
In some cases, the candidate polypeptide agents to be screened are
antibodies. The term “antibody” or “antibody moiety” is intended to include any
polypeptide chain-containing molecular structure with a specific shape that fits to and
recognizes an epitope, where one or more non-covalent binding interactions stabilize the
complex between the molecular structure and the epitope. The specific or selective fit of
a given structure and its specific epitope is sometimes referred to as a “lock and key” fit.
The archetypal antibody molecule is the immunoglobulin, and all types of
immunoglobulins, IgG, IgM, IgA, IgE, IgD, etc., from all sources, e.g. human, rodent,
rabbit, cow, sheep, pig, dog, other mammal, chicken, other avians, etc., are considered
to be “antibodies.” Antibodies utilized herein may be either polyclonal antibodies or
monoclonal antibodies. Antibodies are typically provided in the media in which the cells
are cultured.
Candidate agents may be obtained from a wide variety of sources
including libraries of synthetic or natural compounds. For example, numerous means are
available for random and directed synthesis of a wide variety of organic compounds,
including biomolecules, including expression of randomized oligonucleotides and
oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial,
fungal, plant and animal extracts are available or readily produced. Additionally, natural
or synthetically produced libraries and compounds are readily modified through
conventional chemical, physical and biochemical means, and may be used to produce
combinatorial libraries. Known pharmacological agents may be subjected to directed or
random chemical modifications, such as acylation, alkylation, esterification, amidification,
etc. to produce structural analogs.
Candidate agents are screened for biological activity by administering the
agent to at least one and usually a plurality of samples, sometimes in conjunction with
samples lacking the agent. The change in parameters in response to the agent is
measured, and the result evaluated by comparison to reference cultures, e.g. in the
presence and absence of the agent, obtained with other agents, etc. In instances in
which a screen is being performed to identify candidate agents that will prevent, mitigate
or reverse the effects of a toxic agent, the screen is typically performed in the presence
of the toxic agent, where the toxic agent is added at the time most appropriate to the
results to be determined. For example, in cases in which the protective/preventative
ability of the candidate agent is tested, the candidate agent may be added before the
toxic agent, simultaneously with the candidate agent, or subsequent to treatment with the
candidate agent. As another example, in cases in which the ability of the candidate
agent to reverse the effects of a toxic agent is tested, the candidate agent may be added
subsequent to treatment with the candidate agent. As mentioned above, in some
instances, the sample is the humanized M-CSF mouse that has been engrafted with
cells, i.e. candidate agent provided to the humanized M-CSF mouse that has been
engrafted with cells. In some instances, the sample is the cells to be engrafted, i.e. the
candidate agent is provided to cells prior to transplantation.
If the candidate agent is to be administered directly to the mouse, the
agent may be administered by any of a number of well-known methods in the art for the
administration of peptides, small molecules and nucleic acids to mice. For example, the
agent may be administered orally, mucosally, topically, intradermally, or by injection, e.g.
intraperitoneal, subcutaneous, intramuscular, intravenous, or intracranial injection, and
the like. The agent may be administered in a buffer, or it may be incorporated into any of
a variety of formulations, e.g. by combination with appropriate pharmaceutically
acceptable vehicle. "Pharmaceutically acceptable vehicles" may be vehicles approved
by a regulatory agency of the Federal or a state government or listed in the U.S.
Pharmacopeia or other generally recognized pharmacopeia for use in mammals, such as
humans. The term "vehicle" refers to a diluent, adjuvant, excipient, or carrier with which a
compound described herein is formulated for administration to a mammal. Such
pharmaceutical vehicles can be lipids, e.g. liposomes, e.g. liposome dendrimers; liquids,
such as water and oils, including those of petroleum, animal, vegetable or synthetic
origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like, saline; gum
acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition,
auxiliary, stabilizing, thickening, lubricating and coloring agents may be used.
Pharmaceutical compositions may be formulated into preparations in solid, semi-solid,
liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments,
solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols. The
agent may be systemic after administration or may be localized by the use of regional
administration, intramural administration, or use of an implant that acts to retain the
active dose at the site of implantation. The active agent may be formulated for
immediate activity or it may be formulated for sustained release. For some conditions,
particularly central nervous system conditions, it may be necessary to formulate agents
to cross the blood-brain barrier (BBB). One strategy for drug delivery through the blood-
brain barrier (BBB) entails disruption of the BBB, either by osmotic means such as
mannitol or leukotrienes, or biochemically by the use of vasoactive substances such as
bradykinin. A BBB disrupting agent can be co-administered with the agent when the
compositions are administered by intravascular injection. Other strategies to go through
the BBB may entail the use of endogenous transport systems, including Caveolin-1
mediated transcytosis, carrier-mediated transporters such as glucose and amino acid
carriers, receptor-mediated transcytosis for insulin or transferrin, and active efflux
transporters such as p-glycoprotein. Active transport moieties may also be conjugated to
the therapeutic compounds for use as described herein to facilitate transport across the
endothelial wall of the blood vessel. Alternatively, drug delivery of agents behind the BBB
may be by local delivery, for example by intrathecal delivery, e.g. through an Ommaya
reservoir (see e.g. US Patent Nos. 5,222,982 and 5385582, incorporated herein by
reference); by bolus injection, e.g. by a syringe, e.g. intravitreally or intracranially; by
continuous infusion, e.g. by cannulation, e.g. with convection (see e.g. US Application
No. 20070254842, incorporated here by reference); or by implanting a device upon which
the agent has been reversably affixed (see e.g. US Application Nos. 20080081064 and
20090196903, incorporated herein by reference).
If the agent(s) are provided to cells prior to transplantation, the agents are
conveniently added in solution, or readily soluble form, to the medium of cells in culture.
The agents may be added in a flow-through system, as a stream, intermittent or
continuous, or alternatively, adding a bolus of the compound, singly or incrementally, to
an otherwise static solution. In a flow-through system, two fluids are used, where one is a
physiologically neutral solution, and the other is the same solution with the test
compound added. The first fluid is passed over the cells, followed by the second. In a
single solution method, a bolus of the test compound is added to the volume of medium
surrounding the cells. The overall concentrations of the components of the culture
medium should not change significantly with the addition of the bolus, or between the two
solutions in a flow through method.
A plurality of assays may be run in parallel with different agent
concentrations to obtain a differential response to the various concentrations. As known
in the art, determining the effective concentration of an agent typically uses a range of
concentrations resulting from 1:10, or other log scale, dilutions. The concentrations may
be further refined with a second series of dilutions, if necessary. Typically, one of these
concentrations serves as a negative control, i.e. at zero concentration or below the level
of detection of the agent or at or below the concentration of agent that does not give a
detectable change in the phenotype.
An analysis of the response of cells in the humanized M-CSF mouse to
the candidate agent may be performed at any time following treatment with the agent.
