U.S. patent application number 10/267923 was filed with the patent office on 2003-09-18 for transgenic mammals lacking expression of erythropoietin or of erythropoietin receptor, transgenic mammals expressing chimeric erythropoietin receptors, constructs for producing the transgenic mammals and uses therefor.
This patent application is currently assigned to Arris Pharmaceutical Corporation. Invention is credited to Giebel, Lutz B., Liu, Xin, Lodish, Harvey F., Matthews, David, Ross, Michael J., Wu, Hong.
Application Number | 20030177515 10/267923 |
Document ID | / |
Family ID | 27410707 |
Filed Date | 2003-09-18 |
United States Patent
Application |
20030177515 |
Kind Code |
A1 |
Wu, Hong ; et al. |
September 18, 2003 |
Transgenic mammals lacking expression of erythropoietin or of
erythropoietin receptor, transgenic mammals expressing chimeric
erythropoietin receptors, constructs for producing the transgenic
mammals and uses therefor
Abstract
Transgenic nonhuman mammals, such as transgenic mice, which lack
erythropoietin expression, in which the erythropoietin receptor is
deleted, which carry a heterologous erythropoietin receptor (e.g.,
a chimeric receptor); constructs useful for producing such
transgenic nonhuman mammals, embryonic stem cells containing the
constructs, a method of producing the transgenic nonhuman mammals
and a method of identifying erythropoietin mimics or mimetics.
Inventors: |
Wu, Hong; (Brookline,
MA) ; Liu, Xin; (Brookline, MA) ; Lodish,
Harvey F.; (Brookline, MA) ; Giebel, Lutz B.;
(San Mateo, CA) ; Ross, Michael J.; (Hillsborough,
CA) ; Matthews, David; (San Francisco, CA) |
Correspondence
Address: |
FINNEGAN, HENDERSON, FARABOW, GARRETT & DUNNER
LLP
1300 I STREET, NW
WASHINGTON
DC
20005
US
|
Assignee: |
Arris Pharmaceutical
Corporation
South San Francisco
CA
94080
|
Family ID: |
27410707 |
Appl. No.: |
10/267923 |
Filed: |
October 8, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10267923 |
Oct 8, 2002 |
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08839605 |
Apr 15, 1997 |
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6485964 |
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08839605 |
Apr 15, 1997 |
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08540366 |
Oct 6, 1995 |
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08540366 |
Oct 6, 1995 |
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08467234 |
Jun 6, 1995 |
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08467234 |
Jun 6, 1995 |
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08407462 |
Mar 20, 1995 |
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Current U.S.
Class: |
800/18 ;
435/320.1; 435/354; 435/69.1; 530/350; 536/23.5 |
Current CPC
Class: |
A01K 2217/075 20130101;
A01K 2207/15 20130101; A01K 67/0276 20130101; C12N 2800/30
20130101; A01K 2267/01 20130101; A01K 2227/105 20130101; A01K
2267/0381 20130101; C12N 15/8509 20130101; A01K 2267/03 20130101;
A01K 2217/05 20130101; A01K 2217/00 20130101 |
Class at
Publication: |
800/18 ;
435/69.1; 435/320.1; 435/354; 536/23.5; 530/350 |
International
Class: |
A01K 067/027; C07H
021/04; C12P 021/02; C12N 005/06; C07K 014/72 |
Claims
We claim:
1. A transgenic nonhuman mammal wherein expression of
erythropoietin is lacking.
2. The transgenic nonhuman mammal of claim 1 which is a transgenic
mouse.
3. The transgenic mouse of claim 2 which is a knockout mouse
wherein the erythropoietin gene was deleted by homologous
recombination between the mouse erythropoietin gene and DNA
comprising the 2.5 Kb PstI-PstI fragment of the 5' end of the mouse
erythropoietin gene and the 5.0 Kb HindIII-KpnI fragment of the
mouse erythropoietin gene.
4. The transgenic mouse of claim 2 wherein the erythropoietin
receptor is deleted.
5. The transgenic mouse of claim 4 derived from a cross between a
transgenic mouse lacking erythropoietin expression and a transgenic
mouse wherein the erythropoietin receptor is deleted.
6. A transgenic nonhuman mammal wherein the erythropoietin receptor
is deleted.
7. The transgenic mammal of claim 6 which is a transgenic
mouse.
8. The transgenic mouse of claim 7 which is a knockout mouse
wherein the erythropoietin receptor was deleted by homologous
recombination between the mouse erythropoietin receptor gene and
DNA comprising the 4 Kb SalI-BamHI fragment derived from the 5' end
of the mouse erythropoietin receptor gene and the 2.7 Kb XbaI-EcoRI
fragment derived from the 3' end of the mouse erythropoietin
receptor gene.
9. The transgenic mouse of claim 8 wherein expression of
erythropoietin is lacking.
10. A transgenic nonhuman mammal wherein expression of
erythropoietin is lacking and the erythropoietin receptor is
deleted.
11. A transgenic nonhuman mammal wherein the endogenous
erythropoietin receptor is replaced by a chimeric erythropoietin
receptor.
12. The transgenic nonhuman mammal of claim 11 which is a
transgenic mouse, wherein the chimeric erythropoietin receptor is a
human/mouse erythropoietin receptor.
13. The transgenic mouse of claim 12 wherein the human/mouse
erythropoietin receptor comprises an extracellular domain, a
transmembrane domain and an intracellular domain, wherein the
extracellular domain is the human erythropoietin receptor
extracellular domain, the transmembrane domain is the mouse
erythropoietin receptor transmembrane domain or the human
erythropoietin receptor transmembrane domain and the intracellular
domain is the mouse erythropoietin receptor intracellular
domain.
14. A DNA construct comprising: a) the 2.5 Kb PstI-PstI fragment of
the 5' end of the erythropoietin gene, b) DNA encoding a selectable
marker and c) the 5.0 Kb HindIII-KpnI fragment of the
erythropoietin gene, wherein the DNA encoding the selectable marker
is positioned between the fragment of a) and the fragment of
c).
15. A DNA construct comprising: a) the 4 Kb SalI-BamHI fragment
derived from the 5' end of the mouse erythropoietin receptor gene,
b) DNA encoding a selectable marker and c) the 2.7 Kb XbaI-EcoRI
fragment derived from the 3' end of the mouse erythropoietin
receptor gene, wherein the DNA encoding the selectable marker is
positioned between the fragment of a) and the fragment of c).
16. The plasmid comprising pEPO-M1.
17. The plasmid comprising pEPOR-M1.
18. Mouse embryonic stem cells containing the DNA construct of
claim 14.
19. Mouse embryonic stem cells containing pEPO-M1.
20. Mouse embryonic stem cells containing the DNA construct of
claim 15.
21. Mouse embryonic stem cells containing pEPOR-M1.
22. A method of identifying an agent that is active in stimulating
human EPO receptor, which comprises: a) administering an agent to a
transgenic mammal, wherein said mammal carries an EPO receptor with
a human extracellular domain; and b) assessing the effect of the
agent on erythropoiesis, wherein if the agent stimulates production
of red blood cells, then the agent is active in stimulating human
EPO receptor.
23. A method of identifying an agent which is an erythropoietin
mimic or mimetic, comprising the steps of: a) administering an
agent to a transgenic mouse, wherein the transgenic mouse lacks
erythropoietin and carries a chimeric erythropoietin receptor
comprising a human erythropoietin receptor extracellular domain, a
mouse erythropoietin receptor transmembrane domain and a mouse
erythropoietin intracellular domain; and b) assessing the effect of
the agent on erythropoiesis, wherein if erythropoiesis is
stimulated, the agent is an erythropoietin mimic or mimetic.
Description
RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S. Ser. No.
08/467,234, filed Jun. 6, 1995, entitled "Transgenic Mammals
Lacking Expression of Erythropoietin or of Erythropoietin Receptor,
Transgenic Mammals Expressing Chimeric Erythropoietin Receptors,
Constructs for Producing the Transgenic Mammals and Uses Therefor",
which is a continuation-in-part of U.S. Ser. No. 08/407,462, filed
Mar. 20, 1995, by Hong Wu, Xin Liu and Harvey F. Lodish.
BACKGROUND OF THE INVENTION
[0002] Erythropoietin (EPO), a 34 kd glycoprotein hormone produced
primarily by the kidney, is the principal factor regulating
erythropoiesis and plays a key role in stimulating erythrocyte
formation in higher organisms. It serves as both mitogen and
survival factor which acts on erythroid progenitors, such as
erythroid colony-forming units (CFU-E), to promote these cells to
proliferate and, possibly, to maturate. EPO is necessary for the
replacement of erythrocytes which must occur on an ongoing basis
because mature erythrocytes cannot grow or divide and have a
limited lifespan. EPO can be used therapeutically for treatment of
anemia.
[0003] The function of EPO in erythropoiesis is mediated by its
cellular receptor (EPOR), a 507 amino acid polypeptide with a
single transmembrane domain. Although the EPOR is expressed in a
cell type-restricted fashion, i.e., in cells of erythroid lineage,
its transcripts have also been detected in non-erythroid cell types
such as megakaryocyte, mast cells, umbilical vein endothelial cells
and cells with neuronal characteristics.
SUMMARY OF THE INVENTION
[0004] The present invention relates to transgenic nonhuman mammals
and their progeny, such as transgenic mice and their progeny, in
which expression of erythropoietin is lacking (e.g., deleted); the
endogenous (naturally occurring) erythropoietin receptor is
deleted; the endogenous erythropoietin receptor is replaced by a
heterologous erythropoietin receptor (i.e., one not present in the
corresponding nontransgenic or wild type mammal); erythropoietin
expression is lacking and the erythropoietin receptor is deleted;
or erythropoietin expression is lacking and the erythropoietin
receptor is replaced by a heterologous erythropoietin receptor.
Transgenic mammals in which EPO expression is lacking or the EPOR
is deleted (i.e., transgenic nonhuman animals carrying null
mutations in the Epo gene or EpoR gene) are produced by knocking
out (deleting or otherwise disabling) the endogenous EPO gene (Epo)
or the endogenous EPO receptor gene (EpoR), respectively, using
methods described herein and known to those in the field.
