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RED CELLS
From the Division of Hematology, Department of Internal
Medicine, Washington University School of Medicine, St Louis, MO, and
the Howard Hughes Medical Institute, Harvard Medical School, Boston,
MA.
Patients with paroxysmal nocturnal hemoglobinuria (PNH) have blood
cells deficient in glycosyl phosphatidylinositol (GPI)-linked proteins
owing to a somatic mutation in the X-linked PIGA gene. To
target Piga recombination to the erythroid/megakaryocytic
lineage in mice, the Cre/loxP system was used, and Cre was
expressed under the transcriptional regulatory sequences of
GATA-1. Breeding of GATA1-cre (G) transgenic mice with mice
carrying a floxed Piga (L) allele was associated
with high embryonic lethality. However, double-transgenic (GL) mice
that escaped early recombination looked healthy and were observed for
16 months. Flow cytometric analysis of peripheral blood cells showed
that GL mice had up to 100% of red cells deficient in GPI-linked
proteins. The loss of GPI-linked proteins on the cell surface occurred
late in erythroid differentiation, causing a proportion of red cells to
express low residual levels of GPI-linked proteins. Red cells with
residual expression of GPI-linked proteins showed an intermediate
sensitivity toward complement and thus resemble PNH type II cells in
patients with PNH. Recombination of the floxed Piga allele
was also detected in cultured megakaryocytes, mast cells, and
eosinophils, but not in neutrophils, lymphocytes, or nonhematopoietic
tissues. In summary, GATA1-Cre causes high-efficiency Piga
gene inactivation in a GATA-1-specific pattern. For the first time,
mice were generated that have almost 100% of red cells deficient in
GPI-linked proteins. These animals will be valuable to further
investigate the consequences of GPI-anchor deficiency on
erythroid/megakaryocytic cells.
(Blood. 2001;98:2248-2255) In patients with paroxysmal nocturnal
hemoglobinuria (PNH), a somatic mutation in the X-linked
PIGA gene causes a proportion of blood cells to be deficient
in all proteins that are linked to the membrane via a glycosyl
phosphatidylinositol (GPI) molecule.1,2 PIGA
encodes a protein subunit of the N-acetyl glucosaminyltransferase, an
enzyme essential in GPI-anchor biosynthesis.3 The lack of GPI-linked proteins on blood cells is responsible for some of the
clinical symptoms seen in patients with PNH. For example, intravascular
hemolysis and hemoglobinuria are caused by the lack of CD55
decay accelerating factor (DAF) and CD59 membrane inhibitor of reactive
lysis (MIRL) on red cells. CD55 and CD59 are GPI-linked complement-regulatory molecules. Their absence increases the
sensitivity of PNH red cells toward complement-mediated lysis, which,
when the complement becomes activated, leads to intravascular hemolysis and hemoglobinuria.
PNH is believed to be a disorder of the hematopoietic stem cell. Cells
deficient in GPI-linked proteins are found not only within the red
cells but also within the granulocytes, monocytes, platelets, and
lymphocytes (reviewed by Rosse4). An inherited form of
PNH has never been described. Indeed, Piga gene inactivation in murine embryonic stem cells is lethal in very early
embryonic development.5,6 To study the functional role of
GPI-linked proteins in hematopoiesis, we therefore generated a mouse
line in which Piga gene inactivation can be targeted
specifically to hematopoietic cells by means of the Cre/loxP
system.7 Targeting Piga gene inactivation to
the early embryo, we previously reported on mice that were mosaic for
PIGA GATA-1 was the first transcription factor shown to be necessary for
erythroid-specific gene expression.9-11 It is expressed at
low levels in early CD34+ hematopoietic stem
cells12 and at high levels in erythroblasts, megakaryocytes, mast cells, and eosinophils.13-16
Experiments using targeted gene mutation showed that GATA-1 is required
for terminal differentiation in both primitive and definitive
erythropoiesis17 and plays an important role in
megakaryocyte proliferation and differentiation.18
Transcriptional regulatory sequences necessary for expression in
primitive and definitive erythroid cells have been
identified.19,20 Recent expression studies using
lacZ, Scl, and Gfp complementary DNAs
in a transgene under these GATA-1 regulatory sequences suggested that
GATA-1 regulatory sequences are able to mediate tissue-specific gene
expression in transgenic mice.19,21,22 Here we used the
transcription regulatory elements of GATA-1 to drive the expression of
the Cre recombinase. GATA1-Cre transgenic animals were crossbred with
mice carrying a floxed Piga allele. For the first time we
generated offspring with up to 100% of red cells deficient in
GPI-linked proteins. Owing to the time point of loxPiga gene
recombination, a proportion of red cells resembled PNH type II cells
with a residual expression of GPI-linked proteins. Recombination of the
floxed Piga allele was also found in megakaryocytes, mast
cells, and eosinophils, but not in any other blood cell lineage or in
nonhematopoietic tissue.