For example, the cells may be analyzed 1, 2, or 3 days, sometimes 4, 5, or 6 days,
sometimes 8, 9, or 10 days, sometimes 14 days, sometimes 21 days, sometimes 28
days, sometimes 1 month or more after contact with the candidate agent, e.g. 2 months,
4 months, 6 months or more. In some embodiments, the analysis comprises analysis at
multiple time points. The selection of the time point(s) for analysis will be based upon the
type of analysis to be performed, as will be readily understood by the ordinarily skilled
artisan.
The analysis may comprise measuring any of the parameters described
herein or known in the art for measuring cell viability, cell proliferation, cell identity, cell
morphology, and cell function, particularly as they may pertain to cells of the immune
cells. For example, flow cytometry may be used to determine the total number of
hematopoietic cells or the number of cells of a particular hematopoietic cell type.
Histochemistry or immunohistochemistry may be performed to determine the apoptotic
state of the cells, e.g. terminal deoxynucleotidyl transferase dUTP nick end labeling
(TUNEL) to measure DNA fragmentation, or immunohistochemistry to detect Annexin V
binding to phosphatidylserine on the cell surface. Flow cytometry may also be employed
to assess the proportions of differentiated cells and differentiated cell types, e.g. to
determine the ability of hematopoietic cells to differentiate in the presence of agent.
ELISAs, Westerns, and Northern blots may be performed to determine the levels of
cytokines, chemokines, immunoglobulins, etc. expressed in the engrafted humanized M-
CSF mice, e.g. to assess the function of the engrafted cells. In vivo assays to test the
function of immune cells, as well as assays relevant to particular diseases or disorders of
interest such as diabetes, autoimmune disease, graft v. host disease, AMD, etc. may
also be performed. See, e.g. Current Protocols in Immunology (Richard Coico, ed. John
Wiley & Sons, Inc. 2012) and Immunology Methods Manual (I. Lefkovits ed., Academic
Press 1997), the disclosures of which are incorporated herein by reference.
So, for example, a method is described for determining the effect of an
agent on a human pathogen, comprising exposing an engrafted humanized M-CSF
-/- -/-
mouse, e.g. an engrafted Rag2 IL2rg hM-CSF mouse, to an effective amount of a
human pathogen, the effective amount of a pathogen being the amount of pathogen
required to produce an infection in the mouse; allowing the pathogen to infect the
mouse; measuring a parameter of the infection over time in the presence of the agent;
and comparing that measurement to the measurement from an engrafted humanized M-
CSF mouse not exposed to the agent. The agent is determined to be an antipathogenic,
e.g. anti-S. typhi, agent if it reduces the amount of the agent in blood or a tissue of the
mouse by at least half following a single administration or two or more administrations of
the agent over a selected period of time.
As another example, a method is described for determining if a pathogen
isolate or strain of interest is drug resistant, e.g. multidrug resistant. In these methods,
-/- -/-
an engrafted humanized M-CSF mouse, e.g. an engrafted Rag2 IL2rg hM-CSF
mouse, is exposed to an effective amount of a human pathogen isolate or strain of
interest, the effective amount of the pathogen being the amount of pathogen required to
produce an infection in the mouse; the pathogen is allowed to infect the mouse; a
parameter of the infection, e.g. the titer of the isolate or strain of interest in the blood or
tissue of the mouse, the ability of the isolate or strain of interest to maintain an infection
in the mouse, or the ability of the isolate or strain of interest to reproduce in the mouse at
a point in time after administration of the drug, is measured in the presence of the drug;
and that measurement is compared to the measurement from an engrafted humanized
M-CSF mouse infected with pathogen not exposed to the agent. Examples of drugs of
interest include amoxicillin, ampicillin, cefotaxime, ceftriaxone, ceftazidime,
chloramphenicol, ciprofloxacin, co-trimoxazole, ertapenem, imipenem, fluoroquinolones
(e.g., ciprofloxacin, gatifloxacin, ofloxacin), streptomycin, sulfadiazine, sulfamethoxazole,
tetracycline, and a combination thereof. In a specific embodiment, the administration of
the drug or combination of drugs is at least a week, 10 days, two week, three weeks, or
four weeks after an infection-producing exposure to the isolate or strain of interest.
Other examples of uses for the subject mice are described elsewhere
herein. Additional applications of the genetically modified and engrafted mice described
in this disclosure will be apparent to those skilled in the art upon reading this disclosure.
REAGENTS, DEVICES AND KITS
Also described are reagents, devices and kits thereof for practicing one or
more of the above-described methods. The subject reagents, devices and kits thereof
may vary greatly.
In some embodiments, the reagents or kits will comprise one or more
agents for use in the methods described. For example, the kit may comprise a
humanized M-CSF mouse. The kit may comprise reagents for breeding humanized M-
CSF mice, e.g. primers and, in some instances, reagents for genotyping humanized M-
CSF mice. The kit may comprise human hematopoietic cells or an enriched population
of human hematopoietic progenitor cells for transplantation into the humanized M-CSF
mouse, or reagents for preparing a population of hematopoietic cells or an enriched
population of hematopoietic cells from a human for transplantation into a humanized M-
CSF mouse. Other reagents may include reagents for determining the viability and/or
function of hematopoietic cells, e.g. in the presence/absence of candidate agent, e.g.
one or more antibodies that are specific for markers expressed by different types of
hematopoietic cells, or reagents for detecting particular cytokines, chemokine, etc..
Other reagents may include culture media, culture supplements, matrix compositions,
and the like.
In addition to the above components, the subject kits will further include
instructions for practicing the subject methods. These instructions may be present in the
subject kits in a variety of forms, one or more of which may be present in the kit. One
form in which these instructions may be present is as printed information on a suitable
medium or substrate, e.g., a piece or pieces of paper on which the information is printed,
in the packaging of the kit, in a package insert, etc. Yet another means would be a
computer readable medium, e.g., diskette, CD, etc., on which the information has been
recorded. Yet another means that may be present is a website address which may be
used via the internet to access the information at a removed site. Any convenient means
may be present in the kits.
EXAMPLES
The following examples are put forth so as to provide those of ordinary
skill in the art with a complete disclosure and description of how to make and use the
present invention, and are not intended to limit the scope of what the inventors regard as
their invention nor are they intended to represent that the experiments below are all or
the only experiments performed. Efforts have been made to ensure accuracy with
respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors
and deviations should be accounted for. Unless indicated otherwise, parts are parts by
weight, molecular weight is weight average molecular weight, temperature is in degrees
Centigrade, and pressure is at or near atmospheric.
