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HEMATOPOIESIS
From the Departments of Laboratory and Internal
Medicine, University of California, San Francisco, and the Department
of Molecular Oncology, Genentech, San Francisco, CA.
Mpl is the thrombopoietin (TPO) receptor. The current molecular
understanding of how Mpl activation stimulates proliferation of
megakaryocyte-lineage cells is based largely on the engineered expression of Mpl in nonmegakaryocyte-lineage cell lines. However, the
relevance of these findings to Mpl signaling in primary
megakaryocyte-lineage cells remains largely unknown. Therefore, a
system was developed to study Mpl function in primary
mpl Thrombopoietin (TPO), the Mpl ligand, is the
primary physiologic regulator of megakaryocytopoiesis.1-3
Genetic disruption of TPO or mpl leads to an 85%
to 90% reduction in circulating platelets.4-6 Like other
members of the hematopoietic receptor superfamily, Mpl lacks intrinsic
tyrosine kinase activity and is thought to be activated by TPO-induced
homodimerization.7-9 The Mpl extracellular domain contains
2 cytokine receptor homology domains, each characterized by 2 pairs of
membrane-distal cysteines and a membrane-proximal WSXWS motif. The
121-amino acid intracellular domain of the 625-amino acid murine Mpl
contains membrane-proximal Box1 and Box2 motifs characteristic
of members of the hematopoietic receptor superfamily. There are 5 intracellular tyrosines.
The current understanding of how TPO engagement of Mpl activates
intracellular signaling pathways and regulates megakaryocytopoiesis comes largely from the heterologous expression of wild-type and mutant
Mpl in nonmegakaryocyte-lineage immortalized cell
lines.10-15 These studies suggest that the
membrane-proximal intracellular domain provides the primary
proliferative signal that requires activation of JAK/STAT signaling,
whereas the membrane-distal portion of the intracellular domain is
thought to be more important for maturation, possibly by prolonged
activation of the Raf/MAPK/Erk signaling pathway (reviewed in Drachman
et al16). However, this simple model of Mpl activity must
be re-evaluated in light of the recent demonstration that mice lacking
the membrane-distal half of the Mpl intracellular domain have full
megakaryocyte-lineage maturation, including normal platelet counts and
agonist-induced fibrinogen binding.17 The problems of
translating intracellular signaling data from various immortalized cell
lines to physiologically relevant primary cells and the related need to
confirm cell line findings in primary cells have been emphasized by
others.15,16,18-21 We therefore sought to develop a new
system for studying Mpl signaling in primary megakaryocyte-lineage
cells that lack endogenous Mpl expression.
Avian retroviruses fail to replicate in mammalian cells because of a
block at viral entry and a block in viral assembly. However, expression
of the subgroup A avian leukosis virus (ALV-A) receptor, TVA, on the
surfaces of mammalian cells confers susceptibility to ALV-A
infection.22,23 Furthermore, tissue-specific expression of
TVA in transgenic mice allows for selective infection of TVA-expressing cells by avian retroviral vectors.24-27 Herein we describe
a new transgenic mouse in which 815 bp human GPIIb 5'-regulatory
sequence has been used to direct megakaryocyte-lineage restricted TVA
expression. After crossing to an mpl Mice
Screening transgenic mice
Cell lines