Mice
DNA analysis
Blood cell analysis
Flow cytometric analysis Flow cytometric analysis of fetal and adult blood cells was performed as described.6 Bone marrow cells from mouse femora were washed in phosphate-buffered saline plus 2% bovine calf serum and analyzed by flow cytometry. The following chromophore-labeled monoclonal antibodies against GPI-linked surface antigens were used: CD24 (30-F1), Ly6G (RB6-8C5), and CD48 (HM48-1). Alexa Fluor 488-labeled mutant aerolysin (FLAER) that binds to the GPI-anchor protein complex26 was a generous gift from Thomas Buckley (University of Victoria, BC, Canada). Lineage specificity was determined by means of chromophore-labeled monoclonal antibodies against CD11b (M1/70), B220 (RA3-6B2), TcR chain (H57-597), CD4
(GK1.5), CD8 (53-6.7), Ter119 (TER-119), CD71 (C2), c-kit (CD117, 2B8),
and CD34 (RAM34). Lineage commitment was determined with a mixture of
phycoerythin-labeled antibodies against CD3e (145-2C11), CD4, CD5
(53-7.3), CD8, B220, CD11b, and Ter119. All antibodies were purchased
from Pharmingen (San Diego, CA).
Analysis of primitive red cells and fetal liver hematopoiesis Fetuses at embryonic day (E) 10.5 to 13.5 were isolated. Red blood cells from the umbilical cord were collected and analyzed for the expression of the GPI-linked protein CD24 by flow cytometry.6 Fetal livers were disintegrated, the cells resuspended in 2% fetal calf serum-Iscoves modified Dulbecco medium and analyzed for the expression of CD24.Cell cultures For methylcellulose cultures, 104/mL fetal liver cells or 2 × 104/mL bone marrow cells were plated in 1% methylcellulose Methocult M3434 (Stemcell Technologies, Vancouver, BC, Canada) and cultured for 7 days. For erythroid burst-forming unit (BFU-E) colonies, 2 × 104 fetal liver cells and 5 × 104 bone marrow cells were plated in Methocult M3230 (Stemcell Technologies) supplemented with 50 ng/mL rat stem cell factor (rat SCF) and 3 U/mL erythropoietin (both a gift from Amgen, Thousand Oaks, CA). After 8 days of culture, colonies were counted and individually analyzed by flow cytometry.Cultures enriched in megakaryocytes were obtained by plating 0.8 × 106/mL bone marrow cells in Myelocult M5300 medium (Stemcell Technologies) supplemented with 5 µg/mL recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) (a generous gift from Amgen) for 10 days. Cytospin preparations were stained with May-Grünwald-Giemsa27 and for acetylcholinesterase activity.28 DNA was isolated from the bulk culture and analyzed by PCR. Eosinophil cultures were obtained by plating 0.4 × 106/mL bone marrow cells in Myelocult M5300 medium supplemented with 10 ng/mL recombinant murine interleukin 3 (rmIL-3) (R&D Systems, Minneapolis, MN) and 10 ng/mL recombinant murine interleukin 5 (rmIL-5) (R&D Systems) for 7 to 10 days. Cytospin preparations from 10-day cultures were stained with May-Grünwald-Giemsa and Luxol Fast Blue.29 DNA was isolated from the bulk culture and analyzed by PCR. Liquid cultures for granulocytes were performed in Myelocult M5300 medium supplemented with 50 ng/mL rat SCF and glycosylated recombinant human granulocyte colony-stimulating factor (r-metHuG-CSF) (generously provided by Amgen). DNA was isolated from the bulk culture and analyzed by PCR. Mast cell cultures were obtained as described.30 In brief, bone marrow cells were cultured in RPMI 1640 supplemented with 1 mM sodium pyruvate, 1 mM nonessential amino acids, 5.5 × 105 2-mercaptoethanol, 0.075% sodium bicarbonate, 2 mM L-glutamine, 10% fetal bovine serum, 15% Wehi-3 conditioned medium, and 5% pokeweed mitogen-stimulated spleen-conditioned medium (Stemcell Technologies). A homogeneous population of mast cells was obtained after 4 weeks as determined by Toluidine Blue staining and the expression of CD117. DNA was isolated from the bulk culture and analyzed by PCR. Complement-sensitivity test Complement sensitivity was assessed as previously described.8 Acidified human serum was used as a source of complement. The hemoglobin released was determined by measuring absorbance at 412 nm.