Colony Stimulating Factor- 1(CSF-1) or Macrophage Colony Stimulating
Factor (M-CSF) is one of the early cytokines that was discovered to promote
hematopoiesis. In the hematopoietic system, M-CSF is believed to act specifically on
myeloid progenitors, starting from the common myeloid progenitor (CMP) stage, and to
favor the differentiation of CMPs into the monocyte/macrophage lineage (Sherr, C.J. et
al. (1988) Macrophage colony-stimulating factor, CSF-1, and its proto-oncogene-
encoded receptor, Cold Spring Harb. Symp. Quant. Biol. 53 Pt 1:521-530). In addition,
M-CSF is necessary for the survival, adhesion and motility of macrophages (Pixley, F.J.,
and Stanley, E.R. (2004) CSF-1 regulation of the wandering macrophage: complexity in
action, Trends Cell Biol. 14:628-638; Socolovsky, M. et al. (1998) Cytokines in
hematopoiesis: specificity and redundancy in receptor function, Adv. Protein Chem.
52:141-198; Stanley, E.R. et al. (1997) Biology and action of colony--stimulating factor-
1, Mol. Reprod. Dev. 1997;46:4-10). Other than its key role in myeloid differentiation,
M-CSF is vital for the differentiation of osteoclasts, for the differentiation, survival and
proliferation of the cells of female reproductive tract, and for the formation of placenta
(Pixley et al. (2004); Socolovsky et al. (1998); Stanley et al. (1997)). M-CSF is produced
by a variety of cells including fibroblasts, bone marrow (BM) stromal cells, activated T
cells and macrophages, and secretory epithelial cells. M-CSF signals through the M-
CSF receptor (Fms; CD115) and ligation of its receptor by M-CSF results in tyrosine
phosphorylation of Fms and subsequent phosphorylation of several host cell proteins,
such as Grb2, Shc, Sos1 and p85 (Pixley et al. (2004); Stanley et al. (1997);
Rohrschneider, L.R. et al. (1997) Growth and differentiation signals regulated by the M-
CSF receptor, Mol. Reprod. Dev. 46:96-103; Yeung, Y.G. and Stanley, E.R. (2003)
Proteomic approaches to the analysis of early events in colony-stimulating factor-1
signal transduction, Mol. Cell. Proteomics 2:1143-1155).
The inventors hypothesized that the defective human myeloid
differentiation in the humanized mice might be due to the lack of specific signals that
promote myeloid differentiation. To validate this, the inventors engineered a new
generation of humanized mice to secrete human M-CSF at physiological levels from the
appropriate tissues. Analysis of these humanized M-CSF mice revealed normal
expression, both qualitatively and quantitatively, of human M-CSF. Analysis of
humanized M-CSF mice engrafted with human CD34 cells indicated augmented
frequencies of human monocytes/macrophages in various tissues. Furthermore, human
monocytes/macrophages obtained from these mice exhibited enhanced functional
properties.
Humanized M-CSF mice described herein show augmented frequencies
and functions of human myeloid cells. Insertion of human M-CSF into the mouse M-CSF
locus of Balb/c mice deficient for recombination activating gene 2 (Rag2; Genbank
Accession No. 1.NM_009020.3) and gamma chain (γc, also known as “Interleukin 2
receptor, gamma chain” or IL2RG; Genbank Accession No. 1.NM_013563.3) (Balb/c
-/- -/-
γc mice) resulted in faithful expression of human M-CSF in these mice both
Rag2
qualitatively and quantitatively. Intra-hepatic transfer of human fetal liver-derived
+ h/h
hematopoietic stem and progenitor cells (CD34 ) in humanized M-CSF (M-CSF )
newborn pups resulted in more efficient differentiation and enhanced frequencies of
human monocytes/macrophages in the bone marrow, spleen, and peripheral blood. In
addition, M-CSF mice exhibited sustained abilities to support human
monocyte/macrophage differentiation even after 20 weeks of transplantation. Moreover,
M-CSF mice contain resident human monocytes/macrophages within various tissues,
including liver and lungs, unlike control unmodified mice. Human
monocytes/macrophages obtained from the humanized M-CSF mice also show
augmented functional properties such as migration, phagocytosis, activation and
responses to LPS.
Example 1: Cell Preparations, Analytical Methods, and Assays
CD34 cell isolation and transplantation. Human fetal liver samples
were obtained from the human fetal liver tissue repository at the Albert Einstein College
of Medicine, Bronx, NY and from the Advance Biosciences Resources, Inc., Alameda,
CA. All experiments involving human tissues were performed under the approval of the
Yale Human Investigations Committee.
For isolating human CD34 cells, fetal liver samples were rinsed once
with PBS and cut into small pieces, treated with collagenase D (100 ng/mL) at 37 °C for
45 minutes. Single cell suspensions were prepared and the mononuclear cells were
isolated using density gradient centrifugation (lymphocyte separation medium, MP
biomedicals). CD34 cells were isolated after treating the cells with anti-human CD34
microbeads followed by MACS™ technique (Miltenyi Biotech).
For transplantation, new born pups (day 1 of birth) were sublethally
irradiated with two separate doses (2 x 150 cGy) 4 hours apart and 1 x 10 to 2 x 10
purified human CD34 cells in 20 uL of PBS were injected into the liver using a 22-gauge
needle (Hamilton Company, Reno, NV).
Mesenchymal stroma cell (MSC) Isolation and Culture. Long bones of
mice were isolated and the BM cells were flushed out. Bones were cut into pieces and
digested with a cocktail of collagenase D and P (25 ng/mL) for 45 minutes at 37°C.
Suspension cells were isolated and plated in the presence of MSC culture medium
- + +
(Stem Cell Technologies). After 2 weeks of culture, CD45 Sca1 CD90 cells were
isolated and cultured.
Antibodies and Flow Cytometry. Single cell suspensions were
analyzed by flow cytometry using FACS Calibur or LSRII and CELLQUEST™ software,
FACS DIVA™ software (BD Biosciences, San Jose, CA) or FLOWJO™ software (Tree
Star, Inc., Ashland, OR), respectively. Cell sorting of defined subpopulations was
performed using a FACS ARIA™ cell sorter (BD Biosciences, San Jose, CA).
The following human antibodies were used in the study: CD11b, CD14,
CD33, CD34, CD38, CD40, CD45, CD80, CD86, CD90 and HLA-DR.
The following mouse antibodies were used in this study: CD11b, CD40,
CD45, CD80, CD86, F4/80, Gr1, H2K and IA .
Cell culture. For murine macrophage differentiation, BM cells were
plated in 6 well plates in the presence of DMEM with 10% FCS and necessary
supplements (2mM L-Glutamine, 1% Penicillin-Streptomycin and 1mM nonessential
amino acids). Cells were treated with either recombinant murine M-CSF (10 ng/mL) or
recombinant human M-CSF (10 ng/mL) for 7 days. Cell culture supernatant was
removed every third day and culture was replaced with fresh medium and cytokines.