Transgenic mammals in which the endogenous EPOR is replaced by a
heterologous EPOR can carry a chimeric EPOR, such as a human/mouse
chimeric EPOR, or an EPOR of another species, such as the human
EPOR present in a mouse. Transgenic mammals in which EPO expression
is lacking and the EPOR is lacking or is a heterologous EPOR are
produced by breeding mice which lack EPO expression (EPO gene
knockout mice) with mice in which, respectively, the EPOR is
lacking or is a heterologous EPOR. For example, transgenic mice in
which EPO expression is lacking and the mouse EPOR (mEPOR) is
replaced with the human EPOR (hEPOR) are produced by breeding mice
which are EPO gene knockouts with mice carrying the hEPOR gene. The
transgenic mice or other nonhuman transgenic mammal can be
heterozygous or homozygous for the knockout or deletion.
[0005] A further subject of the present invention is mammalian,
nonhuman embryonic stem (ES) cells or cell lines in which the EPO
gene or the EPOR gene has been knocked out (deleted or otherwise
disabled) by the methods described herein and embryonic stem cell
lines in which the EPOR gene has been replaced by a gene encoding a
heterologous EPOR, which is a chimeric (e.g., human-mouse) EPOR or
an EPOR which is not chimeric and is an EPOR from another animal
(e.g., a gene encoding the hEPOR replacing the mEPOR).
[0006] The present invention also relates to nucleic acid
constructs, particularly DNA constructs, useful for producing the
transgenic nonhuman mammals, such as transgenic mice, described
herein. It also relates to methods of identifying or designing
agents or drugs, such as small molecules, which mimic the effects
or activity of EPO. In a method of the present invention, a
candidate agent is administered, for example, to a transgenic mouse
and the effect of the agent on erythropoiesis is assessed. The
transgenic mouse can, for example, lack EPO expression and carry a
human/mouse chimeric EPOR in which the extracellular domain is the
human EPOR extracellular domain, the transmembrane domain is either
the mouse or the human EPOR transmembrane domain and the
intracellular domain is the mouse EPOR intracellular domain. If,
after the candidate agent is administered to the transgenic animal,
the animal displays effects associated with administration of EPO
(e.g., enhanced erythropoiesis, treatment of anemia), the agent is
EPO-like in its effects; such an agent is referred to as an EPO
mimic or EPO mimetic. An agent shown to have EPO-like effects in a
transgenic animal in which the EPOR extracellular domain is human
is particularly desirable because it has a demonstrated effect on
the human EPOR. The present invention further relates to EPO mimics
or mimetics and their use. Such EPO mimics can be used
prophylactically or therapeutically in any context in which EPO can
be used (e.g., to prevent or treat anemia, such as the anemia
associated with renal failure).
BRIEF DESCRIPTION OF THE DRAWINGS
[0007] FIG. 1 is a schematic representation of inactivation of the
mouse erythropoietin gene (Epo) by homologous recombination in
embryonic stem cells.
[0008] FIG. 2 is a schematic representation of inactivation of the
mouse erythropoietin receptor gene (EpoR) by homologous
recombination in embryonic stem cells.
DETAILED DESCRIPTION OF THE INVENTION
[0009] Definitions
[0010] Unless otherwise stated, the following terms used in this
specification and the claims have the meanings given below:
[0011] "Transgenic" means containing genetic material other than
that which occurs naturally in a specific nonhuman mammal.
[0012] "Transgene" means a DNA construct which is introduced into a
specific nonhuman mammal.
[0013] "Functional transgene" means a transgene which is added to
cellular DNA or replaces a specific endogenous gene and is a
functional heterologous gene, such as a or chimeric gene or is a
regulatory element.
[0014] "Progeny" means offspring or succeeding generations
thereof.
[0015] "Heterozygous" means bearing different alleles at a
specified genetic locus.
[0016] "Homozygous" means bearing identical alleles at a specified
genetic locus.
[0017] "Null mutation" means a mutation which annuls the function
of a specified gene.
[0018] "Endogenous" means pertaining to material which is natural
to a specified nonhuman mammal.
[0019] "Heterologous" means pertaining to material which is
introduced into or unnatural to a specific nonhuman mammal.
[0020] "Chimeric" means pertaining to a combination of materials
from different sources, such as a combination of endogenous and
heterologous material.
[0021] "Erythropoietin mimetic" means a molecule other than natural
erythropoietin which mimics the pharmacological activity of natural
erythropoietin.
[0022] "Nonhuman mammal" means any mammal other than human and
includes but is not limited to mouse.
[0023] "Transgenic nonhuman mammal" means a nonhuman mammal bearing
a transgene and includes the progeny thereof.
[0024] "Genetic Material" means any material which can encode for
and express protein, including but not limited to genomic DNA,
cDNA, degenerate forms of endogenous DNA and combinations
thereof.
[0025] The present invention relates to transgenic nonhuman mammals
in which EPO and/or EPOR expression is lacking. That is, the
invention relates to a transgenic nonhuman animal carrying a null
mutation in the Epo gene and/or EpoR gene (an EPO or EPOR knockout
transgenic nonhuman animal). In one embodiment, the endogenous Epo
gene in mice has been knocked out. As described in Examples 1 and
3, an example of a transgenic mouse that carries a null mutation
for the Epo gene is a transgenic mouse comprising a first 2.5 Kb
PstI-PstI fragment from the 5' end of the murine Epo gene, a second
5.0 Kb HindIII-KpnI fragment from the 3' end of the murine Epo gene
and a DNA fragment encoding a selectable marker positioned between
the first and second fragments. In a further embodiment, the
expression or production of the EPOR gene has been knocked out. As
described in Examples 2 and 3, an example of a transgenic mouse
that carries a null mutation for the EPOR gene is a transgenic
mouse comprising a first 4 Kb SalI-BamHI fragment of the 5' end of
mouse EPOR gene, a second 2.7 Kb XbaI-EcoRI fragment from the 3'
end of mouse EPOR gene and DNA encoding a selectable marker
positioned between the first and second fragments. As used herein,
the term "knocked out" refers to deletion of all of an endogenous
gene or deletion or alteration of a portion of the endogenous gene
with the result that expression or production of the encoded
polypeptide is partially or completely suppressed.
[0026] A knockout construct is a nucleic acid sequence, such as a
DNA construct, which, when introduced into a cell, results in
suppression (partial or complete) of expression of a polypeptide or
protein encoded by endogenous DNA in the cell. A cell or animal in
which expression of a polypeptide is suppressed completely is
referred to as a cell or animal in which expression is lacking (a
cell or animal carrying a null mutation). The knockout construct is
introduced into a cell, such as an ES cell, using known methods
such as electroporation. In the ES cell the construct integrates
into genomic DNA (e.g., through homologous recombination),
resulting in removal or disruption of endogenous DNA, which, as a
result, is not transcribed. The resulting ES cell, which now has
the targeted gene (e.g., the Epo or EpoR gene) knocked out, is
introduced into a developing embryo, with which it integrates.
[0027] The present invention also relates to transgenic nonhuman
animals in which the EPOR is a heterologous EPOR (an EPOR not
present in the corresponding nontransgenic animal). In one
embodiment, a transgenic nonhuman mammal carries a functional
transgene (i.e., DNA which either encodes a product which alters
EPO or EPOR expression, or is itself a sequence which alters EPO or
EPOR expression, such as a promoter or enhancer) for a chimeric
EPOR comprising genetic material coding for essentially human EPOR
extracellular domain. For example, a transgenic nonhuman animal
carries a functional transgene for a chimeric EPOR comprising
genetic material coding for essentially human EPOR extracellular
domain, genetic material coding for essentially human and/or murine
EPOR transmembrane domain and genetic material coding for
essentially murine EPOR intracellular domain. As described in
Examples 3 and 4, an example of a transgenic mouse that carries a
functional transgene for chimeric EPOR is a transgenic mouse
comprising genetic material essentially equivalent to exons I, VI
and VII of murine EpoR gene and genetic material essentially
equivalent to exons II, III, IV and V of human EpoR gene. As
defined herein, "essentially" as in "essentially human EPOR
extracellular domain", "essentially murine EPOR intracellular
domain", "genetic material essentially equivalent to exons I and
VII of murine EpoR gene" and the like, means that the protein or
genetic material referred to is functionally the same as the
protein or genetic material endogenous to the identified species.
For example, the phrase "essentially human EPOR extracellular
domain" refers to any protein, regardless of the kind and/or number
of amino acid substitutions or deletions therein, which retains the
pharmacological characteristics of human EPOR extracellular domain.
Similarly, the phrase "genetic material essentially equivalent to
exons I and VII of murine EpoR gene" refers to genetic materials
that codes for a protein that is functionally the same as the
protein expressed by endogenous murine exons I and VII.
[0028] In a second embodiment, the transgenic nonhuman animal
carries a functional transgene for an EPOR in which the entire EPOR
is an EPOR present in another animal (e.g., another strain of
mouse; human).
[0029] In a particular embodiment the transgenic nonhuman mammal
carries a functional transgene for EPO under the control of a
promoter/enhancer system and a functional transgene for a
heterologous EPOR (e.g., comprising genetic material coding for
essentially human EPOR extracellular domain). For example, such a
transgenic mouse bears a functional transgene for EPO under the
control of a tetracycline promoter/enhancer system (Khun, R., et
al., Science, 265:1427-1429 (1995)) and a functional transgene for
a chimeric EPOR comprising genetic material coding for essentially
human EPOR extracellular domain, genetic material coding for
essentially human and/or murine EPOR transmembrane domain and
genetic material coding for essentially murine EPOR intracellular
domain. In a particular embodiment, a transgenic mouse carries a
functional transgene for EPO under the control of a tetracycline
promoter/enhancer system and a functional transgene for a chimeric
EpoR gene and genetic material essentially equivalent to exons II,
III, IV and V of human EpoR gene.