Generation of GATA1-cre × loxPiga offspring To restrict Piga gene inactivation in vivo to hematopoietic cells, we crossbred mice that had a floxed Piga allele (L mice, Figure 1A) with transgenic mice expressing Cre under the regulatory sequences required for erythroid- and megakaryocyte-specific expression of the mouse GATA-1 gene (G mice, Figure 1B).19 Of 168 newborn mice, 16 carried a GATA1-cre and a loxPiga transgene (GL mice). Table 1 summarizes the numbers of GL offspring obtained and expected from the first generation of GL breeding. Interestingly, double-transgenic mice were born only from crosses in which the GATA1-cre transgene was provided by the mother and the loxPiga allele came from the father (47% of expected). No offspring carrying both transgenes were obtained from the opposite breeding. Analysis of embryos collected at different stages of development showed embryos with complex developmental abnormalities and extensive recombination of the floxed Piga allele in nonhematopoietic tissues as determined by Southern blot analysis (data not shown). Highly efficient excision of floxed DNA sequences in early embryogenesis has been observed previously in offspring of G mice bred with Rosa26 carrying a floxed-galactosidase-neomycin
phosphotransferase fusion gene and was believed to be caused by leaky
Cre expression in early embryogenesis.31 In these
experiments, Cre-mediated DNA excision was equally efficient whether
the target gene was of paternal or maternal origin. Piga
maps to the X chromosome. We find the loxPiga allele escapes
early excision only when it is on the paternal X chromosome, suggesting
some difference in Cre accessibility between paternal and maternal X
chromosomes during the crucial period in embryogenesis.
Double-transgenic mice that escaped early recombination were born
alive, looked healthy, and were indistinguishable from littermates that
carried only one of the transgenes. Owing to the breeding strategy, GL
mice of the first generation were all females. Male mice hemizygous
(G+/ GL mice have circulating red blood cells that are deficient in GPI-linked proteins Circulating red blood cells from GL mice were analyzed for the expression of GPI-linked proteins. Figure 2A shows representative examples from 2 newborn and two 4-month-old GL animals. Animals heterozygous for loxPiga (G+/ L+/) have 2 populations of red cells, 1 with normal expression of CD24 and 1 deficient in CD24. In contrast, males hemizygous
(G+/L+) and females homozygous for the
loxPiga allele (G+/L+/+) showed
close to 100% of red blood cells deficient in CD24. This is consistent
with Piga's being on the X chromosome. In male offspring, the inactivation of the one Piga allele is sufficient to
cause the loss of GPI-linked proteins. In females, the inactivation of
the Piga allele when it is on the active X chromosome will lead to the loss of GPI-linked proteins on the cell surface. In females
heterozygous for L, owing to random X-inactivation, this is expected to
occur in about half of the cells. Long-term follow-up showed a decrease
in the proportion of red cells deficient in GPI-linked proteins in
G+/L+/ animals within the first 4 months of
life (40% ± 13% at birth [no. = 15] to 31% ± 7% at the
age of 4 months [no. = 13]; P < .05). Thereafter, the
proportion of GPI-anchor-deficient red blood cells was stable over the
observation period of 16 months (Figure 2B). In
G+/L+ and G+/L+/+
mice, the proportion of red blood cells deficient in GPI-linked proteins increased in the first months of life and remained stable at
99% ± 0.5% (no. = 8) throughout the entire observation period of
up to 8 months (Figure 2B).
The deficiency of CD24 on the affected red blood cells was not complete in all cells, but showed a wide distribution ranging from almost normal to complete deficiency (Figure 2A). The proportion of red cells with a residual expression of CD24 and the level of residual expression were highest after birth but decreased in the first 4 months of life. Thereafter, only a small proportion of red blood cells had a residual expression of GPI-linked proteins, the remainder being completely deficient in CD24. Figure 2C shows the decrease in mean fluorescence intensity of the affected red cell population in GL mice over time. GATA1-Cre mediates loxPiga gene inactivation in primitive erythropoiesis To test whether GATA1-Cre mediates loxPiga gene recombination during primitive erythropoiesis, blood samples from genotyped fetuses between E10.5 and E13.5 were analyzed for the expression of CD24. At E10.5, all circulating red blood cells are nucleated primitive red blood cells derived from erythropoiesis of the yolk sac, whereas at E13.5 about 50% of circulating red blood cells are primitive red blood cells and 50% are definitive red cells. In all GL fetuses analyzed (no. = 12), GPI-linked proteins were deficient on red cells derived from primitive and definitive hematopoiesis. Figure 2D shows 2 representative G+/L+/ fetuses,
each with primitive and definitive red cells deficient in
GPI-linked proteins.