For human macrophage studies, such as activation, phagocytosis and
+ + +
migration, 2 x 10 CD45 CD14 CD33 cells of the spleens were sorted and cultured in
vitro in the DMEM with 15 % human AB serum and necessary supplements (2mM L-
Glutamine, 1% Penicillin-Streptomycin and 1mM nonessential amino acids).
Activation, Phagocytosis and Migration Assays. For LPS stimulation
in vivo, mice were injected i.p. with LPS (100 ng/g body weight). For LPS stimulation in
vitro, LPS (10 ng/mL) was added to the cells and cultured for either 1 or 2 days. For
poly I:C stimulation in vitro, cells were cultured in the presence of poly I: C (10 ug/mL) for
either 6 or 12 hours.
Phagocytosis assay was performed using the commercially available
VYBRANT™ phagocytosis assay kit (Invitrogen) according to the manufacturer’s
instructions.
Migration assays were performed using a commercially available QCM™
chemotaxis cell migration assays kit (Millipore) according to the manufacturer’s
instructions.
RNA extraction and Real time PCR. Total RNA was isolated using
commercially available kit systems (RNEASY™ Mini kit, Qiagen). cDNA was
synthesised using oligo dT primer and expand reverse transcriptase (Roche). The PCR
reaction was performed in duplicates using 7500 real time PCR systems and power
SYBR™ Green PCR master mix (Applied Biosystems) according to the manufacturer’s
instructions using the following gene specific primer pairs: Human CSF1 (sense: 5′-
TACTGTAGCCACATGATTGGGA-3′ (SEQ ID NO:1) and antisense: 5′-
CCTGTGTCAGTCAAAGGAAC-3′ (SEQ ID NO:2)), Mouse csf1 (sense: 5′-
CGACATGGCTGGGCTCCC-3′ (SEQ ID NO:3) and antisense: 5′ -
CGCATGGTCTCATCTATTAT-3′ (SEQ ID NO:4), Human IFNa (sense:5′-
GTACTGCAGAATCTCTCCTTTCTCCTG-3′ (SEQ ID NO:5) and antisense: 5′-
GTGTCTAGATCTGACAACCTCCCAGGCACA-3′ (SEQ ID NO:6)), Human IFNb
(sense:5′-TTGTGCTTCTCCACTACAGC-3′ (SEQ ID NO:7) and antisense: 5′-
CTGTAAGTCTGTTAATGAAG-3′ (SEQ ID NO:8)), Mouse hprt primers (sense: 5′-
AAGGACCTCTCGAAGTGTTGGATA (SEQ ID NO:9) and antisense: 5′-
CATTTAAAAGGAACTGTTGACAACG-3′ (SEQ ID NO:10)) and Human HPRT primers
(sense: 5′-CTTCCTCCTCCTGAGGAGTC-3′ (SEQ ID NO:11) and antisense: 5′-
CCTGACCAAGGAAAGCAAAG-3′ (SEQ ID NO:12)). For normal PCR, DNA of the target
cells was extracted using a commercially available kit (DNEASY™ blood and tissue kit,
Qiagen) and PCR analysis was performed using gene specific primer pairs.
ELISA. For cytokine quantification studies, either blood serum or cell
culture supernatants were collected and subjected to the ELISA using commercially
available human IL6 and human TNF ELISA kits (Ray Biotech, Inc., GA) according to the
manufacturer’s instructions.
Histology. Solid organs were fixed in 4% PFA. Fixed organs were
embedded in paraffin (Blue RiBbon; Surgipath Medical Industries). Blocks were
sectioned and the 5-μm sections were stained with H&E stain, followed by placement of
coverslips by routine methods. Sections were maintained without any medium. Digital
light microscopic images were recorded, at room temperature, with a Zeiss Axio
Imager.A1 microscope (with 2× and 10× objective lenses), AxioCam MRc5 camera, and
AxioVision 4.7.1 imaging software (Carl Zeiss Microimaging LLC).
Statistical analysis. Data are presented as mean ± SEM. Statistical
significance was assessed using a 2-sided Student t test. P values > 0.05 were
considered to be nonsignificant and P values <0.05 were represented as *.
Example 2: Genetically Modified Mice for Engraftment
Human M-CSF Knockin Strategy. A targeting construct for replacing
the mouse M-CSF nucleic acid sequence with human M-CSF nucleic acid sequence
(VELOCIGENE® Allele Identification Number 5093) in a single targeting step was
constructed using VELOCIGENE® technology as described previously (Valenzuela et al.
(2003) High-throughput engineering of the mouse genome coupled with high-resolution
expression analysis, Nat. Biotechnol. 21:652-659). Mouse and human M-CSF DNA
were obtained from bacterial artificial chromosome (BAC) RPCI-23, clone 373B18 and
from BAC RPCI-11, clone 101M23 respectively. In brief, a linearized targeting construct
generated by gap repair cloning containing mouse M-CSF upstream and downstream
homology arms flanking a 17.5 kb human M-CSF sequences extending from exon 2 to
633nt downstream of non-coding exon 9, and a floxed drug selection cassette was
+/- -/-
electroporated into RAG2 γc mouse embryonic stem (ES) cells, which was made
from a commercially available V17 ES cell line (BALB/c x 129 F1). Mouse ES cells
carrying a heterozygous deletion of the M-CSF gene were identified by Loss-of-Allele
screening with 2 TaqMan qPCR assays that recognized sequences in intron 2 (TUF
primer, 5′-CCAGGAATGTCCACTATGGATTC-3′ (SEQ ID NO:13); TUP probe, 5′
ACTGCTCCTTGACCCTGCTCTGACTCA-3 ′ (SEQ ID NO:14); TUR primer, 5′-
TGGGCTGACTTCCCAAAGG-3′ (SEQ ID NO:15)) and in the 3′ flanking sequence (TDF
primer, 5′-TTAGGTGCTAGTAGGCTGGAAAGTG-3′ (SEQ ID NO:16); TDP probe, 5′-
TGCAATCGCAGCTTCTCTCCTTACTAGGCT-3 (SEQ ID NO:17)′; TDR primer, 5′-
AATAGGAAGAACGAACAGGTCTAATACC-3′ (SEQ ID NO:18)) of the mouse Csf1
gene. Simultaneous replacement of the mouse gene with the human CSF1 gene was
confirmed by Gain-of-Allele TaqMan assays that detected one copy of a sequence in
intron 2 of CSF1 (forward primer, 5′-GCTGCTTGCCTGGGTTAGTG-3′ (SEQ ID NO:19);
probe, 5′-TGCCCAGGAACATCAACCACTGATTCTG-3′ (SEQ ID NO:20); reverse
primer, 5′-GAGGGACAGCAGACCTCAGAAG-3′ (SEQ ID NO:21)) and one copy of the
neomycin resistance (neor) cassette (forward primer, 5′-GGTGGAGAGGCTATTCGGC-
3′ (SEQ ID NO:22); probe, 5′-TGGGCACAACAGACAATCGGCTG-3′ (SEQ ID NO:23);
reverse primer, 5′-GAACACGGCGGCATCAG-3′ (SEQ ID NO:24); see Fig. 8. The qPCR
assay that recognizes the CSF1 sequence does not amplify DNA from the mouse
genome. The same assays were used to confirm the genotypes of mice derived from the
targeted ES cells. Cre-mediated excision of the drug selection cassette was confirmed
with the neo TaqMan assay. All primer-probe sets were supplied by Biosearch
Technologies. Probes were labeled with 6-carboxy-fluorecein (FAM) on their 5′ ends
and BHQ-1 on their 3′ ends.