[0030] A replacement construct is a nucleic acid sequence, such as
a DNA construct, which comprises DNA encoding a functional
heterologous EPOR (a functional transgene) and, when introduced
into a cell, such as an ES cell, integrates into genomic DNA and is
expressed, resulting in production of the heterologous EPOR. A
replacement construct comprises transcriptional and processing
elements which allow expression of the encoded heterologous EPOR in
cells of the transgenic mammal. For example, a replacement
construct is typically comprised of DNA from some portion of the
gene (exon sequence, intron sequence and/or promoter sequence) to
be suppressed and a marker sequence used to detect the presence of
the construct in the cell. The ES cell containing the replacement
construct is introduced into a developing embryo, with which it
integrates.
[0031] Genomic constructs containing DNA which when introduced into
cells results in a null mutation for EPO or EPOR (knockout
constructs) can be prepared by producing DNA which encompasses the
5' end promoter region of the Epo gene or EpoR gene, the 3' end
untranscribed region of the Epo gene or the EpoR gene and a
selectable marker. In addition, the construct has additional
components necessary and sufficient for expression of the encoded
products in the cell. For example, a genomic construct can be
prepared by (1) isolating a first DNA fragment encompassing the 5'
end promoter region of the Epo gene or EpoR gene, respectively and
introducing the fragment into a suitable vector, (2) isolating a
fragment of DNA encoding for a selectable marker and inserting the
marker adjacent to the first fragment and (3) isolating a second
DNA fragment encompassing the 3' end untranscribed region of the
Epo gene or EpoR gene and inserting the second fragment adjacent to
DNA encoding a selectable marker. Alternatively, the null mutation
constructs can be constructed by introducing the DNA segments of
the gene into a vector which contains the selectable marker. The
DNA fragments encompassing the 3' and 5' end fragments may vary,
but must be of sufficient size to effect homologous recombination
to the endogenous gene. Alternatively, DNA constructs containing
null mutations for Epo and EpoR genes can be prepared by cloning a
fragment of DNA encompassing the entire Epo gene or EpoR gene,
respectively, into a suitable vector and then introducing a
selectable marker in the transcriptional region of the gene.
[0032] The cloning procedures can be carried out by methods known
to those of ordinary skill in the art. The DNA fragments derived
from 3' and 5' end regions of the Epo gene can be obtained from a
restriction map generated by Southern blot analysis of genomic DNA
clones corresponding to the Epo locus with probes derived from 5'
end promoter and 3' end untranslated regions of EPOR cDNA. In a
similar fashion, the DNA fragments derived from 3' and 5' end
regions of the EpoR gene can be obtained from genomic DNA clones
corresponding to the EpoR locus.
[0033] Suitable selectable markers include, but are not limited to,
neomycin, puromycin and hygromycin. A preferred selectable marker
is the PGK-neo gene, which confers resistance to the antibiotic
G418. A preferred PGK-neo gene is derived from the plasmid
pGEM7(KJ1)R, which contains a PGK-neo gene derived from plasmid
pKJ1 and modified by replacing the mutant coding region of neo with
wild-type neo sequences by swapping the Eagl-Ncol fragments between
pKJ1 and pSV.sub.2-neo. Suitable vectors include, but are not
limited to, Bluescript, pBR322, pGEM7, preferably Bluescript.
Details for preparing the constructs for Epo and EpoR null
mutations are provided in the Examples.
[0034] Genomic constructs containing a functional transgene for an
EPOR (replacement construct) can be prepared in a variety of ways.
For example, a replacement construct can comprise genetic material
encoding human EPOR extracellular domain can be prepared by cloning
a first chimeric construct comprising genetic material essentially
equivalent to exons II, III, IV and V, and optionally exons I and
VI, of human EpoR gene and introns from murine EpoR gene into a
vector containing a construct comprising the exons and introns of
murine EpoR gene to produce a second chimeric construct comprising
genetic material essentially equivalent to exons I and VI of murine
and/or human EpoR gene, exons II, III, IV and V of human EpoR gene
and introns and exon VII, VIII of murine EpoR gene. The second
chimeric construct is then subcloned into a vector containing a
genomic construct of the murine EpoR gene. Exon I of the EPOR codes
for the first 13 amino acids of the extracellular domain. Of these
13 amino acids, only residues 2 and 4 differ between the murine and
human sequences. Accordingly, genetic material that is essentially
equivalent to murine exon I can be used in the human/mouse chimeric
constructs of this invention. The cloning and subcloning procedures
are preformed by methods known to those of ordinary skill in the
art.
[0035] The chimeric construct comprising genetic material
essentially equivalent to exons II, III, IV and V and optionally
exons I and VI of human EpoR gene and introns from murine EPOR can
be prepared by ligating together in the appropriate sequence, a
number of chimeric DNA fragments which each comprise one or more of
the desired human exons and appropriate murine introns. The
ligation can be effected by methods known to those of ordinary
skill in the art.
[0036] For example, suitable chimeric DNA fragments are prepared by
"sticky feet" PCR mutagenesis (Clackson, et al., PCR: A Practical
Approach M. J. McPherson, P. Quirke and G. R. Taylor, eds. IRL
Press, Washington, D.C.). The method is carried out by first
amplifying exons from human genetic material (cDNA or genomic DNA)
using PCR primers comprised of the 5' and 3' ends of human exons
and attendant region of the native murine intron to give exons with
regions of the native murine intron region at either end to serve
as the "sticky feet". The amplified exons are then used as
double-stranded mutagenesis primers which are annealed separately
or together to template DNA in a site-directed mutagenesis
reaction. (See Example 3).
[0037] In an alternative method, suitable chimeric DNA fragments
can be prepared by oligonucleotide directed mutagenesis, which is
carried out using synthetic single-stranded DNA molecules designed
to direct the mutation of murine codons to human codons in the
exons and performing a series of oligonucleotide-directed
mutagenesis reactions to change the desired endogenous exons to
their human analogs. Alternatively, suitable chimeric DNA fragments
can be prepared by PCR assembly, by amplifying exons from human
genetic material (cDNA or genomic DNA) and murine introns from
murine genomic DNA using synthetic oligonucleotides designed such
that there is some overlap at the intron/exon boundaries and then
splicing the PCR products together by combining them in subsequent
PCR reactions.
[0038] Genomic DNA clones corresponding to the Epo or EpoR locus
can be isolated by methods known to those of ordinary skill in the
art. For example, murine genomic clones corresponding to the Epo or
EpoR locus can be isolated by screening a 129 (J1) genomic library
(Wu, H., et al., Proc. Natl. Acad. Sci., USA, 91:2519-2823 (1994))
with .sup.32P-labelled murine EPO or EPOR cDNA fragments. Genomic
DNA can be prepared from positive clones according to standard
methods (e.g., Sambrook, et al., Molec. Cloning: A Laboratory
manual 1:2.60-2.80 (1989)).
[0039] The ES cells carrying the knockout or replacement construct
are introduced into embryos using known methods. For example, they
are microinjected into eggs according to methods described by Li et
al. and Bradley. (Li, E. et al., Cell, 69:915-926 (1992); Bradley,
A., Teratocarcinomas and Embryonic Stem Cells: A Practical
Approach, E. J. Robertson (ed.), Oxford IRL Press, pp. 113-151
(1987)) The resulting developing embryo (carrying the knockout
construct, the replacement construct or both constructs) is
introduced (implanted) into an appropriate female and allowed to
develop into offspring. Prior to introduction into the female, the
resulting embryo can, optionally, be incubated in vitro. The
nonhuman mammal which develops from the embryo into which the
embryonic stem cell has been introduced is a transgenic nonhuman
mammal in which EPO expression is suppressed, the EPOR is not
present, a chimeric EPOR or an EPOR of another species has replaced
the endogenous EPOR or EPO expression is suppressed and a chimeric
EPOR or EPOR of another species is present. The term "transgenic
nonhuman mammal" as used herein includes the mammal which develops
from the embryo into which the embryonic stem cell was introduced
and its progeny (all future generations derived or descending from
a particular mammal which contains a knockout construct or a
replacement construct in its genomic DNA).
[0040] As described herein, targeting vectors or constructs have
been produced and used to knock out a mammalian Epo gene and a
mammalian EpoR gene. Specifically, knockout constructs were
produced and introduced into mouse ES cells by electroporation,
resulting in knockout of the endogenous mouse EPO (mEPO) gene and
the mouse EPOR (mEPOR) gene. Constructs can also be introduced by
other means, such as microinjection. Positive clones (ES cells in
which the Epo gene or the EpoR gene is knocked out) were identified
and injected into mouse embryos, which were introduced into an
appropriate female, in which they developed into offspring.
Chimeric mice were backcrossed to nonchimeric mice and germline
transmission of the mutant allele was detected. As described in the
examples, the mutant allele was transmitted to the germ line as a
result of some of the crosses. The positive mice (heterozygous for
the deletion) were crossbred to generate homozygotes. Homozygosity
for the EPO deletion or the EPOR deletion was lethal. Homozygous
animals can be kept alive by, for example, administering EPO or a
blood substitute. Alternatively, animals can be maintained as
heterozygotes and then crossbred to produce homozygous animals for
the assays.
[0041] In one embodiment of the present invention, the transgenic
nonhuman mammal carries a chimeric (e.g., human/mouse) EPOR in
place of the endogenous (e.g., mouse) EPOR. In this embodiment, the
chimeric EPOR can be produced by transfecting ES cells carrying the
mEPOR knockout with a construct comprising DNA encoding the
chimeric EPOR and DNA homologous to endogenous sequences. The
construct DNA encoding the chimeric EPOR is introduced into the
endogenous DNA, preferably at the location at which the mEPOR would
have occurred (i.e., if it had not been knocked out). In one
embodiment, the chimeric EPOR includes a human EPOR extracellular
domain, a mouse EPOR transmembrane domain, and a mouse EPOR
intracellular domain. Alternatively, the transmembrane domain can
be a mammalian transmembrane domain such as the human EPOR
transmembrane domain. Further, the transmembrane domain can be a
chimeric domain in which both mouse and human portions of the
transmembrane domain of the EPO receptor are present. ES cells
containing the construct encoding the chimeric EPOR are introduced
into embryos, using known methods (e.g., electroporation,
microinjection). The resulting embryos containing the constructs
are introduced into an appropriate female and allowed to develop to
term. The resulting offspring carry the chimeric EPOR and can be
bred to produce animals homozygous for the chimeric EPOR.