Recombination of the loxPiga allele occurs late in erythroid differentiation To investigate the stage in erythroid maturation when CD24 expression is lost, liver cells of E12.5 to E13.5 fetuses and bone marrow cells of adult mice were analyzed for the expression of CD24 and Ter119.32,33 On fetal liver cells, which at the age of E12.5 to E13.5 consist of 80% erythroid cells, a decreased expression of CD24 was detected only on Ter119high but not on Ter119low or Ter119neg cells (Figure 3, top panel). Similarly, on bone marrow cells, CD24 deficiency was detected only on cells that simultaneously expressed Ter119 at high levels (Figure 3, bottom panel). No CD24-deficient cells were detected in progenitor cells expressing c-kit+ or on CD34+Lin cells (data
not shown). This suggests that the loss of CD24 occurs at the time of
Ter119 expression. In mice hemizygous or homozygous for
loxPiga, which had almost 100% of circulating red blood
cells deficient in CD24, a small proportion (6%) of
Ter119+CD24+ cells was detected in the bone
marrow (data not shown), suggesting that the final loss of CD24 occurs
as these cells mature into circulating red cells.
To further investigate the time point of loxPiga gene
recombination, single BFU-Es grown in methylcellulose from bone marrow or fetal liver cells were analyzed for the expression of CD24. Flow
cytometric analysis of 86 BFU-Es from 7 GL mice showed that 26.7% had
reduced expression of CD24. The deficiency of CD24 on BFU-Es ranged
from a partial (20.9% of BFU-Es) to an almost complete deficiency of
CD24 (5.8% of BFU-Es), suggesting that the loss of CD24 on red cells
occurs mainly after the BFU-E maturation stage (Figure
4).
GATA1-Cre mediates recombination of the loxPiga allele in megakaryocytes, mast cells, and eosinophils but not in granulocytes, monocytes, or nonhematopoietic tissues To test whether GATA1-Cre mediates recombination of the loxPiga allele in all blood cell lineages that express GATA-1 at high levels, liquid cultures were obtained from fetal liver cells and bone marrow cells from adult mice and cultured under conditions that support the growth of megakaryocytes, mast cells, eosinophils, or granulocytes.Megakaryocytes were obtained from fetal liver or bone marrow cell
cultures in the presence of PEG-rHuMGDF (Figure
5A-B). A PCR amplification product
specific for
Homogeneous mast cell cultures were obtained by culturing bone marrow
cells for 4 to 6 weeks in conditioned medium enriched in IL-3 and
SCF30 (Figure 5C-D). The loss of the loxPiga
allele and amplification of the product specific for
Cultures enriched in eosinophils were obtained from bone marrow cells
in the presence of IL-3 and IL-5 (Figure 5E-F). PCR analysis specific
for No recombination of the loxPiga allele was detected in granulocyte cultures or in monocyte/macrophage cultures either by PCR or by flow cytometric analysis (Figure 5F-G and data not shown). No or very low numbers (in 2 mice) of granulocytes, B cells, or T cells
deficient in GPI-linked proteins were detected (Figure 2B). Southern
blot and PCR analysis with DNA isolated from a variety of tissues from
3 representative GL animals showed recombination of the
loxPiga allele only in bone marrow and spleen cells but not
in any other tissue (representative data are displayed in Figure
6). The sole exception was DNA isolated
from the testis of a G+/
Red blood cells with residual expression of GPI-linked proteins have an intermediate sensitivity toward complement-mediated lysis To assess the complement sensitivity of red blood cells with a residual expression of GPI-linked proteins, we incubated red cells from G+/L+/+ animals with serial dilutions of
acidified human serum.8 Red cells from healthy mice and
from mice whose blood cells completely lack GPI-linked
proteins34 were used as controls. At all complement concentrations, more GL red cells were lysed when compared with normal
red cells, but fewer GL red cells were lysed when compared with red
cells that completely lack GPI-linked proteins (Figure 7A). This indicates that red blood cells
with a residual expression of GPI-linked proteins have an intermediate
sensitivity to complement.