Antibodies and cytokines Fluorescein isothiocyanate (FITC)-conjugated F4/80 is from Serotec (Raleigh, NC), and anti-Flag M2 monoclonal antibody is from Sigma (St Louis, MO). FITC-conjugated antibodies to CD41, Gr-1, CD3, and B220 and phycoerythrin-conjugated antibodies to Ter-119, and all respective isotype controls, are from Pharmingen (San Diego, CA). Antibodies to detect phosphorylated Erk 1/2 (p44/42), total Erk 1/2 (p44/42), phosphorylated (Tyr705) Stat-3, total Stat-3, phosphorylated (Ser473) Akt, and total Akt are from New England Biolabs (Beverly, MA). Antibody to Stat-5b, which also detects Stat-5a, is from Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish peroxidase (HRP)-conjugated donkey antirabbit antibody is from Amersham Pharmacia (Piscataway, NJ), and HRP-conjugated swine antigoat antibody is from Boehringer Mannheim (Indianapolis, IN). Murine stem cell factor (SCF), murine IL-3 (IL-3), and murine IL-11 (IL-11) are from PeproTech (Rocky Hill, NJ). Murine IL-6 (IL-6) is from R&D Systems (Minneapolis, MN). Genentech (San Francisco, CA) generously provided human thrombopoietin (h-TPO).Plasmid constructs Murine c-mpl cDNA was removed from plasmid pSK-c-mpl28 as a 2-kb EcoR1-Bcl1 fragment and ligated into the pCla12 shuttle vector.29 The FLAG sequence (DYKDDDDK) was generated by PCR and inserted between Mpl residues 26 and 27, immediately downstream of the amino-terminal Mpl leader sequence, generating pCla12-FLAGMpl. Using Cla1 sites, the 2-kb FLAGMpl sequence was cloned into the replication competent avian retroviral vector RCAS.29 Mpl-Y582F was generated using a 2-step PCR procedure. Primer pairs were: 5'-CCG AGC CTG CAC TGG AGG GA-3' and 5'-CAG TCC TCT GAA GTC CAT CTG-3'; and 5'-CAG ATG GAC TTC AGA GGA CTG-3' and 5'-ATA AGC TTG GGC TGC AGG-3'. The final PCR product was digested with AvrII and NcoI to generate a 209-bp fragment containing the Y582F mutation that was cloned into pCla12-FLAGMpl to generate pCla12-FLAGMpl-Y582F. Mpl- 50 was also generated using
PCR mutagenesis. The oligo pair was 5'-CCG AGC CTG CAC TGG AGG GA-3'
and 5'-CAT GCC ATG GTT ATC AGG GAC ACA GAG GTA AAG GAG-3', which
introduced 3 consecutive stop codons after Mpl residue 575. The
Blp1-Nco1 Mpl fragment from plasmid
pMT21myc-c-mpl7 (kindly provided by Marion Dorsch and Stephen P. Goff, Columbia University, New York, NY) was introduced into
pCla12-FLAGMpl and pCla12-FLAGMpl-Y582F to
generate pCla12-FLAGMpl-7 and
pCla-12FLAGMpl-7, Y582F, respectively. All Mpl sequences
were transferred into the avian RCAS vector using the Cla1
cloning site to generate RCAS-Mpl, RCAS-Mpl-7, RCAS-Mpl-Y582F,
RCAS-Mpl-50, RCAS-Mpl-7, Y582F. All Mpl mutations were
confirmed by DNA sequencing. The FLAG sequence is present on all Mpl
sequences in RCAS to facilitate immunologic detection with anti-FLAG
antibody, but the FLAG designation is left off the names of the RCAS
vectors to simplify the nomenclature. RCAS-GFP expresses an enhanced
green fluorescent protein, and RCAS-PURO expresses the puromycin
resistance gene.27
Virus production DF1 cells at 40% to 60% confluence were transfected with RCAS vectors using the calcium phosphate precipitation method as described.27 Fresh media were added 12 to 16 hours after transfection. After 5 days in culture, the virus-producing DF1 cells were subjected to FACS analysis for the expression of each FLAG-tagged Mpl construct or GFP. The RCAS system typically produces viral titers of 5 × 106 or greater.29Bone marrow cultures and infections Murine bone marrow cells were harvested using standard procedures and cultured in Iscove modified Dulbecco medium (IMDM) supplemented with 10% horse serum (Gibco-BRL), 100 U penicillin G sodium/mL, and 0.1 mg streptomycin sulfate/mL as described.27 For cells from an mpl+/+ background, the media were supplemented with 10 ng/mL SCF, 10 ng/mL IL-3, and 250 ng/mL h-TPO, whereas h-TPO was not included in the media for cells harvested from an mpl / background. Unfractionated bone marrow