Alternatively, they can be bred with animals which are Epo gene
knockouts, to produce progeny which are heterozygous or homozygous
for the chimeric EPOR and heterozygous or homozygous for deletion
of the Epo gene.
[0042] In a further embodiment, the entire endogenous EPOR can be
replaced by an EPOR from a different type of animal (e.g., the
mEPOR can be replaced by the hEPOR). In this case, the resulting
EPOR is not chimeric, but an EPOR from another animal. The
replacement EPOR can be an EPOR present in a type of animal other
than that of the transgenic animal or can be an EPOR not known to
occur in nature and designed in such a manner that it functions as
an EPOR and, optionally, has enhanced binding affinity or other
additional advantageous characteristics.
[0043] Transgenic mammals from any species of rodent, including
without limitation, rabbits, rats, hamsters, and mice, can be
produced, as can other nonhuman transgenic mammals, such as dog,
cat, pig, sheep, cow, and primates. In most cases, the embryonic
stem cells (ES cells) used to produce the transgenic mammal will be
of the same species as the transgenic mammal to be generated. Thus,
for example, mouse embryonic stem cells will usually be used for
generation of knockout mice. Transgenic mammals can be prepared
using methods known to those of skill in the art. See, for example,
Hogan et al. (Ed.), Manipulating the Mouse Embryo: A Laboratory
Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y. (1986).
[0044] Embryonic stem cells are typically selected for their
ability to integrate into and become part of the germ line of a
developing embryo so as to create germ line transmission of the
knockout construct. Thus, any ES cell line that can do so is
suitable for use herein. For example, the J1 ES cell line described
in the Examples can be used. Alternatively, suitable cell lines
which can be used include, but are not limited to, the 129J ES or
the murine stem cell line D3 (American Type Culture Collection
catalog no. CRL 1934). The cells are cultured and prepared for DNA
insertion using methods well known to the skilled artisan, such as
those set forth by Robertson (in: Teratocarcinomas and Embryonic
Stem Cells: A Practical Approach, E. J. Robertson, ed. IRL Press,
Washington, D.C. [1987]) and by Bradley et al. (Current Topics in
Devel. Biol., 20:357-371 [1986]) and by Hogan et al. (Manipulating
the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y. [1986]).
[0045] Insertion of the knockout construct or the replacement
construct into the ES cells can be accomplished using a variety of
methods well known in the art, including, for example,
electroporation, microinjection, and calcium phosphate treatment. A
preferred method of insertion is electroporation. For insertion of
the DNA sequence, the knockout construct DNA or replacement
construct DNA is added to the ES cells under appropriate conditions
for the insertion method chosen. If the cells are to be
electroporated, the ES cells and construct DNA are exposed to an
electric pulse using an electroporation machine (electroporator)
and following the manufacturer's guidelines for use. After
electroporation, the cells are allowed to recover under suitable
incubation conditions. The cells are then screened for the presence
of the knockout construct or the replacement construct.
[0046] Each knockout construct DNA or replacement construct DNA to
be inserted into the cell is first typically linearized if the
knockout construct has been inserted into a vector. Linearization
is accomplished by digesting the DNA with a suitable restriction
endonuclease selected to cut only within the vector sequence and
not within the knockout construct sequence.
[0047] Screening for cells which contain the knockout construct or
replacement construct (homologous recombinants) can be done using a
variety of methods. For example, as described herein, cells can be
processed as needed to render DNA in them available for
hybridization with a nucleic acid probe designed to hybridize only
to cells containing the construct. For example, cellular DNA can be
probed with .sup.32P labelled DNA which locates outside the
targeting fragment. This technique can be used to identify those
cells with proper integration of the knockout construct or the
replacement construct. The DNA can be extracted from the cells
using standard methods, such as those described by Sambrook, J. et
al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).
The DNA can then be probed on a Southern blot with a probe or
probes designed to hybridize in a specific pattern to genomic DNA
digested with (a) particular restriction enzyme(s).
[0048] Once appropriate ES cells are identified, they are
introduced into an embryo using standard methods. They can be
introduced using microinjection, for example. Embryos at the proper
stage of development for integration of the ES cell to occur are
obtained, such as by perfusion of the uterus of pregnant females.
For example, mouse embryos at 3-4 days development can be obtained
and injected with ES cells using a micropipet. After introduction
of the ES cell into the embryo, the embryo is introduced into the
uterus of a pseudopregnant female mouse. The stage of the
pseudopregnancy is selected to enhance the chance of successful
implantation. In mice, 2-3 days pseudopregnant females are
appropriate.
[0049] Germline transmission of the mutant allele can be determined
using standard methods. Offspring resulting from implantation of
embryos containing the ES cells described above are screened for
the presence of the desired alteration (e.g., EPO knockout, EPOR
knockout, heterologous EPOR). This can be done, for example, on the
basis of coat color, if a coat color selection strategy is used.
Alternatively, DNA obtained from offspring (e.g., tail DNA) can be
assessed for the knockout construct, using known methods (e.g.,
Southern analysis, dot blot analysis, PCR analysis). See, For
example, Sambrook, J. et al., Molecular Cloning: A Laboratory
Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y. (1989). Transgene expression can also be
assessed (e.g., to determine if a replacement construct is
expressed) by known methods, such as Northern analysis or PCR
analysis, to detect transgene in RNA levels. Offspring identified
as mosaics can be crossed with one another to produce homozygous
knockout animals. As described in the Examples, homozygosity for
the Epo gene knockout is lethal. Animals homozygous for the Epo
gene deletion (i.e., animals carrying a null mutation in the Epo
gene) can be kept alive during development by administration of
mouse EPO in utero and by subcutaneous administration of EPO after
birth. Alternatively, animals homozygous for the EpoR gene deletion
can be maintained through administration of a blood substitute. It
is also possible to produce transgenic mice which carry the
human/mouse EPOR replacement and the mouse Epo gene (and, thus,
mouse EPO, which triggers the human EPOR). This would maintain the
mouse, which can be made anemic when needed, such as by treatment
in a hyperbaric chamber. High oxygen pressure will turn off EPO
production and the mouse will become anemic. The resulting anemic
mouse is useful for the assay described herein.
[0050] In an alternative embodiment, animals homozygous for a null
mutation in the Epo gene and a functional chimeric transgene for
EpoR are kept alive through development by the exogenous EPO
transgene and after birth by tetracycline-induced deletion of
exogenous EPO transgene. While tetracycline also is known to
inhibit ribosome synthesis, analogs of tetracycline are available
which do not have such properties.
[0051] DNA constructs for a functional transgene for EPO under the
control of a tetracycline promoter/enhancer system can be prepared
by inserting Loxp fragments (Kuhn, R., et al., Science,
269:1427-1429 (1995)) in front of the murine EPO cDNA and
generating transgenic mice according to standard methods (Sambrook,
et al., Molec. Cloning: A Laboratory Manual 1:2.60-280).
[0052] Transgenic mammals (e.g., transgenic mice) of the present
invention are useful to identify agents (e.g., small organic
molecules, nucleic acids, peptides) which are active in stimulating
human EPO receptor and agents which are EPO-like in their activity
(e.g., agents which regulate erythropoiesis and stimulate
erythrocyte formation), and particularly to identify agents which
mimic human EPO (EPO mimetics). They can also be used to identify
artificial blood substitutes. In one embodiment, an agent that is
active in stimulating human EPO receptor can be identified by
administering a test compound or candidate agent to a transgenic
mammal (e.g., mouse) which carries an EPO receptor with a human
extracellular domain. If the agent stimulates production of red
blood cells, then the agent is active in stimulating human EPO
receptor. In another embodiment, an EPO mimic or mimetic can be
identified by administering a candidate agent to a transgenic
mammal which carries an EPO receptor or to a transgenic mouse or
other mammal which is an EPO knockout carrying a human/mouse EPOR
in which the EPOR extracellular domain is human. If the agent
stimulates erythropoiesis in the animal (e.g., as evidenced by
increased erythrocyte number), it is an EPO mimic or mimetic and,
particularly, a hEPO mimic or mimetic. Alternatively, an EPO
knockout mouse in which the EPOR is the mEPOR can be used for the
assay. An agent identified by this means can be further tested to
assess its ability to bind the hEPOR.
[0053] In one embodiment, the method for identifying agents capable
of stimulating EPORs to promote erythropoiesis comprises (A)
administering a test agent to a transgenic mouse homozygous for a
functional transgene comprising genetic material coding for
essentially human EPOR extracellular domain; and (B) measuring the
erythropoietic effect of the test agent. In addition, the transgene
can comprise genetic material coding for essentially human EPOR
extracellular domain, genetic material coding for essentially human
and/or murine EPOR transmembrane domain and any genetic material
coding for essentially murine EPOR intracellular domain. Further,
the transgene can comprise any genetic material essentially
equivalent to exons I, VI and VII of murine EpoR gene and any
genetic material essentially equivalent to exons II, III, IV and V
of human EpoR gene.
[0054] In another embodiment, the method for identifying agents
capable of simulating EPORs to promote erythropoiesis comprises (A)
administering a test agent to a transgenic mouse homozygous for a
null EPO mutation and homozygous for a functional transgene
comprising any genetic material coding for essentially human EPOR
extracellular domain; and (B) measuring the erythropoietic effect
of the test agent. In addition, the null transgene mutation can
comprise a first 2.5 Kb PstI-PstI fragment of the 5' end of the
murine Epo gene, a second 5 Kb HindIII-KpnI fragment of the murine
Epo gene and a DNA fragment encoding a selectable marker positioned
between the first and second fragments, and the functional
transgene comprises genetic material coding for essentially human
EPOR extracellular domain, genetic material coding for essentially
human and/or urine EPOR transmembrane domain and any genetic
material coding for essentially murine EPOR intracellular domain.