G+/
The deficiency of GPI-linked proteins on the cell surface is the hallmark of blood cells in patients with PNH. The lack, or decreased expression, of these proteins is believed to be responsible for the clinical symptoms of patients with this disease. A mouse that has blood cells deficient in GPI-linked proteins and closely mimics the human disease would provide a powerful tool for studying the pathophysiology of PNH and the role of GPI-linked proteins on blood cells. Here we report on the generation of mice that have almost 100% of red blood cells deficient in GPI-linked proteins. In contrast to our previously reported mice, which were mosaic for a nonfunctional Piga gene and had only a small proportion of red cells deficient in GPI-linked proteins,8 the mice described here show highly efficient Piga gene recombination limited to hematopoietic cells of the erythroid/megakaryocytic lineage. Owing to the time point of loxPiga gene recombination, a proportion of red blood cells in our GL mice copied the phenotype that in humans has previously been described as PNH type II cells. GATA1-Cre-mediated Piga gene inactivation was associated with a high embryonic lethality owing to leaky Cre expression in early embryogenesis leading to loxPiga gene recombination in multiple nonhematopoietic tissues. However, a proportion of GL mice escaped early embryonic gene recombination, were born alive, looked healthy, and were fertile. All double-transgenic mice born alive had circulating red blood cells deficient in GPI-linked proteins. In males hemizygous and females homozygous for the loxPiga allele, the proportion of red cells deficient in GPI-linked proteins was almost 100% in all animals tested, suggesting that GATA1-Cre-mediated recombination of the loxPiga allele in erythroid cells occurs at very high efficiency throughout adult life. A decreased expression of GPI-linked proteins was also detected on circulating primitive red cells of GL fetuses, indicating that recombination of the loxPiga allele also occurs in primitive erythropoiesis originating from the blood cell islands of the yolk sack. Red cells in patients with PNH have been classified as PNH type I, II, and III cells depending on their sensitivity toward complement.35 PNH type I cells have a normal sensitivity toward complement, whereas PNH type III cells are highly sensitive. PNH type II cells are complement sensitive as well, but to a lesser extent.35 In humans, complement sensitivity of red cells depends directly on the expression level of CD59 and CD55. Thus, type I cells have a normal, and type II cells a decreased, expression of CD55 and CD59, while type III cells completely lack these 2 proteins. Point mutations in the PIGA gene that only impair but do not abrogate the function of the glycosyltransferase are mainly responsible for the PNH type II phenotype of red cells from patients with PNH.36 In GL mice, Cre-mediated DNA excision of Piga exon 2 completely abolishes PIGA function.8 The residual expression of GPI-linked proteins on red blood cells from GL mice was therefore a surprise. The expression of GPI-linked proteins ranged from close to normal levels to the complete lack of these proteins (Figure 2A). As in humans, in GL mice, red cells with a residual expression of GPI-linked proteins have an intermediate sensitivity to complement when compared with healthy red cells and with red cells that completely lack GPI-linked proteins (Figure 7A). Thus, GL red blood cells with residual expression of GPI-linked proteins phenocopy PNH type II cells from patients with PNH. The loss of GPI-anchored proteins on GL blood cells after Piga gene inactivation depends on the half-life of the PIGA protein, the half-life of the anchor precursors, and the half-life of the GPI-linked proteins on the cell surface. Investigation of the time point of Piga recombination in erythroid maturation revealed that the loss of GPI-linked proteins in GL mice occurs rather late in erythropoiesis, after the BFU-E stage, in cells that already express Ter119, which is a late erythroid differentiation marker.33 Thus, although the red cells in GL mice copy the PNH type II phenotype from patients with PNH, the molecular mechanism that gives rise to these cells is very different. In our animals, the PNH type II phenotype is caused by continuous Piga gene inactivation in late erythroid cells that do not undergo sufficient cell divisions to completely lose the expression of GPI-linked proteins. However, clinical symptoms in PNH are caused by the deficiency of GPI-linked proteins on blood cells. Thus, despite the difference in the molecular mechanisms of Piga gene inactivation, GL mice will be a valuable tool for further investigating the consequence of GPI-anchor deficiency specifically on red blood cells and possibly also on platelets. PCR analysis of DNA isolated from cultured megakaryocytes, mast cells, and eosinophils demonstrated that GATA1-Cre mediates loxPiga recombination in these blood cell lineages. Previous experiments using the GATA-1 transcription regulatory sequences showed transgene expression in megakaryocytes but not in mast cells or eosinophils.19 A possible explanation might be a short time period of transgene expression during eosinophil or mast cell maturation, which might be missed in assaying for the transgene product. In contrast, the irreversibility of loxP-dependent DNA excision and the sensitivity of the assay to screen for Piga gene inactivation in our animals provide us with a very sensitive tool to trace the earliest time point of transgene expression during maturation in an individual blood cell lineage. GATA-1 expression has been found in CD34+ hematopoietic progenitor cells enriched for hematopoietic stem cells.12 However, no evidence was found that would suggest that in GL mice recombination of the loxPiga allele occurs in hematopoietic stem cells. The small proportion of GPI-anchor-deficient white blood cells in 2 GL animals is most likely due to low-level mosaicism caused by incomplete recombination in early embryogenesis. However, low-level Piga gene inactivation in a committed pluripotent hematopoietic progenitor cell in these animals cannot be excluded. The lack of GPI-anchor-deficient cells within myeloid cells, T cells, and B cells in the majority of GL mice suggests either that GATA1-Cre expression is not sufficient or that additional expression regulatory sequences are required to cause recombination of the loxPiga allele in multipotent progenitor cells. GL mice looked healthy with no obvious signs of anemia, hemoglobinuria, or thrombosis. However, in all GL mice, red blood cell values, although within normal limits, were lower than red blood cell values from age- and sex-matched control mice (Figure 7B). Furthermore, in GL mice heterozygous for loxPiga, the proportion of mature red blood cells deficient in GPI-linked proteins was below the expected 50%. In contrast, the proportion of reticulocytes deficient in GPI-linked proteins was around 50% (Figure 2B). These findings suggest that red cells deficient in GPI-linked proteins have a decreased half-life in circulation. The most likely explanation is that this is due to the increased sensitivity of GL red cells to complement. PNH patients with only PNH type II cells have mild hemolysis and are rarely transfusion dependent. Similarly, the anemia in our mice is very mild and the reticulocytosis is moderate. In summary, findings in our GL mice demonstrate that recombination of the loxPiga allele occurs at high efficiency in a pattern specific for GATA-1 expression. Circulating red cells from GL mice share many characteristics of red cells from patients with PNH. GL mice thus promise to be an interesting tool for modeling the human disease in order to study the functional role of GPI-anchor deficiency in hemolysis and thrombosis.