cells were cultured for 48 hours before they were infected with avian
retrovirus expressing either GFP, the puromycin-selectable marker, or
one of the Mpl constructs by co-culture of virus-producing DF1 cells in
6-well plates. Infections were performed for 8 hours daily for 3 days. At 48 hours after infection, cells were selected in 250 or 500 ng/mL
h-TPO when Mpl constructs were introduced or in 1.5 µg/mL puromycin
after infection with RCAS-PURO. To assess TVA expression on
megakaryocytes from mpl/ mice, maximal
megakaryocyte-lineage maturation was achieved by culturing the bone
marrow in a serum-free medium supplemented with IL-11 (10 ng/mL), IL-6
(10 ng/mL), and h-TPO (50 ng/mL).30
FACS analysis and cell staining FACS analysis for surface markers, immunohistochemical staining for CD41 and von Willebrand factor (vWf), and Wright-Giemsa staining were performed as previously described.27 Isotype controls were used in all experiments.Immunoprecipitation and Western blot analysis For 14 to 16 hours, 1 to 2 × 107 cells were starved of serum and cytokines and then exposed for 10 minutes to 250 ng/mL h-TPO (stimulated) or buffer (unstimulated). Cells were then immediately lysed in 300 µL lysis buffer (50 mM Tris, pH 8.1, containing 150 mM NaCl, 1% NP-40, 1 µg/mL each of leupeptin, pepstatin, and aprotinin, 1 mM phenylmethylsulfonyl fluoride, and 2 mM sodium orthovanadate). Lysates were centrifuged for 10 minutes at 10 000g. For Western blots, 20 µg of clarified total protein (Bradford assay; Bio-Rad, Hercules, CA) was mixed with 2 × sodium dodecyl sulfate (SDS) sample buffer, incubated at 100°C for 5 minutes, and subjected to SDS-polyacrylamide gel electrophoresis (PAGE). For immunoprecipitations, 4 to 5 µg primary antibody was incubated with 1 mg clarified total protein and incubated overnight at 4°C. This was followed by a 2-hour incubation with protein G-coated Sepharose beads, boiling in SDS sample buffer and then SDS-PAGE. Proteins were transferred to a nitrocellulose membrane using an E&K semi-dry blotter at 1- to 2-mA/cm2 gel for 1 hour. Membranes were washed once in phosphate-buffered saline containing 0.1% Tween-20 and were blocked for 1 hour at room temperature. Membranes were incubated with primary antibodies for 1 hour at RT or O/N at 4°C, washed 3 × with 0.1% Tween-20 in phosphate-buffered saline, incubated for 45 to 60 minutes at room temperature with a 1:10 000 dilution of the appropriate HRP-conjugated secondary antibody, washed three times, and developed using Supersignal West Pico Chemiluminescent substrate kit (Pierce, Rockford, IL).Cell proliferation RCAS-infected cells expressing the Mpl constructs were washed and plated at 1 × 105 cells/mL in a 12-well culture plate in the presence of the indicated cytokines. Cells were plated in triplicate for each cytokine condition. Cell counts were determined by hemocytometry.Apoptosis assay Cells expressing the Mpl constructs were cultured at a starting density of 1 × 106 cell/mL in a 12-well tissue culture plate in IMDM containing varying h-TPO concentrations. Apoptosis was determined after 3 days in culture using the Annexin V-FITC Apoptosis Detection kit (Oncogene Research Products) according to the manufacturer's protocol. Apoptotic cells were defined by FACS analysis as those that stained positively for propidium iodide, annexin V-FITC, or both.