Correctly targeted ES cells were further electroporated with a transient
Cre-expressing vector to remove the drug selection cassette. Targeted ES cell clones
without drug cassette were introduced into an 8-cell stage mouse embryo by the
VELOCIMOUSE® method (Poueymirou et al. (2007)). VELOCIMICE® (F0 mice fully
derived from the donor ES cell) bearing the humanized M-CSF gene (VG 5093) were
identified by genotyping for loss of mouse allele and gain of human allele using a
modification of allele assay (Valenzuela et al. (2003)).
-/- -/- m/m -/- -/-
Mouse Maintenance. Balb/c-Rag2 γc M-CSF , Balb/c-Rag2 γc M-
h/m -/- -/- h/h
CSF and Balb/c-Rag2 γc M-CSF mice were kept under specific pathogen-free
conditions in the animal care facility at Yale University. All mouse experiments were
approved by the Institutional Animal Care and Use Committee of Yale University.
Making Humanized M-CSF Mice. To validate whether physiologic
expression of human M-CSF in a mouse results in improved differentiation of human
-/- -/-
macrophages in the humanized mice, the Balb/c Rag2 γc mice were engineered to
-/- -/-
express human M-CSF. The Balb/c strain with Rag2 γc deficiency serves as
successful model system for the study of the human immune system in mice (Traggiai E
et al. (2004) Development of a human adaptive immune system in cord blood cell-
transplanted mice, Science 304:104-107). In order to circumvent supra-physiological
expression of human M-CSF in these mice, a strategy to replace mouse M-CSF coding
sequence with the human counterpart was adopted. A construct ( for replacing,
in a single targeting step, the majority of the M-CSF open reading frame with human M-
CSF coding sequence (VELOCIGENE® Allele Identification Number 5093), was
constructed using the VELOCIGENE® technology as described previously (Valenzuela
et al. (2003)). Of note, the promoter and other regulatory elements (such as 5’UTR) of
the mouse were preserved in this vector. The linearized targeting vector was
+/- -/-
electroporated into the Balb/c x 129 Rag 2 γc embryonic stem cells. Correctly
targeted ES cells were further electroporated with a transient Cre-expressing vector to
remove the drug selection cassette. Targeted ES cell clones without drug cassette were
introduced into an 8-cell stage mouse embryo by the VELOCIMOUSE® method
(Poueymirou et al. (2007)). VELOCIMICE® (F0 mice fully derived from the donor ES
cell) bearing the humanized M-CSF gene (VG 5093) were identified by genotyping for
the loss of the mouse allele and gain of the human allele using a modification of allele
assay (Valenzuela et al. (2003)). Through sequential intercrossing of progenies, Balb/c
-/- -/-
Rag2 γc mice chimeric mice and germline transmitted mice with mouse and human
m/h h/h
M-CSF (M-CSF ; heterozygous knockin) and human M-CSF only (M-CSF ;
homozygous knockin) were generated.
Characterization of Humanized M-CSF Mice. Expression of human M-
CSF in the humanized M-CSF mice was evaluated. Organs from either M-CSF or M-
mice were harvested and analyzed for murine and human M-CSF mRNA
expression using primers that are species specific. As shown in and 1B, M-
CSF is expressed in the majority of the analyzed organs including BM, spleen, blood,
liver, brain, lung, testis and kidney. However, thymus and skin did not show detectable
expression of M-CSF. Of note, the expression pattern of mouse and human M-CSF was
m/m h/h
comparable between M-CSF and M-CSF mice, respectively. Next, expression
m/m m/h h/h
levels of mouse and human M-CSF in M-CSF , M-CSF , and M-CSF mice were
quantified. Bone marrow mesenchymal stromal cells (MSCs) were isolated and the
expression levels of M-CSF mRNA were quantified using Realtime-PCR () and
M-CSF protein (secreted) was quantified using ELISA (). M-CSF mice
expressed only mouse M-CSF, M-CSF mice expressed both mouse and human M-
CSF and M-CSF mice expressed only human M-CSF. Expression levels of human M-
CSF was comparable with mouse M-CSF. In line with these data, analysis of CSF-1 in
serum revealed comparable expression levels of CSF-1 protein in m/m, h/m, and h/h
mice (Fig. 1E). Hemizygocity does not lead to decreased gene and protein expression
levels, indicating that gene-dosage levels seem not to be limiting for this cytokine.
To investigate whether replacing mouse M-CSF with human M-CSF
results in deleterious effects, especially on the bone and hematopoiesis, M-CSF mice
were analyzed at various ages. Earlier studies have documented that mice with
op/op -/-
defective M-CSF signaling (Csf1 and Csf1r ) exhibit tooth eruption failure and bone
defects (Dai, X.M. et al. (2002) Targeted disruption of the mouse colony-stimulating
factor 1 receptor gene results in osteopetrosis, mononuclear phagocyte deficiency,
increased primitive progenitor cell frequencies, and reproductive defects, Blood 99:111-
120; Felix, R. et al. (1990) Macrophage colony stimulating factor restores in vivo bone
resorption in the op/op osteopetrotic mouse, Endocrinology 127:2592-2594; Wiktor-
Jedrzejczak, W. et al. (1990) Total absence of colony-stimulating factor 1 in the
macrophage-deficient osteopetrotic (op/op) mouse, Proc. Natl Acad. Sci. USA 87:4828-
4832; Yoshida, H. et al. (1990) The murine mutation osteopetrosis is in the coding region
of the macrophage colony stimulating factor gene, Nature 345:442-444). In contrast, M-
op/op
CSF mice revealed normal teeth and bone properties. Further, unlike the Csf1 and
Csf1r mice, the total cell content of the BM (), frequencies of myeloid cells in
the BM, spleen (SP) and peripheral blood (PB) () and the frequencies of
, M-
macrophages in the BM and SP () were comparable among the M-CSF
h/m h/h
CSF and M-CSF mice. In line with this observation, the frequencies of the HSC
compartment (including long term-HSC, short term-HSCs and multipotent progenitors)
and myeloid progenitor compartment (including common myeloid progenitors,
granulocyte monocyte progenitor and megakaryocyte erythrocyte progenitors) were
m/m h/m h/h
comparable among the M-CSF , M-CSF and M-CSF mice (.