We thank Patty Sipes for the excellent care of our mouse colony;
Martin Rogers for technical support; Peter Besmer, Memorial Sloan
Kettering Cancer Institute (New York, NY), for advice in mast cell
cultures; Dan Link and Philip J. Mason for stimulating discussions;
Hurry Mead and Yann Echelard, Genzyme Transgenics (Boston, MA), for
their kind provision of chicken
Submitted April 3, 2001; accepted May 30, 2001.
Supported by The Barnes Jewish Hospital Foundation, Howard Hughes Medical Institute, the Mallinckrodt Foundation, the McDonnell Foundation, and grant RO1-HL63208 from the National Institutes of Health (M.B.). S.H.O. is an investigator of the Howard Hughes Medical Institute. P.K. is the recipient of a fellowship from the Akademische Nachwuchsförderung ZH, Switzerland.
M.J. and P.K. contributed equally to this work.
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: Monica Bessler, Division of Hematology, Department of Internal Medicine, Washington University School of Medicine, 660 S Euclid Ave, Box 8125, St Louis, MO 63110-10093; email: mbessler{at}im.wustl.edu.
1. Takeda J, Miyata T, Kawagoe K, et al. Deficiency of the GPI anchor caused by a somatic mutation of the PIG-A gene in paroxysmal nocturnal hemoglobinuria. Cell. 1993;73:703-711[CrossRef][Medline] [Order article via Infotrieve]. 2. Bessler M, Mason PJ, Hillmen P, et al. Paroxysmal nocturnal haemoglobinuria (PNH) is caused by somatic mutations in the PIG-A gene. EMBO J. 1994;13:110-117[Medline] [Order article via Infotrieve].
3.
Miyata T, Takeda J, Iida Y, et al.
The cloning of PIG-A, a component in the early step of GPI-anchor biosynthesis.
Science.
1993;259:1318-1320 4. Rosse WF. Paroxysmal nocturnal hemoglobinuria as a molecular disease. Medicine (Baltimore). 1997;76:63-93[CrossRef][Medline] [Order article via Infotrieve]. 5. Kawagoe K, Takeda J, Endo Y, Kinoshita T. Molecular cloning of murine pig-a, a gene for GPI-anchor biosynthesis, and demonstration of interspecies conservation of its structure, function, and genetic locus. Genomics. 1994;23:566-574[CrossRef][Medline] [Order article via Infotrieve]. 6. Rosti V, Tremml G, Soares V, Pandolfi PP, Luzzatto L, Bessler M. Embryonic stem cells without pig-a gene activity are competent for hematopoiesis with the PNH phenotype but not for clonal expansion. J Clin Invest. 1997;100:1028-1036[Medline] [Order article via Infotrieve].
7.
Sauer B, Henderson N.
Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage P1.
Proc Natl Acad Sci U S A.
1988;85:5166-5170
8.
Tremml G, Dominguez C, Rosti V, et al.
Increased sensitivity to complement and a decreased red cell life span in mice mosaic for a nonfunctional Piga gene.
Blood.
1999;94:2945-2954
9.
Evans T, Reitman M, Felsenfeld G.
An erythrocyte-specific DNA-binding factor recognizes a regulatory sequence common to all chicken globin genes.
Proc Natl Acad Sci U S A.
1988;85:5976-5980
10.
Wall L, deBoer E, Grosveld F.
The human beta-globin gene 3' enhancer contains multiple binding sites for an erythroid-specific protein.
Genes Dev.
1988;2:1089-1100 11. Martin DI, Tsai SF, Orkin SH. Increased gamma-globin expression in a nondeletion HPFH mediated by an erythroid-specific DNA-binding factor. Nature. 1989;338:435-438[CrossRef][Medline] [Order article via Infotrieve].