Human GPIIb regulatory sequence directs megakaryocyte-lineage-restricted TVA expression The tv-a transgene was detected by Southern blot analysis of tail DNA from one (9552) of 13 pups born from fertilized oocytes injected with a GPIIb-tva950 expression cassette (Figure 1A). RT-PCR of bone marrow RNA from transgenic and nontransgenic 9552 offspring demonstrated a 420-bp fragment indicative of tv-a mRNA only in mice carrying the transgene (Figure 1B, lanes 1 and 2 vs lanes 3 and 4). The 420-bp fragment did not result from DNA contaminating the RNA preparation, as evidenced by the absence of a band when reverse transcriptase was not included in the RT-PCR cDNA synthesis reaction (Figure 1B, lane 1 vs lane 2). Peripheral blood counts from the transgenic mice did not differ from their nontransgenic littermates, and overt health problems were not observed in mice followed up for 1 year.Immunohistochemical staining demonstrated TVA expression on bone marrow megakaryocytes from GPIIb-tva+ mice but not from their nontransgenic littermates (Figure 1C). TVA was not detected on mature erythrocytes or leukocytes or on their readily identifiable precursors, but occasional cells smaller than mature megakaryocytes stained positively for TVA (Figure 1C). Total bone marrow grown for 5 days in IL-3, SCF, and TPO, and then dual-stained for CD41 and TVA, demonstrated that essentially all (more than 97%) CD41-expressing cells also expressed TVA (Figure 1D). We could not convincingly detect TVA expression in the absence of CD41 expression, and all mature megakaryocytes expressed TVA. TVA expression is readily detected on splenic megakaryocytes, but we were unable to detect TVA in the splenic lymphoid tissue, the liver, small intestine, heart, and kidney of transgenic mice (data not shown). To further clarify whether functional TVA expression was limited to the megakaryocyte-lineage cells, total bone marrow was harvested and infected with RCAS-PURO, an avian retroviral vector that expresses a puromycin-resistance gene. FACS analysis of cells after RCAS-PURO infection and puromycin selection demonstrate a surface phenotype consistent with a pure megakaryocyte lineage. They express CD41 but do not express Ter119, CD3, B220, Gr-1, or F4/80. Taken together, these data strongly suggest that the 815-bp regulatory sequence of the human GPIIb gene used in our transgene construct directed megakaryocyte-lineage restricted, functional TVA expression. Virally introduced Mpl confers TPO-responsive proliferation in
mpl / background. Cultured bone marrow from
GPIIb-tva+mpl/ mice demonstrated
the expected approximately 10-fold reduction in megakaryocytes and
TVA-expressing cells compared with marrow from
GPIIb-tva+ mice (Figure
2A).
Unfractionated bone marrow from
GPIIb-tva+mpl
The membrane-proximal intracellular MPL sequence is required for TPO-induced proliferation in primary megakaryocyte-lineage cells Deletion of the membrane-proximal 10 amino acids of the Mpl intracellular domain (Mpl-7; Figure 3) unmasks a
JAK/STAT-independent TPO-induced proliferation in immortalized,
nonmegakaryocyte-lineage cells.31,32 To better understand
the biologic significance of these observations, we sought to determine
whether the same Mpl mutant has a similar phenotype in primary
megakaryocyte-lineage cells. Unfractionated bone marrow from
GPIIb-tva+mpl/ mice was infected
with RCAS-Mpl, RCAS-Mpl-7, and RCAS-GFP, and cells were selected in
TPO. Unlike what happens in immortalized cells,31,32
Mpl-7 failed to confer a TPO-induced proliferative signal in primary
megakaryocyte-lineage cells (Figure 4A),
even at h-TPO concentrations up to 500 ng/mL. Because others have
described enhanced TPO-stimulated proliferation associated with an Mpl
Y582F mutation,14 we added a Y582F mutation to Mpl-7 to
ask whether the doubly mutated Mpl transmits a TPO-induced
proliferative signal in primary megakaryocyte-lineage cells. Bone
marrow cells from GPIIb-tva+mpl/
mice infected with RCAS-Mpl-7, Y582F (Figure 3) also failed to
proliferate in response to TPO (Figure 4A).