Further, the functional transgene comprises any genetic material
essentially equivalent to exons I, VI and VII of murine EpoR gene
and genetic material essentially equivalent to exons II, III, IV
and V of human EpoR gene.
[0055] In another embodiment, the method for identifying agents
capable of stimulating EPOR to promote erythropoiesis comprises (A)
administering a test agent to a transgenic mouse homozygous for a
functional transgene for EPO under the control of a tetracycline
promoter/enhancer system and homozygous for a functional transgene
for EPOR comprising genetic material coding for essentially human
EPOR extracellular domain; and (B) measuring the erythropoietic
effects of the test agent.
[0056] In another embodiment, the method for identifying agents
capable of stimulating EPORs to promote erythropoiesis comprises
(A) administering a test agent to a transgenic mouse homozygous for
a functional transgene for EPO under the control of the
above-described promoter/enhancer system and homozygous for a
functional transgene for EPOR comprising any genetic material
coding for essentially human EPOR extracellular domain, any genetic
material coding for essentially human and/or murine EPOR
transmembrane domain and any genetic material coding for
essentially murine EPOR intracellular domain. Further, the
functional transgene for EPOR can comprise any genetic material
essentially equivalent to exons I, VI and VII of murine EpoR gene
and any genetic material essentially equivalent to exons II, III,
IV and V of human EpoR gene.
[0057] The EPO receptor in the methods of the present invention can
be a complete human EPO receptor or a chimeric receptor. For
example, the EPO receptor can have a mouse or human intracellular
domain. Further, the EPO receptor can have a mouse transmembrane
domain, a human transmembrane domain, or a chimeric transmembrane
domain (e.g., a transmembrane domain which includes both mouse and
human portions of the EPO receptor transmembrane domain).
[0058] In addition, in the methods of the present invention, a
transgenic mammal (e.g., mouse) can be used which produces EPO or
does not produce EPO. In the method in which the transgenic mammal
produces EPO, the methods of the present invention can further
comprise maintaining the transgenic mammal under high-oxygen
conditions sufficient to depress EPO production, reducing oxygen
availability and then administering the test compound or agent. A
transgenic mammal which does not produce EPO can be a transgenic
mammal missing genetic information encoding mouse EPO receptor. For
example, an EPO knockout mouse, which is homozygous for EPO
knockout or an EPO knockout mouse which is a cross-bred mouse in
which one ancestral line is heterozygous for mouse EPO knockout and
one ancestral line is heterozygous for the EPO receptor, can be
used in the methods of the present invention. Further, the
transgenic mammal used in the assays of the present invention can
be heterozygous for the EPO receptor and for mouse EPO receptor
production, or homozygous for EPO receptor production.
[0059] The present invention will now be illustrated by the
following Examples, which are not intended to be limiting in any
way.
EXAMPLES
Example 1
Construction of Targeting Vector for EPO Knock Out
[0060] Genomic DNA clones corresponding to the Epo locus were
isolated from a 129(J1) genomic library (Wu, H. et al., Proc. Natl.
Acad. Sci., USA, 91:2819-2823 (1994)). A restriction map was
generated by Southern blot analysis with probes derived from the
EPO cDNA, the 5' end promoter region and the 3' end untranscribed
region. Targeting vector pEPO-M1 was constructed by first inserting
the 2.5 Kb PstI-PstI fragment from the 5' end of the Epo locus
(gene) into a Bluescript vector, forming pEPO-M1-5'. Then a 1.8 Kb
EcoRI-HindIII fragment from the plasmid pGEM7(KJ1)R (Rudnicki, M.
A. et al., Cell, 71:383-390 (1992)) containing PGKneopA sequences
was inserted into the EcoRI and HindIII sites of the pEPO-M1-5'
vector forming pEPO-M1-neo) Finally, a 5.0 Kb HindIII-KpnI fragment
from the 3' region of the Epo gene was inserted into the HindIII
and KpnI sites of the pEPO-M1-neo vector.
[0061] Electroporation, Isolation of ES Clones and Southern
Analysis
[0062] J1 ES cells were cultured essentially as described (Li, E.
et al., Cell, 69:915-926 (1992)). To introduce the targeting vector
into the endogenous Epo gene, 25 .mu.g of pEPO-M1 plasmid was
linearized at the NotI site of the vector sequence and
electroporated into 1.times.10.sup.7 J1 ES cells in a volume of 0.8
ml at 400V and 25 .mu.F by use of Bio-Rad Gene Pulser. After 24
hours of culture, the ES medium was supplemented with 400 .mu.g/ml
of G418 (GIBCO/BRL) and 400 ES clones were isolated after 7-10 days
of selection. Individual clones were expanded, and genomic DNAs
were prepared as described (Laird, P. W. et al., Nucleic Acids
Res., 19:4293 (1991)). DNAs were digested with EcoRV and resolved
on a 0.7% agarose gel. After transferring, filters were hybridized
with .sup.32P labeled 1.7 Kb HindIII fragment which locates outside
of the targeting vector. A 10.0 Kb band corresponding to the
targeting allele was detected in 10.8% of ES clones isolated.
[0063] The murine Epo gene is encoded by five exons within 2.7 kb
of genomic DNA (McDonald et al., 1986; Shoemaker and Mitsock,
1986). The translational start site (ATG) is located in the first
exon. A replacement-type targeting vector, pEpo-M1, that contains
2.5 kb upstream and 5.0 kb downstream flanking sequences of the Epo
gene, and the PGKneopA cassette for positive selection was
constructed. Exons 2-5 were completely deleted so that no EPO
protein could be produced from the mutant allele. After
electroporation and drug selection, G418-resistant (G418') clones
from embryonic stem (ES) cells were isolated. DNAs from individual
clones were prepared and digested with EcoRV. After gel separation,
filters were hybridized with an external probe from the 3' flanking
region. A 10.0 kb band corresponding to the targeted allele was
detected in 10.8% of ES clones isolated.
Example 2
Construction of Targeting Vector for EPOR Knock Out
[0064] Genomic DNA clones corresponding to the EpoR locus were
isolated from a 129(J1) genomic library (Wu, H. et al., Proc. Natl.
Acad. Sci., USA, 91:2819-2823 (1994)). A restriction map was
generated by Southern blot analysis with probes derived from the
EPOR cDNA, the 5' end promoter region and the 3' end untranscribed
region. Targeting vector pEPO-M1 was constructed by first replacing
a BglII-BamHI fragment in the plasmid pGEM7(KJ1)R (Rudnicki, M. A.
et al., Cell, 71:383-390 (1992)) with a 4 Kb SalI-BamHI fragment
derived from the 5' end of the EpoR locus, yielding pEpoR-M1-5',
and then inserting 2.7 Kb XbaI-EcoRI* (site in the polylinker)
fragment derived from the 3' end of the EpoR gene into the XbaI and
EcoRI sites of the plasmid pEpoR-M1-5'.
[0065] Electroporation, Isolation of ES Clones and Southern
Analysis
[0066] J1 ES cells were cultured essentially as described (Li, E.
et al., Cell, 69:915-926 (1992)). To introduce the targeting vector
into the endogenous EpoR gene, 25 .mu.g of pEPOR-M1 plasmid was
linearized at the SalI site, and electroporated into
1.times.10.sup.7 J1 ES cells in a volume of 0.8 ml at 400V and 25
.mu.F by use of a Bio-Rad Gene Pulser. After 24 hours of culture,
the ES medium was supplemented with 400 .mu.g/ml of G418
(GIBCO/BRL), and 400 ES clones were isolated after 7-10 days of
selection. Individual clones were expanded, and genomic DNAs were
prepared as described (Laird, P. W. et al., Nucleic Acids Res.,
19:4293 (1991)). DNAs were digested with EcoRV and resolved on a
0.7% agarose gel. After transferring, filters were hybridized with
.sup.32P labeled 1 Kb EcoRV-SalI fragment which locates outside of
the targeting vector. A 4.2 Kb band corresponding to the targeting
allele was detected in 13.5% of ES clones isolated.
[0067] The EpoR gene is encoded by eight exons with the
transmembrane domain encoded by exon 6. The minimum promoter is
located 0.5 kb upstream of the transcription initiation site
(Youssouflan et al., 1993). To silence the EpoR gene completely,
the EpoR-M-1 plasmid, in which a 4.7 kb BamHI-XbaI fragment from
the EpoR locus, including 1.6 kb from the 5' flanking region and
exons 1-6, was deleted and replaced with PGKneopA for position
selection. Among 400 G418' ES clones isolated, homologous
recombination occurred at a frequency of 13.5%.
Example 3
Generation of Germline Chimeras
[0068] Five lines of positive ES clones containing the EPO null
mutation were injected into Balb/c or C57BL/6 embryos. Chimeric
mice were backcrossed to Balb/c or C57BL/6 mice, and germline
transmission of the mutant allele was detected by Southern blot
analysis of tail DNA from F1 offspring with agouti coat color. Four
lines have transmitted the mutant allele to the germ line.
[0069] Six lines of positive ES clones containing the EPOR null
mutation were injected into Balb/c or C57BL/6 embryos. Chimeric
mice were backcrossed to Balb/c or C57BL/6 mice, and germline
transmission of the mutant allele was detected by Southern blot
analysis of tail DNA from Fl offspring with agouti coat color. Two
lines have transmitted the mutant allele to the germ line.
[0070] Epo.sup.-/- and EpoR.sup.-/- Mice Are Embryonic Lethal
[0071] Targeted ES clones carrying null mutations in the Epo or
EpoR genes were injected into C57BL/6 or BALB/c blastocysts, and
the resulting chimeric mice were backcrossed. Germline transmission
of the mutant allele was detected by Southern blot analysis of tail
DNA from F1 offspring. Four and two independently isolated ES
clones transmitted the mutant Epo and EpoR alleles, respectively,
to the germline.
[0072] Heterozygotes carrying deletions in either the Epo or EpoR
genes appeared normal and were viable and fertile. The levels of
blood hemoglobin, hematocrit, erythrocytes, and leukocytes and the
protein composition of plasma were within normal ranges.