12.
Sposi NM, Zon LI, Care A, et al.
Cell cycle-dependent initiation and lineage-dependent abrogation of GATA-1 expression in pure differentiating hematopoietic progenitors.
Proc Natl Acad Sci U S A.
1992;89:6353-6357 13. Martin DI, Zon LI, Mutter G, Orkin SH. Expression of an erythroid transcription factor in megakaryocytic and mast cell lineages. Nature. 1990;344:444-447[CrossRef][Medline] [Order article via Infotrieve]. 14. Romeo PH, Prandini MH, Joulin V, et al. Megakaryocytic and erythrocytic lineages share specific transcription factors. Nature. 1990;344:447-449[CrossRef][Medline] [Order article via Infotrieve].
15.
Yamamoto M, Ko LJ, Leonard MW, Beug H, Orkin SH, Engel JD.
Activity and tissue-specific expression of the transcription factor NF-E1 multigene family.
Genes Dev.
1990;4:1650-1662
16.
Zon LI, Yamaguchi Y, Yee K, et al.
Expression of mRNA for the GATA-binding proteins in human eosinophils and basophils: potential role in gene transcription.
Blood.
1993;81:3234-3241 17. Pevny L, Simon MC, Robertson E, et al. Erythroid differentiation in chimaeric mice blocked by a targeted mutation in the gene for transcription factor GATA-1. Nature. 1991;349:257-260[CrossRef][Medline] [Order article via Infotrieve]. 18. Shivdasani RA, Fujiwara Y, McDevitt MA, Orkin SH. A lineage-selective knockout establishes the critical role of transcription factor GATA-1 in megakaryocyte growth and platelet development. EMBO J. 1997;16:3965-3973[CrossRef][Medline] [Order article via Infotrieve].
19.
McDevitt MA, Fujiwara Y, Shivdasani RA, Orkin SH.
An upstream, DNase I hypersensitive region of the hematopoietic-expressed transcription factor GATA-1 gene confers developmental specificity in transgenic mice.
Proc Natl Acad Sci U S A.
1997;94:7976-7981 20. Onodera K, Takahashi S, Nishimura S, et al. GATA-1 transcription is controlled by distinct regulatory mechanisms during primitive and definitive erythropoiesis. Dev Biol. 1997;94:4487-4492.
21.
Visvader JE, Fujiwara Y, Orkin SH.
Unsuspected role for the T-cell leukemia protein SCL/tal-1 in vascular development.
Genes Dev.
1998;12:473-479
22.
Takahashi S, Shimizu R, Suwabe N, et al.
GATA factor transgenes under GATA-1 locus control rescue germline GATA-1 mutant deficiencies.
Blood.
2000;96:910-916 23. Chung JH, Whiteley M, Felsenfeld G. A 5' element of the chicken beta-globin domain serves as an insulator in human erythroid cells and protects against position effect in Drosophila. Cell. 1993;74:505-514[CrossRef][Medline] [Order article via Infotrieve]. 24. Hogan B, Beddington R, Constantini F, Lacy E. Production of transgenic mice. In: Hogan B, ed. dington R, Constantini F, Lacy E, eds. Manipulating the Mouse Embryo: A Laboratory Manual. 2nd ed. Plainview, NY: Cold Spring Harbor Laboratory Press; 1994:217-252.
25.
Keller P, Tremml G, Rosti V, Bessler M.
X inactivation and somatic cell selection rescues female mice carrying a Piga-null mutation.
Proc Natl Acad Sci U S A.
1999;96:7479-7483
26.
Brodsky RA, Mukhina GL, Li S, et al.
Improved detection and characterization of paroxysmal nocturnal hemoglobinuria using fluorescent aerolysin.
Am J Clin Pathol.
2000;114:459-466 27. Dacie JV, Lewis SM. Practical Haematology. 8th ed. Edinburgh, Scotland: Churchill Livingstone; 1995. 28. Karnovsky MJ, Roots LJ. A "direct-coloring" thiocholine method for cholinesterases. Histochem Cytochem. 1964;12:219-221[Medline] [Order article via Infotrieve]. 29. Nicola NA, Medcalf D, Johnson GR, Burgess AW. Preparation of colony stimulating factors from human placenta conditioned medium. Leuk Res. 1978;2:313-322[CrossRef].
30.
Yee NS, Langen H, Besmer P.
Mechanism of kit ligand, phorbol ester, and calcium-induced down-regulation of c-kit receptors in mast cells.
J Biol Chem.
1993;268:14189-14201
31.
Mao X, Fujiwara Y, Orkin SH.
Improved reporter strain for monitoring Cre recombinase-mediated DNA excisions in mice.