A possible explanation for the normal hematopoiesis and bone
development in the M-CSF mice might be that human M-CSF is cross reactive with
mouse cells. To validate this, total BM cells from M-CSF were isolated and cultured in
the presence of either recombinant murine M-CSF or recombinant human M-CSF.
Whereas BM cells cultured in the absence of cytokine failed to survive, cells cultured in
the presence of either human or mouse M-CSF showed comparable levels of in vitro
differentiation (). Analysis of these in vitro differentiated macrophages for the
expression of co-stimulatory molecules and MHC indicated comparable levels of these
molecules in the presence of either human or mouse M-CSF (). Consistent with
our findings, previous studies documented that human M-CSF is active in mouse target
cells, whereas mouse M-CSF is not cross-reactive with human cells (Sieff, C.A. (1987)
Hematopoietic growth factors, J. Clin. Invest. 79:1549-1557).
Example 3: Differentiation of Human Monocytes/Macrophages in
Humanized M-CSF mice
To evaluate the impact of M-CSF humanization, sub-lethally irradiated
-/- -/- -/- -/- -/- -/-
m/m h/m h/h
newborn Rag2 γc M-CSF , Rag2 γc M-CSF and Rag2 γc M-CSF pups were
transplanted intra-hepatically (i.h) with ~ 2 x 10 purified human fetal liver CD34 cells.
Recipients were then bled at 8 weeks after transplantation to confirm the cells of donor
(based on human CD45 expression) origin. Twelve weeks after transplantation,
recipients were sacrificed and their BM, SP and PB were harvested. Analysis revealed
augmentation of the relative and absolute frequencies of CD14 CD33
monocyte/macrophage lineage cells in the BM, SP and PB of both M-CSF and M-
h/h m/m h/m
CSF mice as compared with M-CSF mice (-C). Although M-CSF mice
exhibited increased frequencies of CD14 CD33 cells, the maximum frequencies of
+ + h/h
CD14 CD33 cells were found in the M-CSF mice. Interestingly, in addition to this
increase, the frequencies of CD14 CD33 cells were also increased in the BM, SP and
h/m h/h
PB of M-CSF and M-CSF mice ().
To analyze whether the human M-CSF knockin mice support sustained
human myelopoiesis, recipients were analyzed at 12, 16 and 20 weeks after
CD33 monocyte/macrophage lineage cells were
transplantation. While human CD14
slightly reduced at 16 weeks and highly reduced after 20 weeks of transplantation in the
m/m + +
M-CSF mice, significant proportions of human CD14 CD33 cells were observed in
h/m h/h
and M-CSF mice at even 16 and 20 weeks. Nevertheless, the
both M-CSF
+ + h/h
maximum frequencies of human CD14 CD33 cells were seen in the M-CSF mice
( and 4B).
Next, whether the humanized M-CSF mice support efficient differentiation
m/m m/h
of human tissue macrophages was assessed. To this end, M-CSF , M-CSF and M-
CSF mice were perfused with PBS and their organs (including liver, lungs and skin)
were harvested. Cells of the peritoneum were obtained by flushing the peritoneal cavity
with PBS. Single cell suspensions were prepared and the frequencies of human
CD14 CD33 cells were calculated. As expected, the frequencies of human
CD14 CD33 cells were significantly increased in the liver, lungs and peritoneum of both
m/h h/h
M-CSF and M-CSF mice. However, analysis of skin explants revealed comparable
+ + m/m m/h
frequencies of human CD14 CD33 cells between M-CSF and M-CSF mice,
although a significant increase of these cells was observed in the skin explants of M-
CSF mice (. Taken together, these data suggest that expression of human M-
CSF in mice improves myeloid/macrophage lineage differentiation of human HSCs.
Example 4: Human Monocyte/Macrophage Function in Humanized
M-CSF Mice
To investigate whether the human CD14 CD33 monocytes/macrophages
in the humanized M-CSF mice functioned normally, both in vivo and in vitro functional
m/m m/h
studies were performed. Sublethally irradiated M-CSF and M-CSF pups were
injected with fetal liver CD34+ cells and 12 weeks after transplantation, donor derived
hematopoiesis was assessed and recipient mice were injected with either LPS or PBS.
Two days after LPS injection, recipients were analyzed for the frequencies of human
CD14 CD33 cells in the spleen. While LPS injection induced only a modest increase of
monocyte/macrophage lineage cells in the M-CSF mice, when compared with the
PBS injected groups, LPS injected M-CSF mice showed a several fold increase of
human CD14 CD33 cells in the spleen (). Next, the abilities of these cells to
produce pro-inflammatory cytokines in response to LPS stimulation in vivo were
examined.
m/m m/h
M-CSF and M-CSF mice engrafted with human CD34+ cells were
injected with LPS. Six hours after injection, mice were bled and the serum levels of
human and mouse IL6 and TNFα were determined by ELISA. Consistent with the
increased frequencies of monocytes/macrophages in the humanized M-CSF mice,
elevated levels of human IL6 and TNFα were detected in the M-CSF mice. Although
the basal levels of these cytokines were higher in the M-CSF mice, LPS stimulation
resulted in augmented levels of human IL6 and TNFα in the serum ( and 6C).
Next, the capacity of monocytes/macrophages (obtained from humanized M-CSF mice)
to secrete pro-inflammatory cytokines in vitro was analyzed. Human CD14 CD33 cells
m/m h/h
were isolated from the spleens of either M-CSF or M-CSF mice, after 12 weeks of
reconstitution with human CD34 cells, and stimulated with LPS in vitro for either 24 or
48 hours. The levels of IL-6 and TNFα cytokines in the cell culture supernatants were
assessed by ELISA. In line with the in vivo data, CD14 CD33 cells purified from M-
CSF mice secreted augmented levels of these cytokines in response to LPS (
and 7B). Similarly, human CD14 CD33 cells isolated from the humanized M-CSF mice
expressed augmented levels of interferon-α and interferon-β mRNA in response to poly
I:C stimulation (). Finally, the phagocytosis, migration and activation properties
of human monocytes/macrophages obtained from the humanized M-CSF mice were
+ + +
analyzed. Human CD14 CD33 cells purified from human CD34 reconstituted, M-
CSF mice exhibited increased phagocytic properties () and displayed
augmented chemotaxis in response to the chemokine Mip3β (). As expected,
human monocytes/macrophages obtained from the M-CSF mice displayed enhanced
activation properties as assessed based on upregulation of co-stimulatory molecules
including CD40, CD80 and CD86, and HLA-DR in response to LPS stimulation in-vitro
(). Overall, human monocytes/macrophages differentiated in the presence of
human M-CSF in the humanized mice exhibit augmented functional properties.