Heterozygotes derived from independent ES clones were then
intercrossed to determine whether mice homozygous for either
mutation were viable. No mice homozygous for the Epo or EpoR null
mutation were found at 3 weeks of age; since no postnatal lethality
was observed, the homozygous Epo.sup.-/- or EpoR.sup.-/- mice were
embryonic lethal. Examination of embryos at successive stages of
embryonic development revealed that homozygous Epo.sup.-/- or
EpoR.sup.-/- embryos died between days 13 and 15 of gestation. No
difference was seen in mice derived from different ES clones or
under different genetic backgrounds.
Example 4
Construction of Human/Murine Chimeric EPOR Target Vector
[0073] The following describes the preparation of a plasmid vector
containing a DNA construct for a functional murine EPOR transgene,
encoding murine introns and Exons I, VI and VII and human Exons II,
III, IV and V of the EPO-receptor gene.
[0074] An 8.3 Kb SalI-XbaI fragment was isolated from a murine EPOR
genomic clone, prepared as in Example 2, and subcloned into the
ClaI and XbaI sites of pBluescript SK(+) cloning vector
(Stratagene, La Jolla, Calif.) to give the plasmid
pBluescript.mEPOR.Sal-Xba. A 2 Kb NarI-XbaI fragment, encompassing
exons II, III, IV, V and VI and the attendant introns of the murine
EpoR gene, was isolated from the plasmid and subcloned into the
ClaI and XbaI sites of pBluescript SK(+) to give
pBluescript.mEPOR.Nar-Xba.
[0075] The cDNA of exons from human EPO receptor was amplified
using the following PCR primers:
1 Exon II: forward primer: 5'-cgccatggctatccccttttctagCGG-
CCTTGCTGGCGGCCCCGGG, (SEQ ID NO: 1) reverse primer:
5'-ggccacccagtcctgaggactcacTCGAGCTGGTAGGAGAGCTG; (SEQ ID NO: 2)
Exon III: forward primer: 5'-ttcttggttcccccgatcgcacagGGATGA-
GCCATGGAAGCTGTG, (SEQ ID NO: 3) reverse primer:
5'-tcgctcccatcccctctcacctacCTACTTCATTGATGTGGATGA; (SEQ ID NO: 4)
Exon IV: forward primer: 5'-caggatccatttcactttctgcagTGCTCCT-
AGACGCCCCCGTGG, (SEQ ID NO: 5) reverse primer:
5'-tgcggctgttctcttagctctcacCCTCTGTACGCTCCCTGCGCC; and (SEQ ID NO:
6) Exon V: forward primer: 5'-tctcctggctccacccgcccccagGTGG-
AGATCCTGGAGGGCCGC, (SEQ ID NO: 7) reverse primer:
5'-caccccgaggaactagggcctcacCGCTAGGCGTCAGCAGCGACA (SEQ ID NO: 8)
[0076] to generate cDNA segments for the exons with a 24-base
5'-end (lower case) corresponding to murine intron sequence. The
amplified cDNA was then purified by agarose gel electrophoresis to
serve as mutagenesis primers in a "sticky feet" mutagenesis
reaction.
[0077] Phagemid particles of the plasmid pBluescript.mEPOR.Nar-Xba
were grown in E. coli CJ236. Single-stranded mutagenesis templates
were obtained and annealed in a single reaction mixture with all
the mutagenesis primers and an additional single-stranded primer:
5'-CTCGAGGTCGACGGTGGCGCCACTTTTGCAAGACC (SEQ ID NO: 9), designed to
recreate the NarI site destroyed when the NarI-XbaI fragment was
cloned into the ClaI site of the pBluescript SK(+). The primers
then were extended around the template and ligated. The resulting
double-stranded DNA was transformed into E. Coli XL-1 and clones
containing the mutations were identified by restriction mapping of
DNA minipreparations, followed by DNA sequencing. one clone (#16)
so isolated contained intact human exons II and II and the
recreated NarI site. A second clone (#37) contained intact human
exon IV and a third clone (#1) contained intact human exon V.
[0078] The final chimeric fragment was constructed by a 3-way
ligation of a ClaI-BGlII fragment containing human exon IV
(obtained from clone #37), a BglII-XbaI fragment containing human
exon V and murine exon VI (obtained from clone #1) and a ClaI-XbI
fragment containing human exons II and III and the reconstructed
NarI site (obtained from clone #16). The chimeric NarI-XbaI
fragment then was subcloned into the plasmid
pBluescript.mEPOR.Sal-Xba, replacing the murine NarI-baI fragment
with its chimeric analog, to give the plasmid
pBluescript.chEPOR.Sal-Xba.
EXAMPLE 5
Generation of Committed Erythroid BFU-E and CFU-E Progenitors Does
Not Require EPO or EPOR
[0079] Committed erythroid progenitor cells that specifically
respond to EPO are detected by the formation of discrete erythroid
colonies following in vitro culture in plasma clots or
methylcellulose and are termed the colony-forming unit-erythroid
(CFU-E) and the burst-forming unit-erythroid (BFU-E) (Gregory and
Eaves, 1977; Gregory and Eaves, 1978). As demonstrated by thymidine
suicide experiments, the CFU-E is a rapid diving cell that is
highly responsive to low concentrations of EPO and gives rise to
erythroblast colonies of 8-49 cells in 7 days (human) or of 8-64
cells in 2 days (mouse). The BFU-E is a more immature cell that
divides less frequently. This cell requires EPO as well as other
growth factors (Emerson et al., 1985), such as interleukin-3
(IL-3), granulocyte/macrophage colony-stimulating factor (GM-CSF),
and stem cell factor (SCF) to develop into grouped clusters of
erythroblasts or larger colonies (bursts) of greater than 500
erythroblasts by 15 days (human) or 7-10 days (mouse) in culture
(Gregory and Eaves, 1977, 1978). Development from the earliest
BFU-E to the latest CFU-E is a continuous process, with an
intermediate progenitor often termed the mature BFU-E (Gregory and
Eaves, 1977, 1978). The sensitivity of the progenitors to EPO is
transient. Beyond the late basophilic erythroblast stage, the level
of the EPOR drops, and the cells are no longer dependent on EPO for
continued maturation (Koury and Bondurant, 1988). While EPO is
thought to be required for proliferation from BFU-E to CFU-E and
subsequent proliferation of the CFU-Es, the role of EPO in
regulating erythroid differentiation is less defined.
[0080] Several aspects of the in vivo function of EPO and the EPOR
were investigated. Specifically, whether maternal EPO can support
fetal erythropoiesis; whether EPO plays a similar role in primitive
and definitive erythropoiesis; whether EPO is an essential factor
in determining the commitment of cells to the erythroid lineage;
and whether EPO is crucial for proliferation and differentiation of
some or all types of erythroid progenitor cells were investigated.
To address these questions, the mouse strains carrying null
mutations in the Epo or EpoR genes by gene targeting in embryonic
stem cells described in Examples 1 and 2 were used. It is shown
that heterozygous mice lacking one allele of the Epo or EpoR genes
are viable and fertile and have normal erythrocytes and leukocytes.
Homozygous mice, however, die by embryonic days 13-15 (E13-E15)
with severe anemia. By culturing fetal livers from both types of
homozygous embryos, it was shown that BFU-E and CFU-E progenitors
were present. Thus, neither EPO nor the EPOR is required for
erythroid lineage commitment or for the proliferation and
differentiation of BFU-E to CFU-E progenitors. The results reveal
an essential role for EPO in regulating definitive erythropoiesis
by controlling processes such as proliferation, survival, and
irreversible terminal differentiation of the late progenitors
(CFU-E).
[0081] Methods and Materials
[0082] Histological Analysis of Embryos
[0083] Embryos were dissected free of uterine muscle and decidua,
and the placenta and yolk sac were saved for genotyping. For
histology, the embryos were paced in 10% buffered formalin for
24-48 hours and then in successive ethanol and xylene baths and
finally embedded in Paraplast Plus (Oxford) with an Autotechnicon
mono-embedder (Technicon). Embryos were sectioned by using a
Reichert-Jung microtome and stained with hematoxylin and eosin.
[0084] Progenitor Cell Assays
[0085] Individual fetal livers were dissected free in Iscove's
modified Dulbecco's medium (IMDM), disaggregated into single cell
suspensions, passed through a Cell Strainer (70 .mu.m, Falcon), and
then washed three times in IMDM medium. Cells were diluted 1:20 in
2% acetic acid to lyse nonnucleated mature erythrocytes, and then
the remaining cells were counted. Cells from each fetal liver were
placed in triplicate in .alpha.-methylcellulose without growth
factor or supplemented with EPO (3U/ml) or a cocktail of growth
factors (3U/ml EPO and 1% pokeweed mitogen-stimulated murine spleen
cell conditioned medium) (Stam-Cell Technologies, Incorporated).
Colony formation was monitored at appropriate times (2-3 days for
CFU-E an 7-10 days for BFU-E), and benzidine-positive colonies were
counted. For retroviral infection, fetal liver cells were
resuspended in medium containing a recombinant spleen focus-forming
virus expressing EpoR (SFFVEpoR) (Pharr, et al., 1993) 4 .mu.g/ml
Polybrene and kept on ice for 2-3 hours. Following infection, cells
were washed once and placed in .alpha.-methycellulose as described
above.
[0086] Treatment of Mice with mEPO
[0087] Pregnant Epo.sup.-/- females, 6-8 weeks old, were injected
subcutaneously every day for a total of 8 days with 3,000 U/kg mEPO
(in phosphate-buffered saline containing 0.025% bovine serum
albumin, provided by Amgen, Incorporated). Blood for hematocrit
determinations was collected directly into hematocrit tubes
(Baxter) from the retroorbital sinus under anesthesia. Baseline
hematocrit determinations were performed prior to the first
injection.