Proc Natl Acad Sci U S A.
1999;96:5037-5042 32. Ikuta K, Kina T, MacNeil I, et al. A developmental switch in thymic lymphocyte maturation potential occurs at the level of hematopoietic stem cells. Cell. 1990;62:863-874[CrossRef][Medline] [Order article via Infotrieve]. 33. Kina T, Ikuta K, Takayama E, et al. The monoclonal antibody TER-119 recognizes a molecule associated with glycophorin A and specifically marks the late stages of murine erythroid lineage. Br J Haematol. 2000;109:280-287[CrossRef][Medline] [Order article via Infotrieve]. 34. Keller P, Payne JL, Tremml G, et al. Fes-Cre targets PIGA inactivation to hematopoietic stem cells in the bone marrow. J Exp Med. In press. 35. Rosse WF. Variations in the red cells in paroxysmal nocturnal haemoglobinuria. Br J Haematol. 1973;24:327-342[Medline] [Order article via Infotrieve]. 36. Bessler M, Mason PJ, Hillmen P, Luzzatto L. Mutations in the PIG-A gene causing partial deficiency of GPI-linked surface proteins (PNH II) in patients with paroxysmal nocturnal haemoglobinuria. Br J Haematol. 1994;87:863-866[Medline] [Order article via Infotrieve].
© 2001 by The American Society of Hematology.
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  A. B. Cantor, H. Iwasaki, Y. Arinobu, T. B. Moran, H. Shigematsu, M. R. Sullivan, K. Akashi, and S. H. Orkin Antagonism of FOG-1 and GATA factors in fate choice for the mast cell lineage J. Exp. Med., March 17, 2008; 205(3): 611 - 624. [Abstract] [Full Text] [PDF]
|
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 |
|
 C. Pondarre, B. B. Antiochos, D. R. Campagna, S. L. Clarke, E. L. Greer, K. M. Deck, A. McDonald, A.-P. Han, A. Medlock, J. L. Kutok, et al. The mitochondrial ATP-binding cassette transporter Abcb7 is essential in mice and participates in cytosolic iron-sulfur cluster biogenesis Hum. Mol. Genet., March 15, 2006; 15(6): 953 - 964. [Abstract] [Full Text] [PDF]
|
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H. Iwasaki, S.-i. Mizuno, R. Mayfield, H. Shigematsu, Y. Arinobu, B. Seed, M. F. Gurish, K. Takatsu, and K. Akashi Identification of eosinophil lineage-committed progenitors in the murine bone marrow J. Exp. Med., June 20, 2005; 201(12): 1891 - 1897. [Abstract] [Full Text] [PDF]
|
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 N. Suzuki, S. Imagawa, C. T. Noguchi, M. Yamamoto, and U. Klingmuller Do {beta}-globin, GATA-1,or EpoR regulatory domains specifically mark erythroid progenitors in transgenic reporter mice? Blood, November 1, 2004; 104(9): 2988 - 2989. [Full Text] [PDF]
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M. Jasinski, P. Pantazopoulos, R. P. Rother, N. van Rooijen, W.-C. Song, H. Molina, and M. Bessler A novel mechanism of complement-independent clearance of red cells deficient in glycosyl phosphatidylinositol-linked proteins Blood, April 1, 2004; 103(7): 2827 - 2834. [Abstract] [Full Text] [PDF]
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 K. Hahm, E. Y. M. Sum, Y. Fujiwara, G. J. Lindeman, J. E. Visvader, and S. H. Orkin Defective Neural Tube Closure and Anteroposterior Patterning in Mice Lacking the LIM Protein LMO4 or Its Interacting Partner Deaf-1 Mol. Cell. Biol., March 1, 2004; 24(5): 2074 - 2082. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
10); G mice were C57BL/6.G
(N 
Piga)
Cre-mediated loxP recombination. Cre-mediated
loxPiga gene recombination deletes 662 base pairs of
Piga exon 2 and 1.6 kilobases (kb) of the adjoining intron
2, which completely abrogates the function of PIGA.
.05). (C) Decreasing
levels of residual CD24 expression on the affected red cell population
in GL mice during the observation period. The y-axis represents the
mean fluorescence intensity of red cells deficient in GPI-linked
proteins normalized to the mean fluorescence intensity determined
at birth. (D) Expression of CD24 on red cells from 4 representative
fetuses at E13.5. At this stage in development, 2 circulating red cell
populations can be identified in healthy fetuses by flow cytometry. One
population has high side scatter characteristics (SSChigh)
and a low level of CD24 expression (CD24low), which
corresponds to circulating primitive red cells. Red cells derived from
definitive hematopoiesis are SSClow and
CD24high (left panel). Note that both
E+/
represent
positive and negative PCR controls. For granulocyte cultures
G+/