Generating a mouse with a completely reconstituted and functional
hematopoietic/immune system of human origin has been a great challenge in the field.
−/− −/−
To date, 3 mouse strains (NOD-scid γc , [NSG], NOD/Shi-scid γc [NOG], and Balb/c-
−/− −/−
Rag2 γc ) have been developed. Despite the advantages conferred by each of these
strains, human hematopoiesis is incomplete in these mice.
To overcome this major technical challenge, the mouse CSF-1 gene was
replaced with its human counterpart. This resulted in efficient human macrophage
differentiation in mice that were reconstituted with human hematopoietic stem and
progenitor cells. Analysis of the humanized CSF-1 mice indicated efficient differentiation
of human monocytes/macrophages in the BM, spleen and peripheral blood. Moreover,
human macrophages were detected in several different tissues including, lungs and
liver, in these mice, indicating that the presence of CSF-1 in humanized mice is sufficient
to promote the differentiation of human tissue macrophages. Additionally, functional
studies described herein involving the human monocytes/macrophages, isolated from
m/m h/h h/h
the CSF1 and the CSF1 mice indicate that cells from the CSF1 mice were better
in performing functions such as phagocytosis, migration, activation and cytokine
secretion. Based on these findings, it may be inferred that monocytes/macrophages that
differentiate in the presence of human CSF-1 function better.
VELOCIGENE® genetic engineering technology was used to generate a
−/− −/−
novel line of Balb/c-Rag2 γc mice that express human CSF-1. Accordingly, the
mouse CSF-1 coding region was replaced with the human counterpart without disturbing
the regulatory elements, such as the promoter, of the mouse csf1 gene. This resulted in
a chimeric gene that contained the mouse regulatory elements and the human CSF-1
coding region. Expression studies of these mice indicated that this chimeric gene is
expressed faithfully in both a qualitative and quantitative manner.
The role of CSF-1 in the differentiation of mouse macrophages has been
op/op
well established. Mice that are deficient for either CSF-1 (Csf1 ) or its receptor
−/−)
(Csf1r exhibit severe reduction in macrophage and osteoclast frequencies,
osteopetrosis, tooth eruption failure, developmental defects in various tissues, including
nervous system, male and female fertility, the dermis and synovial membranes. While
these studies have provided very important insights into the roles of CSF-1 in mice, the
significance of CSF-1 in human hematopoiesis remains largely unknown. In this regard,
the mice described herein will serve as a valuable tool, because it will enable improved
understanding of the physiology and functions of cytokines in human hematopoiesis and
hematopoietic cell function. Additionally, this mouse may be used to model disease and
test the effects of agents on the human immune system. This mouse model is a
valuable tools in understanding the pathophysiology and in the treatment of several
human disorders and diseases.
The preceding merely illustrates the principles of the invention. It will be
appreciated that those skilled in the art will be able to devise various arrangements
which, although not explicitly described or shown herein, embody the principles of the
invention and are included within its spirit and scope. Furthermore, all examples and
conditional language recited herein are principally intended to aid the reader in
understanding the principles of the invention and the concepts contributed by the
inventors to furthering the art, and are to be construed as being without limitation to such
specifically recited examples and conditions. Moreover, all statements herein reciting
principles, aspects, and embodiments of the invention as well as specific examples
thereof, are intended to encompass both structural and functional equivalents thereof.
Additionally, it is intended that such equivalents include both currently known equivalents
and equivalents developed in the future, i.e., any elements developed that perform the
same function, regardless of structure. The scope of the present invention, therefore, is
not intended to be limited to the exemplary embodiments shown and described herein.
Rather, the scope and spirit of present invention is embodied by the appended claims.
Paragraphs
The following numbered paragraphs contain statements of broad
combinations of the technical features herein disclosed. These statements are provided
to assist the reader’s understanding of the invention only. The invention is defined by the
claims of this specification which are found on pages 61 to 63.
1. A method of making a humanized M-CSF mouse, the method comprising:
contacting a mouse pluripotent stem cell with a nucleic acid sequence comprising
a coding sequence for a human M-CSF protein;
culturing the pluripotent stem cell under conditions that promote the integration of
the coding sequence into the genome of the pluripotent stem cell such that the
coding sequence is operably linked to the endogenous promoter of the mouse M-
CSF gene at the mouse M-CSF locus;
making a humanized M-CSF mouse from the mouse pluripotent stem cell,
wherein the humanized M-CSF mouse comprises the coding sequence for the
human M-CSF protein; and
maintaining the humanized M-CSF mouse under conditions sufficient for the
humanized M-CSF mouse to express M-CSF RNA encoded by the coding
sequence in bone marrow, spleen, blood, liver, brain, lung, testis and kidney,
wherein the humanized M-CSF mouse is immunocompromised for a mouse
immune system.
2. The method of 1, wherein the humanized M-CSF mouse comprises two copies of
the coding sequence.
3. The method of 1, wherein the humanized M-CSF mouse comprises a null
mutation in at least one mouse M-CSF allele.
4. The method of 3, wherein the null mutation is a deletion of mouse M-CSF exons
2-9.
. The method of 1, further comprising transplanting human cells into the humanized
M-CSF mouse.
6. The method of 5, wherein the human cells are hematopoietic cells.
7. The method of 5, further comprising infecting the humanized M-CSF mouse with a
human pathogen.
8. The method of 7, wherein the human pathogen is selected from a virus, a fungus,
and a bacterium.
9. The method of 8, wherein the bacterium is a mycobacterium or an
enterobacterium.
. The method of 1, wherein the humanized M-CSF mouse comprises a Rag2 gene
knock-out and an IL2rg gene knock-out.
11. A method of screening an agent, the method comprising:
(a) providing or receiving a first humanized M-CSF mouse and a second
humanized M-CSF mouse, each of the first and second humanized M-CSF mice
comprising:
a nucleic acid sequence incorporated into the genome of the humanized M-CSF
mouse, which sequence encodes a human M-CSF protein and is operably linked
to the endogenous promoter of the mouse M-CSF gene at the mouse M-CSF
locus; and
human hematopoietic cells, wherein each of the first and second humanized M-
CSF mice is immunocompromised for a mouse immune system, and wherein
each of the first and second humanized M-CSF mice expresses M-CSF RNA
encoded by the nucleic acid sequence in bone marrow, spleen, blood, liver, brain,
lung, testis and kidney;
(b) contacting the first humanized M-CSF mouse with the agent; and
(c) comparing the first humanized M-CSF mouse contacted with the agent to the
second humanized M-CSF mouse in the absence of the agent to determine the
in-vivo effect of the agent.
12. The method of 11, wherein the comparing comprises comparing the function of
the human hematopoietic cells in the first humanized M-CSF mouse contacted
with the agent to the function of the human hematopoietic cells in the second
humanized M-CSF mouse in the absence of the agent, wherein a modulation in
the function of the human hematopoietic cells in the first humanized M-CSF
mouse relative to the second humanized M-CSF mouse indicates that the agent
modulates human hematopoietic cell function.