[0088] Results
[0089] Definitive Erythropoiesis in Epo.sup.-/- or EpoR.sup.-/-
Fetal Liver is Completely Impaired
[0090] Gross examination of mutant embryos revealed that the most
dramatic consequence of the Epo and EpoR mutations were severe
anemia. No significant differences were observed between wild-type
and heterozygous littermates. Homozygous embryos (Epo.sup.-/-;
EpoR.sup.-/-), however, developed normally until day 13 but
appeared very pale; no red coloring characteristic of the normal
fetal liver could be seen in the Epo.sup.-/- or EpoR.sup.-/-
embryos. Placentas surrounding the homozygous embryos were of
normal size but had fewer erythrocytes. Fetal livers were four to
five times smaller than those in the normal littermates and were
very pale, indicating a significant reduction in erythrocyte
production.
[0091] Histological examination of fetal liver sections obtained
from wild-type and homozygous mutants allowed the evaluation and
comparison of erythropoietic foci within the liver parenchyma.
Erythropoietic islands were numerous in the livers from wild-type
or heterozygous animals but were not identifiable in livers from
Epo.sup.-/- embryos. In the wild-type or heterozygous fetal livers,
erythroid cells at all stages of differentiation could be seen. In
contrast, the only erythroid cells seen in the homozygous fetal
livers were large erythroblasts. All liver sections also showed
yolk sac derived erythrocytes with characteristic abundant
eosinophilic cytoplasm and large nuclei. Many pycnotic nuclei,
characteristic of apoptotic cells, were seen in liver sections from
the Epo.sup.-/- embryos, but few were visible in fetal livers from
wild-type and Epo.sup.-/- embryos. Confirming these results,
cytospin preparations from livers of normal and homozygous mice
were analyzed by Giemsa staining. Erythropoietic cells at all
stages of differentiation, including many fetal liver-derived
nonnucleated erythrocytes (N) and few yolk sac-derived nucleated
erythrocytes (Y), were seen in the wild-type liver preparation,
while only proerythroblasts (E) and yolk sac-derived nucleated
erythrocytes were identifiable in the preparation from the
Epo.sup.-/- homozygotes. The same results were obtained by
analyzing the fetal livers from the EpoR.sup.-/- embryos, compared
with those from the heterozygous and wild-type embryos. These
results indicate that EPO and EPOR are essential for controlling
production of definitive erythrocytes in fetal liver.
[0092] Fetal Livers from Epo.sup.-/- Embryos Have BFU-E and CFU-E
Progenitors
[0093] To determine at which stage of erythropoiesis EPO plays a
crucial role, erythroid progenitors in fetal livers from
Epo.sup.-/- embryos were quantified. Consistent with the anatomical
and histological analyses, livers from Epo.sup.-/- fetuses had
significantly decreased numbers of nucleated cells; an
approximately 6-fold and 17-fold reduction at E12.5 and E13.5,
respectively, compared with wild-type and heterozygous
littermates.
[0094] If the Epo.sup.-/- fetal livers do contain CFU-Es and
BFU-Es, benzidine-positive colonies after 2-3 days (for CFU-E) or
7-10 days (for BFU-E) of cultivation when exogenous EPO is
supplemented should be detected, since these progenitors express
the EPOR on their surface. For this purpose, single cell
suspensions were prepared and plated in methylcellulose culture
supplemented with a cocktail of growth factors including EPO, and
benzidine-positive colonies were counted. Fetal livers from
EPO.sup.-/- embryos contained erythroid progenitors, and the
relative number of both CFU-E and BFU-E progenitors per 10.sup.5
nucleated cells was significantly increased over that in the
wild-type and heterozygous fetal livers. These results suggested
that the homozygous fetal livers were enriched in progenitor cells.
Cytospin preparations of cloned BFU-Es showed only adult-type
definitive erythroid cells. The numbers of
CFU-granulocyte/macrophage and CFU-megakaryocyte in cultures from
homozygous Epo.sup.-/- fetal livers were normal, suggesting that
the function of EPO in vivo is restricted to the erythroid lineage.
Interestingly, in the control fetal livers, progenitor cells
undergo rapid proliferation and differentiation to generate more
mature forms of erythroid cells, and the number of nucleated cells
per fetal liver increased 4-fold from E12.5 to E13.5. On the other
hand, the relative number of progenitors decreases with embryonic
development; a 3-fold and a 2-fold reduction in the numbers of
CFU-E and BFU-E per 10.sup.5 nucleated cells, respectively, were
observed. In contrast, no significant increase in the number of
nucleated cells was observed in livers from Epo.sup.-/- embryos,
suggesting that, in the absence of EPO, fetal liver cells either
become growth-arrested or undergo apoptosis. Interestingly, the
number of BFU-E progenitors in homozygous fetal livers stayed
constant from E12.5 to E13.5, while the number of CFU-Es decreased
almost 2-fold, indicating that in the absence of EPO some of the
CFU-Es underwent apoptosis, as suggested by the histologic
analysis. No differences were seen in either the size or the degree
of hemoglobinization among erythroid colonies derived from the
Epo.sup.-/- homozygotes or their wild-type or heterozygous
littermates.
[0095] Maternal EPO Cannot Support Fetal Erythropoiesis
[0096] It has been suggested that maternal EPO could cross the
placental barrier and support fetal erythropoiesis (Koury et al.,
1988). In particular, in the EPO.sup.-/- embryos the amount might
be sufficient to allow generation of BFU-Es and CFU-Es in the fetal
liver yet insufficient to support CFU-E differentiation. However,
higher concentrations of EPO (3 U/ml) are required for in vitro
differentiation of the earlier BFU-Es than for the later CFU-Es
(0.03 U/ml) (Gregory and Eaves, 1978). The facts that Epo.sup.-/-
embryos died at E13-E15 gestation stages, right after the switch
from primitive to definitive erythropoiesis, and that no
differences were seen between Epo.sup.-/- and EpoR.sup.-/- mice,
also suggested that maternal sources of EPO cannot substantially
affect the definitive erythropoiesis in either Epo.sup.-/- or
wild-type embryos.
[0097] To confirm that material EPO did not affect fetal
erythropoiesis, Epo.sup.-/- heterozygous females, 0-7 days after
detection of the vaginal plug, were injected daily with 3000 U/kg
recombinant murine EPO (mEPO). An approximately 20% increase in
hematocrit was detected in these females after 8 days of injection.
However, no Epo.sup.-/- homozygotes were found in 83 embryos
examined after E15 of gestation, and no improvement in
erythropoiesis was observed in the homozygous embryos at E12-E13
gestation stages. These results strongly suggest that maternal EPO
cannot support fetal erythropoiesis and that the development of
BFU-E and CFU-E progenitors in the Epo.sup.-/- fetal liver is not
due to maternal EPO.
[0098] EpoR.sup.-/- Fetal Livers Also Have BFU-E and CFU-E
Progenitors
[0099] Definitive evidence that neither EPO nor the EPOR is
required for the generation of BFU-E and CFU-E progenitors came
from an analysis of homozygous EpoR.sup.-/- embryos. Direct
detection of CFU-Es and BFU-Es in fetal livers from the Epo.sup.-/-
embryos should not be possible by culture in methylcellulose, since
EPO, acting through the EPOR, is essential for the production of
both types of colonies. Indeed, when fetal liver cells from
EpoR.sup.-/- embryos were cultured with EPO, no CFU-Es or BFU-Es
could be seen. Low levels of CFU-Es and BFU-Es, however, were
detected when cultures were supplemented with a cocktail of growth
factors including EPO. Therefore, fetal liver cells from E12.5
EpoR.sup.-/- embryos with a recombinant retrovirus expressing the
wild-type EpoR (Pharr et al., 1993) were infected, and the cells
were subsequently placed in methylcellulose culture without added
growth factors or were supplemented with a cocktail of growth
factors including EPO. In the absence of added growth factors, no
CFU-Es and BFU-Es were detected after retroviral infection. In the
presence of the cocktail of growth factors, the number of erythroid
progenitors per 10.sup.5 nucleated cells was about the same as that
in normal fetal livers. Since Epo.sup.-/- and EpoR.sup.-/- embryos
died at the same developmental stage and their fetal livers
contained similar numbers of nucleated cells, it was assumed that
the total number of progenitor cells in EpoR.sup.-/- fetal livers
is very similar to that in Epo.sup.-/- fetal livers. On this basis,
the infection efficiencies for BFU-E and CFU-E progenitor cells
were estimated to be 40% and 53%, respectively. Thus, the
generation of BFU-E and CFU-E progenitors in mouse fetal liver
proceeds independently of the production and expression of EPO or
EPOR, and the essential function EPO and EPOR in vivo is to enable
the CFU-E progenitors to survive and to trigger their proliferation
and then irreversible terminal differentiation.
[0100] Primitive Erythropoiesis in the EPO.sup.-/- and EPOR.sup.-/-
Yolk Sac is Partially Impaired
[0101] In early embryogenesis (E7-E11), the yolk sac is the first
site of erythropoiesis. By E12, however, the major site of
erythropoiesis has shifted from the yolk sac to the liver. The fact
that EPO.sup.-/- and EPOR.sup.-/- mutants developed normally and
survived to E13 suggested that primitive erythropoiesis at the yolk
sac stage might be normal. However, both EPO.sup.-/- and
EPOR.sup.-/- embryos at E10-E11 of gestation were very pale,
allowing unequivocal identification of homozygous mutants. Blood
vessels in the yolk sac of EPO.sup.-/- embryos had many fewer
erythrocytes than did the normal embryos. Peripheral blood was
collected from phenotypically normal and homozygous EPO.sup.-/- or
EPOR.sup.-/- embryos at E11-E12 of gestation, and total blood cells
were counted. There was a 5- to 10-fold reduction in the number of
blood cells in either EPO.sup.-/- or EPOR.sup.-/- mutants as
compared with that in wild-type or heterozygous littermates.
Importantly, some primitive erythrocytes were present in the yolk
sac surrounding the EPOR.sup.-/- and EPO.sup.-/- embryos, through
many fewer than in normal embryos. Thus, a low level of EPO and
EPOR-independent erythropoiesis in the yolk sac allows homozygous
EPO.sup.-/- and EPOR.sup.-/- embryos to develop and survive from E7
to E13 of gestation.