To exclude the possibility that the failure of Mpl- Mpl tyrosine-582 has an antiproliferative function in primary megakaryocyte-lineage cells Studies using immortalized, nonmegakaryocyte-lineage cells suggest that Mpl residue Y582 plays a critical role in a roughly 20-amino acid antiproliferative domain within the intracellular portion of Mpl.13,14 To determine whether this is true in primary megakaryocyte-lineage cells, we infected bone marrow from GPIIb-tva+mpl/ mice with
RCAS-Mpl, RCAS-MplY582F, and RCAS-Mpl-50, the latter mutant lacking
Mpl residues 576 to 625 (Figure 3). All 3 Mpl constructs conferred
TPO-dependent proliferation in TVA-expressing primary bone marrow cells
from GPIIb-tva+mpl/ mice (Figure
5A). To directly compare their
proliferative responses to TPO, equal numbers of cells expressing
wild-type or mutant Mpl were cultured, and the total cell number was
monitored over time. Compared with wild-type Mpl, Mpl-Y582F imparts a
marked TPO-dependent proliferative advantage, whereas cells expressing Mpl-50 proliferate similarly to those expressing wild-type Mpl (Figure 5B).
To determine whether changes in apoptosis can explain the
proliferative advantage provided by the point mutation Y582F (Figure 5B), cells were grown at varying TPO concentrations and assayed for
apoptosis using propidium iodide and annexin V staining. At h-TPO
concentrations of 250 ng/mL, the concentration used in Figure 5A, and
h-TPO concentrations of 100 ng/mL, the percentage of apoptotic cells
(annexin V+) was indistinguishable among the different Mpl
constructs (Figure 5C). The Mpl-Y582F proliferative advantage in Figure
5B was, therefore, not due to a decrease in apoptosis. Interestingly,
at 1 ng/mL or less h-TPO, Mpl-Y582F and Mpl- The synergistic effect of SCF on TPO-induced megakaryocyte-lineage proliferation is independent of the C-terminal 50 amino acids of Mpl SCF is synergistic with TPO for colony-forming unit-megakaryocyte colony size and number when bone marrow is cultured in vitro.33 To determine whether SCF is synergistic with TPO in stimulating the proliferation of primary megakaryocyte-lineage cells in our system, GPIIb-tva+mpl/
cells expressing wild-type Mpl, Mpl-50, or MplY582F were grown in media supplemented with either SCF, TPO, SCF and TPO, or no added
cytokines (Figure 6). Although SCF failed
to provide an independent proliferative signal in any of the cells
tested, it provided a similar and marked synergistic effect on
TPO-induced proliferation for all 3 Mpl constructs. The synergistic
effect was therefore independent of any Mpl-stimulated intracellular signaling that originated in the C-terminal 50 amino acids of the
121-amino acid Mpl intracellular domain. Of note,
GPIIb-tva+mpl/ cells expressing
Mpl-7 and Mpl-7, Y582F not only failed to proliferate in response
to TPO, as described above, they also failed to proliferate in media
containing both TPO and SCF (data not shown).
Others have described a synergistic effect between TPO and IL-11 and between TPO and erythropoietin (EPO) when total bone marrow is used to generate megakaryocyte-containing colonies in semisolid (agar) media.33 Neither IL-11 nor EPO was synergistic with TPO in proliferation assays using the early megakaryocyte-lineage cells we describe in this report, and neither cytokine provided a proliferative signal on its own. Primary megakaryocyte-lineage cells provide a new model for studies of Mpl-related intracellular signaling The ability to perform Mpl structure-function studies in a pure population of primary megakaryocyte-lineage cells should help clarify and advance our understanding of MPL-related intracellular signaling and, thereby, our understanding of megakaryocytopoiesis. Hence, we studied TPO-induced activation of 3 intracellular signaling pathways in GPIIb-tva+mpl/ cells expressing
wild-type Mpl, Mpl-50, or MplY582F. Although all 3 Mpl constructs
conferred TPO-inducible proliferation (Figure 6), Mpl-50 uniquely
proliferated in the absence of detectable Stat-5 phosphorylation and
the near-absence of detectable Stat-3 phosphorylation (Figure
7). The point mutation Y582F had no
measurable effect on this signaling cascade. Similar to studies in cell
lines14 and an Mpl knock-in mouse,17
phosphorylation of the PI3 kinase substrate Akt required the Mpl
C-terminus. Furthermore, Mpl tyrosine 582 contributes to Akt
phosphorylation (Figure 7).