13. The method of 12, wherein the first humanized M-CSF mouse and the second
humanized M-CSF mouse are the same mouse, wherein the absence of the
agent occurs prior to the contacting, and wherein a modulation in the function of
the human hematopoietic cells after the contacting relative to the function of the
human hematopoietic cells prior to the contacting indicates that the agent
modulates human hematopoietic cell function.
14. The method of 12, wherein the first humanized M-CSF mouse and the second
humanized M-CSF mouse are different mice.
. The method of 11, comprising exposing the first and second humanized M-CSF
mice to an amount of a human pathogen sufficient to produce an infection in the
first and second humanized M-CSF mice, and measuring a parameter of the
infection in the first humanized M-CSF mouse over time and a parameter of the
infection in the second humanized M-CSF mouse over time, wherein the
comparing comprises comparing the measured parameters to determine the in-
vivo effect of the agent on the human pathogen.
16. The method of 15, wherein the first humanized M-CSF mouse is contacted with
the agent prior to exposing the first and second humanized M-CSF mice to the
human pathogen.
17. The method of 15, wherein the first humanized M-CSF mouse is contacted with
the agent concurrently with the exposure to the human pathogen.
18. The method of 15, wherein the first humanized M-CSF mouse is contacted with
the agent subsequent to exposing the first and second humanized M-CSF mice to
the human pathogen.
19. The method of any one of 11-14, wherein the first and second humanized M-CSF
mice each comprise two copies of the coding sequence.
. The method of any one of 11-14, wherein the first and second humanized M-CSF
mice each comprise the same null mutation in at least one mouse M-CSF allele.
21. The method of any one of 11-14, wherein the null mutation is a deletion of mouse
M-CSF exons 2-9.
22. The method of any one of 11-14, wherein the first and second humanized M-CSF
mice each comprise a Rag2 gene knock-out and an IL2rg gene knock-out.
23. A method as in any one of 1 to 22, substantially as herein described with
reference to any example thereof.
Claims (18)
1. A humanized M-CSF mouse, comprising: a nucleic acid sequence incorporated into the genome of the humanized M-CSF mouse, which sequence encodes a human M-CSF protein and is operably linked to a regulatory sequence 5’ of the mouse M-CSF structural gene locus; wherein the mouse is immunocompromised for a mouse immune system as a result of one or more of the following: sub-lethal irradiation, the mouse is a NOD-SCID mouse, the mouse is a NOD- scid γc−/− (NSG) mouse, and the mouse is a NOD/Shi-scid γc−/− (NOG) mouse; and wherein the mouse expresses M-CSF RNA encoded by the nucleic acid sequence in bone marrow, spleen, blood, liver, brain, lung, testis and kidney.
2. The humanized M-CSF mouse according to claim 1, wherein the mouse comprises two copies of the nucleic acid sequence.
3. The humanized M-CSF mouse according to claim 2, wherein the mouse comprises a null mutation in at least one mouse M-CSF allele.
4. The humanized M-CSF mouse according to claim 3, wherein the null mutation is a deletion of mouse M-CSF exons 2-9.
5. The humanized M-CSF mouse according to any one of claims 1-4, wherein the mouse comprises human cells.
6. The humanized M-CSF mouse according to claim 5, wherein the human cells are hematopoietic cells.
7. The humanized M-CSF mouse according to claim 5 or 6, wherein the mouse comprises an infection by a human pathogen.
8. The humanized M-CSF mouse according to claim 7, wherein the pathogen is selected from a virus, a fungus, and a bacterium.
9. The humanized M-CSF mouse according to claim 8, wherein the bacterium is a mycobacterium or an enterobacterium.
10. A method for making an activated human monocyte and/or activated human macrophage, comprising exposing a humanized M-CSF mouse to an immune stimulant under conditions sufficient for human monocytes and/or macrophages in the mouse to become activated, isolating human monocytes and/or human macrophages from the mouse, wherein the mouse comprises a nucleic acid sequence incorporated into the genome of the mouse, which sequence encodes a human M-CSF protein and is operably linked to the endogenous promoter of the mouse M-CSF gene at the mouse M-CSF locus, wherein the mouse is immunocompromised for a mouse immune system, wherein the mouse comprises human hematopoietic cells, and wherein the mouse expresses M-CSF RNA encoded by the nucleic acid sequence in bone marrow, spleen, blood, liver, brain, lung, testis and kidney.
11. The method of claim 10, wherein the immune stimulant is an endotoxin.
12. The method of claim 11, wherein the endotoxin is LPS.
13. A method of isolating biologically active human M-CSF, the method comprising: isolating biologically active human MCSF from a humanized M-CSF mouse , wherein the mouse comprises a nucleic acid sequence incorporated into the genome of the mouse, which sequence encodes a human M-CSF protein and is operably linked to the endogenous promoter of the mouse M-CSF gene at the mouse M-CSF locus, wherein the mouse is immunocompromised for a mouse immune system, and wherein the mouse expresses M-CSF RNA encoded by the nucleic acid sequence in bone marrow, spleen, blood, liver, brain, lung, testis and kidney.
14. The method of claim 13, wherein the isolating comprises isolating biologically active human MCSF from blood of the mouse.
15. The method of claim 13, wherein, the method comprises: isolating a cell that expresses biologically active human M-CSF from the mouse; culturing the cell under conditions sufficient for the cell to express and secrete biologically active human M-CSF; and isolating the secreted biologically active human M-CSF.
16. A human immune cell isolated from a humanized M-CSF mouse, wherein the mouse comprises a nucleic acid sequence incorporated into the genome of the mouse, which sequence encodes a human M-CSF protein and is operably linked to the endogenous promoter of the mouse M-CSF gene at the mouse M-CSF locus, wherein the mouse is immunocompromised for a mouse immune system, wherein the mouse comprises human hematopoietic cells, and wherein the mouse expresses M-CSF RNA encoded by the nucleic acid sequence in bone marrow, spleen, blood, liver, brain, lung, testis and kidney.
17. The human immune cell of claim 16, wherein the human immune cell is selected from a human monocyte and a human macrophage.
18. The human immune cell of claim 17, wherein the human immune cell is selected from a human NK cell, a human B Cell and a Human T cell.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
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US201161442946P | 2011-02-15 | 2011-02-15 | |
US61/442,946 | 2011-02-15 | ||
NZ708040A NZ708040B2 (en) | 2011-02-15 | 2012-02-14 | Humanized m-csf mice |
Publications (2)
Publication Number | Publication Date |
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NZ724014A NZ724014A (en) | 2018-12-21 |
NZ724014B2 true NZ724014B2 (en) | 2019-03-22 |
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