[0102] Discussion
[0103] In this study, mouse strains in which either the endogenous
EPO or the EPOR gene was inactivated via homologous recombination
in ES cells were generated. The results reveal a pivotal role for
EPO and EPOR in erythropoiesis. Yolk sac hematopoiesis is
significantly reduced and leads to severe anemia. Definitive
erythropoiesis, which begins in the fetal liver and thereafter in
the spleen and bone marrow of adults, is completely impaired,
resulting in embryonic lethality by E13-E15. The deficiency in
erythropoiesis is not at the stem cell or progenitor cell level,
since both BFU-E and CFU-E progenitors are present in the fetal
livers derived from both EPO.sup.-/- and EPOR.sup.-/- mice. The
data indicate that the major function of EPO is to trigger
proliferation and then irreversible terminal differentiation of the
committed late erythroid CFU-E progenitors. EPO may also act to
prevent apoptosis of CFU-Es.
[0104] EPO Does not Cross the Placenta Into the Fetus
[0105] Whether a maternal source of EPO can support fetal
erythropoiesis is controversial. In sheep and monkeys,
administration of EPO to pregnant females causes significant
increase in the level of circulating maternal EPO and in maternal
erythropoiesis (Zanjani, et al., 1993). However, there was no
increase in the level of the fetal plasma EPO or in fetal
eticulocyte production unless EPO was directly injected into the
fetus, suggesting that the fetal rerythrocyte production is not
effected by maternal levels of EPO (Zanjani, et al., 1993). The
study using mice demonstrated the transfer of maternally
administered [.sup.125] EPO into the fetus (Koury, et al., 1988).
Although the small size of the mouse fetus and the short gestation
period in mice did not permit direct assessment of the
physiological significance of transplacental transfer of EPO, this
finding raises the possibility that EPO from a maternal source may
be involved in the regulation of erythropoiesis in the fetus.
[0106] This question was directly addressed here in the study of
EPO.sup.-/- and EPOR.sup.-/- mice. If significant amounts of
maternal EPO do cross the placenta into the fetus, it is expected
that EPO.sup.-/- mice will survive until birth. If maternal EPO
cannot support fetal erythropoiesis, then EPO.sup.-/- mice, like
EPOR.sup.-/- mice, should die during embryogenesis. No EPO.sup.-/-
and EPOR.sup.-/- homozygous mice survived to birth, and both
EPO.sup.-/- and EPOR.sup.-/- mice died at the same period of
gestation with severe anemia. Administration of high doses of rEPO
to pregnant EPO.sup.+/- females significantly increased the
hematocrit of the mothers but had no effect on the erythropoiesis
in the livers of EPO.sup.-/- fetuses or on the survival of the
homozygous embryos. Thus, the results provide definitive
physiological and functional evidence that, in mice, maternal EPO
cannot cross the placenta and regulate fetal erythropoiesis.
[0107] The Roles of EPO and the EPOR in Primitive and Definitive
Erythropoiesis
[0108] In the developing mouse embryos, the yolk sac is the
original site of hematopoiesis, with the first stem cell
colony-forming units (CFU-S) appearing at approximately E7 of
gestation (Moore and Metcalf, 1970). By E12, the fetal liver
becomes the major hematopoietic center. Fetal liver erythropoiesis
and yolk sac erythropoiesis are distinct in several ways. In the
blood islands of the yolk sac, erythrocytes mature as a cohort in a
somewhat asynchronous fashion, which is quite different from the
synchronous differentiation that occurs in the fetal liver
(Tavassoli and Yoffey, 1983). Yolk sac erythropoiesis normally
generates only large nucleated erythrocytes that synthesize
embryonic hemoglobin. Fetal liver erythropoiesis, in contrast,
produces nonnucleated erythrocytes that express adult hemoglobin.
Some experiments have demonstrated that the fetal liver and yolk
sac erythropoiesis differ in their responsiveness to EPO (Cole and
Paul, 1966).
[0109] In EPO.sup.-/- and EPOR.sup.-/- embryos, definitive
erythropoiesis was completely blocked, suggesting that EPO plays a
critical role in regulating the erythrocyte production in the fetal
liver. Primitive erythropoiesis in the yolk sac was, however, only
partially impaired, with a 5- to 10-fold reduction in erythrocyte
production. A small number of primitive erythrocytes are produced
in the complete absence of either EPO or the EPOR, indicating that
some of the erythroid progenitor cells in the yolk sac are able to
proliferate and differentiate independent of either EPO or EPOR.
Thus, the low level of CFU-E and BFU-E colonies detected in the
EPOR.sup.-/- fetal liver prior to retroviral infection could be due
to this small group of yolk sac-derived erythroid progenitors that
migrated to the fetal liver after the blood circulation started at
E9 of gestation. These results are not likely to be due to the
leakiness of the knock-out mutations, since functional proteins are
not likely to be generated from the mutant alleles. On the other
hand, the results raise the very interesting possibility that two
distinct populations of erythroid progenitors are present in the
murine yolk sac, one being EPO- and EPOR-dependent and the other
EPO- and EPOR-independent.
[0110] EPO and EPOR Are Not Required for the Formation of
Definitive Erythroid Progenitor Cells
[0111] The control of erythropoiesis by EPO has been intensively
studied both in vivo and in cell culture. Experimental manipulation
of EPO levels in animals, either through the induction of anemia
that stimulates the endogenous EPO production and increases
circulating levels of erythrocytes, or through direct
administration of EPO, resulted in a significant change in the
number of CFU-Es, but only small nd variable effects in the number
of BFU-Es in the bone marrow (Hara and Ogawa, 1977; Peschle, et
al., 1979). These studies suggest that EPO induces the formation of
CFU-Es from BFU-E progenitors. Administration of large amounts of
EPO in humans led to a significant increase in the number of BFU-Es
in the bone marrow and in the percentage of the BFU-Es active in
DNA synthesis (Dessypris, et al., 1988). Whether these effects were
due to a direct action of EPO to stimulate the development of
BFU-Es and CFU-Es from earlier progenitors or, alternatively, to
support their survival is unknown. In addition, the effects of EPO
may be indirect, possibly through the stimulation of production of
other hematopoietic growth acts or through nonspecific stimulation
of accessory or stromal cells.
[0112] The formation of BFU-Es in cell cultures is affected by a
number of growth factors besides EPO, including SCF, IL-3, and
GM-CSF (Emerson, et al., 1985). It is not known how these facts
function cooperatively with EPO in vivo in controlling
proliferation and differentiation of erythroid progenitors. The
level of EPO responsiveness correlates well with the amount of EPOR
expressed on the surface of the progenitor cells. The human early
BFU-Es have little or no EPOR on their surface and are not
responsive to EPO. After 48-72 hours of growth in the presence of
IL-3, GM-CSF, or SCF, mature (also called late) BFU-Es develop;
they express low levels of EPOR and are weakly responsive to EPO
(Sawada, et al., 1988, 1990). After another 4-5 days in culture,
these cells give rise to a number of CFU-Es that are highly
responsive to EPO (Gregory and Eaves, 1977) and express
approximately 1000 EPORs on their surface (Sawada, et al., 1988).
On the basis of these experiments, EPO was thought to function with
other growth factors, such as IL-3, GM-CS, and SCF, in controlling
the proliferation and differentiation of BFU-E progenitors to
CFU-Es. EPO is clearly the crucial growth factor for the subsequent
proliferation and differentiation of CFU-Es.
[0113] Molecular genetic analysis of mice with a null mutation in
the gene encoding GM-CSF has indicated that GM-CSF is not crucial
for erythropoiesis, or that other factors can compensate for its
function (Dranoff, et al., 1994). SCF, however, is crucial for the
development of BFU-E progenitor cells to the CFU-E stage, since
mice lacking SCF (SI mutants) or its receptor KIT (W mutants)
exhibit a significant reduction of CFU-E progenitors in their fetal
liver and suffer from severe anemia (Nocka, et al., 1989). Since
the survival and proliferation of the CFU-E progenitors depends
absolutely on EPO, these results suggest that the committed
erythroid progenitors cannot proliferate or mature further unless
both the KIT and the EPOR signal transduction pathways are
functional. While KIT binds several intracellular signal
transduction proteins, such as P13K and PL.gamma.C1, it is not
known how it contributes to proliferation and differentiation of
the erythroid cells.
[0114] Recently, it was demonstrated (Wu, et al., 1995) that SCF
could replace EPO in supporting the growth and survival of HCD57
cells, an EPO-dependent erythroid cell line. Interestingly, SCF
supported he proliferation of 32D cells expressing KIT only if they
also expressed EPOR. In HCD57 cells, KIT and the EPOR were
complexed together, and SCF rapidly induced tyrosine
phosphorylation of the EPOR. These results suggested that KIT may
activate the EPO/EPOR signal transduction pathway via tyrosine
phosphorylation of the EPOR and, in turn, the activated EPOR could
induce the further proliferation and maturation of committed
erythroid progenitor cells. Fetal livers from EPOR.sup.-/- embryos
contain CFU-Es and can be rescued by expression of recombinant
EPOR, suggesting that interaction of the KIT and EPOR at or around
the CFU-E stage may be particularly important for triggering
subsequent cell proliferation, differentiation, or both.
[0115] In summary, it has been shown that in the absence of EPO or
EPOR, BFU-E and CFU-E progenitors developed normally in vivo but
failed to undergo terminal differentiation to form mature
erythrocytes. These results suggest that neither EPO nor the EPOR
is required for erythroid lineage commitment or for the
proliferation and differentiation of BFU-E to CFU-E progenitors.
EPO and the EPOR are crucial in vivo for proliferation of CFU-E
progenitors and their survival and irreversible terminal
differentiation into erythrocytes. The results also indicate that
there are no other ligands or receptors that can replace EPO and
the EPOR in controlling definitive erythropoiesis. However, low
levels of EPO- and EPOR-independent erythropoiesis do occur in
primitive erythropoiesis at the yolk sac stage, suggesting that
other mechanisms may play a critical role.
[0116] Equivalents
[0117] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
Sequence CWU 1
1
* * * * *