TPO-stimulation of wild-type Mpl also activates the Ras-Raf-MEK-Erk
intracellular signaling cascade in cell lines and primary megakaryocytes (reviewed in Drachman et al16). To further
characterize our system, we assayed for TPO-induced Erk
activation-phosphorylation in primary megakaryocyte-lineage cells
expressing virally introduced wild-type Mpl, Mpl-Y582F, and Mpl-
Immortalized, nonmegakaryocyte-lineage cell lines have been
central to our current understanding of the mechanism of action of
various hematopoietic cytokine receptors, including
Mpl.10-16,31,32 However, cytokine receptor
structure-function studies, including those designed to help understand
the role of intracellular signaling in specific developmental and
biologic processes, should ideally be performed in primary cells that
naturally express the receptor of interest. This would help overcome
the recurring problem of cell-line dependent
findings13,15,31 and of differences between primary cells
and cell lines,14,17,18 and it would allow study of the
interaction of multiple cytokines that naturally act on the cell type
of interest. The GPIIb-tva+mpl Human GPIIb regulatory sequences can direct megakaryocyte-lineage-restricted functional transgene expression As part of our interest in establishing murine systems that allow for the introduction and expression of genes in primary megakaryocyte-lineage cells,27 815 bp of human GPIIb 5' regulatory sequence was used to generate transgenic mice with megakaryocyte-lineage-restricted TVA expression. Immunohistochemical analysis of multiple tissues using an anti-TVA antibody detected TVA expression only in the bone marrow and spleen. Although bone marrow TVA expression is readily apparent on megakaryocytes, small cells expressing TVA were also detected (Figure 1C). When total bone marrow is infected with RCAS-PURO an avian retrovirus that confers resistance
to puromycinthe puromycin-resistant cells express surface CD41, but
they do not express surface markers for lymphoid (CD3, B220), granulocyte (Gr-1), erythroid (Ter119), or macrophage (F4/80) lineages.
In addition, essentially all TVA-expressing cells co-express CD41
(Figure 1D). The small TVA-expressing bone marrow cells are therefore
most consistent with early megakaryocyte-lineage cells that would be
predicted to express TVA if transgene expression paralleled murine
GPIIb expression.
One major experimental use for the GPIIb-tva+
mice is to cross them to genetically null backgrounds to study
genes important for megakaryocyte-lineage development and megakaryocyte
biology. We therefore generated
GPIIb-tva+mpl Some reports have questioned the long-held view that GPIIb, also
called integrin GPIIb-tva+mpl 7) results in JAK/STAT-independent TPO-inducible proliferation in the Baf/3, 32D, and UT-7 immortalized cell lines engineered to express Mpl-7.31,32 Although
we confirmed the published results in immortalized Baf/3 cells, we
found that Mpl-7 fails to confer a TPO-dependent proliferative
signal in primary megakaryocyte-lineage cells from
GPIIb-tva+mpl/ mice (Figure 4).
Other reports of cell lines engineered to express various Mpl mutants
show a tight correlation between Stat-3 or Stat-5 activation, or both,
and TPO-stimulated proliferation.10,11,14 However, though
TPO-stimulation of wild-type Mpl robustly phosphorylates Stat-3 and
Stat-5 in primary megakaryocyte-lineage cells, TPO stimulation of
Mpl- Numerous studies have reported the activation of STAT family
members after TPO stimulation.18,39-44 The somewhat
confusing nature of the data are cogently summarized by a model in
which Stat-3 and Stat-5 are the key STAT family members activated by TPO-engagement of Mpl, but the specific STAT family member activated changes during the developmental continuum that defines
megakaryocytopoiesis.18 The TPO-induced Stat-3
phosphorylation demonstrated in platelets from knock-in mice expressing
a C-terminal truncated Mpl17 appears greater than what we
see with Mpl- When expressed in cell lines, Mpl Y582 contributes to an
antiproliferative domain within the intracellular portion of
Mpl.13,14 We have demonstrated for the first time that
such a domain functions in primary megakaryocyte-lineage cells (Figure
5B). Although we have not yet identified the molecular mechanism
underlying the Mpl-Y582F phenotype, it is not attributable to an
increase in Akt activation (Figure 7) or to a general increase in
anti-apoptotic signaling (Figure 5C). However, the Y582F mutation
imparts a constitutively active anti-apoptotic phenotype that remains
present at low TPO concentrations (1 ng/mL or less) that may more
closely reflect the TPO concentration seen by megakaryocyte-lineage
cells in a natural physiologic setting (Figure 5C). These findings
provide a cogent reminder of the need to study the effect of Mpl
mutations over a range of TPO concentrations. The highly similar
apoptosis data for Mpl-Y582F and Mpl- The system described allows one to study the interplay of various
physiologically relevant cytokines involved in megakaryocytopoiesis. For example, we demonstrated that SCF cannot independently stimulate the proliferation of primary megakaryocyte-lineage cells, but it has a
marked synergistic effect on TPO-induced proliferation (Figure 6). The
similarity of this finding to what is seen with cultured murine bone
marrow33 helps validate our system. Because we could
introduce Mpl mutants into the primary megakaryocyte-lineage cells from
GPIIb-tva+mpl The expression of wild-type and mutant Mpl protein in
GPIIb-TVA+mpl The GPIIb-tva+ mice described are
one of interest of a series of transgenic mice we have
generated for introducing genes into primary
megakaryocyte-lineage cells. We previously described mice in
which the GP-Ib
We thank John Scarborough (Oregon Health Sciences University,
Portland) for performing the oocyte injections; Paul Bray (Baylor College of Medicine, Houston, TX), Paul Bates (University of
Pennsylvania, Philadelphia), Phil Leder (Harvard Medical School,
Cambridge, MA), Steve Hughes (National Cancer Institute, Frederick,
MD), and Marion Dorsch and Stephen P. Goff (Columbia University, New York, NY), for plasmids, and the staff at Genentech (San Francisco, CA)
for recombinant human TPO and mpl
Submitted July 18, 2000; accepted November 13, 2000.
Supported by grant P50 HL54476 from the National Institutes of Health (NHLBI) Transfusion Medicine Specialized Center for Research.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Andrew D. Leavitt, Departments of Laboratory and Internal Medicine, University of California, San Francisco, 505 Parnassus Ave, Rm L-514, Box 0100, San Francisco, CA 94143-0100; e-mail: leavitt{at}pangloss.ucsf.edu.
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© 2001 by The American Society of Hematology.
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  C. M. Rudin, J. L. Marshall, C. H. Huang, H. L. Kindler, C. Zhang, D. Kumar, P. C. Gokhale, J. Steinberg, S. Wanaski, U. N. Kasid, et al. Delivery of a Liposomal c-raf-1 Antisense Oligonucleotide by Weekly Bolus Dosing in Patients with Advanced Solid Tumors: A Phase I Study Clin. Cancer Res., November 1, 2004; 10(21): 7244 - 7251. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
783 to +32 from a
SacII site to the GPIIb initiation ATG). The tv-a
translation initiation codon is located at the precise position
normally occupied by the initiation codon in GP-IIb (Figure
IIb, is expressed exclusively on
megakaryocyte-lineage cells and platelets. GPIIb expression was
detected on avian intra-embryonic and post-natal multilineage
progenitors.
3 signaling.
J Cell Biol.
1999;147:1419-1429