From www.bloodjournal.org by on March 15, 2011. For personal use only.

2001 97: 2213-2220

doi:10.1182/blood.V97.8.2213

{beta}Minor-globin messenger RNA accumulation in reticulocytes governs

improved erythropoiesis in {beta} thalassemic mice after erythropoietin
complementary DNA electrotransfer in muscles
Selda Samakoglu, Elena Fattori, Stefania Lamartina, Carlo Toniatti, Daniel Stockholm, Jean Michel Heard and
Delphine Bohl

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 From www.bloodjournal.org by on March 15, 2011. For personal use only.

GENE THERAPY

␤Minor-globin messenger RNA accumulation in reticulocytes governs

improved erythropoiesis in ␤ thalassemic mice after erythropoietin
complementary DNA electrotransfer in muscles
Selda Samakoglu, Elena Fattori, Stefania Lamartina, Carlo Toniatti, Daniel Stockholm, Jean Michel Heard, and Delphine Bohl

Mechanisms governing the induction of ef-
fective erythropoiesis in response to eryth-
ropoietin (Epo) oversecretion have been in-
vestigated in ␤ thalassemic C57Bl/6Hbbth
mice. Naked DNA encoding an expression
vector for mouse Epo was introduced into
skeletal muscles by electrotransfer. A tran-
sient increase of serum Epo concentrations
with a proportional augmentation of hemat-
ocrit values was observed. Various parame-
ters relevant to ␤ thalassemia were sur-
veyed in blood samples taken before
treatment, at the peak of Epo secretion, and
when the phenotype reverted to anemia. We
measured globin messenger RNA (mRNA)
levels in reticulocytes by real-time quantita-
tive polymerase chain reaction, globin chain
synthesis levels, and several indicators of
erythrocyte membrane quality, including
bound ␣ chains, bound immunoglobulins,
main protein components, and iron compart-
mentalization. Data indicated that high se-
rum Epo levels primarily affect ␤minor-
globin mRNA accumulation in reticulocytes.
Other changes subsequent to intense Epo
stimulation, like increased ␤minor/␣-globin
chain synthesis ratio, reduced levels of
␣ chains and immunoglobulins bound to
membranes, improved spectrin/band 3 ra-
tio, increased red blood cell survival, and
improved erythropoiesis appeared as con-
sequences of increased ␤minor-globin
mRNA levels. This conclusion is consistent
with models postulating that intense Epo
stimulation induces the expansion and dif-
ferentiation of erythroid progenitors commit-
ted to fetal erythropoiesis. Although pheno-
typic correction was partial in mice, and
comparable achievements will probably be
more difficult to obtain in humans, naked
DNA electrotransfer may provide a safe
and low-cost method for reassessing the
potentials of Epo as an inducer of fetal
erythropoiesis reactivation in patients with
␤ thalassemia. (Blood. 2001;97:2213-2220)
© 2001 by The American Society of Hematology

Introduction
Efficient reactivation of fetal hemoglobin (HbF) in adults would be
a convenient approach to treat hemoglobinopathies secondary to
␤-globin defects. Compounds such as 5-azacytidine, hydroxyurea,
butyric acid, or derivatives have been intensively investigated to
effect that goal.1 So far, clinical benefits appear limited, at least in ␤
thalassemias.1-4
Increased fetal erythropoiesis in response to intense stimulation
by erythropoietin (Epo) was previously observed in animal models
and in erythroid cell cultures. Severe anemia stimulates ␤C-globin
expression in sheep.5 Injections of high doses of recombinant
human Epo (rhuEpo) increased ␤minor-globin (␤min-globin) chain
synthesis in mice6 and the percentage of circulating fetal cells in
normal or anemic baboons.7,8 We showed that continuous secretion
of high quantities of murine Epo from genetically modified
muscles increased ␤min/␣-globin chain synthesis ratio in ␤ thalas-
semic mice and improved the diseased phenotype.9 In humans, a
high concentration of rhuEpo increased the proportion of colonies
containing HbF in erythroid cell cultures.10 However, trials with
rhuEpo in patients with ␤ thalassemia have been relatively
disappointing thus far, although clinical improvements were occa-
sionally observed.4,11-15 A current widely accepted view is that
reactivation of fetal erythropoiesis requires a more intense stimula-
tion in humans than in the animal models that have been explored.
A low threshold may account for clinical responsiveness in certain
individuals, including transfusion-dependent ␤ thalassemic pa-
tients.15 Encouraging results were also obtained in a trial combin-
ing high doses of Epo with hydroxyurea in patients with ␤
thalassemia intermedia.4 Tolerance was good in all reported trials.
Major drawbacks impairing further assessment of this treatment are
cost effectiveness and the theoretical risk of worsening of bone
marrow expansion and bone disease.
Molecular mechanisms governing a reactivation of fetal erythro-
poiesis are imperfectly understood. Smith and colleagues recently
reported that hydroxyurea, 5-azacytidine, or butyric acid modulates
the levels of both ␥- and ␤-globin messenger RNA (mRNA) in
human adult erythroid cells.16 Increased ␤C-globin mRNA amounts
were also observed in anemic sheep5 or in sheep bone marrow
cultures stimulated with high doses of Epo.17 It has been proposed
that a more efficient translation of ␤min-globin mRNA than
␣-globin mRNA would account, at least partly, for the compensa-
tory increase of ␤min-globin synthesis observed in ␤ thalassemic
mice, in which serum Epo concentration is elevated.18 However,
how Epo improves the phenotype of ␤ thalassemic erythrocytes has
not been directly investigated so far. The increased ratio of newly
synthesized ␤min/␣-globin chains that has been observed in ␤
thalassemic mice receiving or secreting high doses of Epo may

From Laboratoire Rétrovirus et Transfert Génétique, CNRS URA 1930; Institut
Pasteur, Paris, France; IRBM, Pomezia, Italy; and Généthon III, CNRS URA1923,
Evry, France.
Submitted July 19, 2000; accepted December 6, 2000.
Supported by grants from the Association Française contre la Myopathie. S.S.
is a fellow from Institut Pasteur, bourse Cantarini.
Reprints: Jean Michel Heard, Laboratoire Rétrovirus et Transfert Génétique,
Institut Pasteur, 28 rue du Dr Roux, 75724, Paris, France; e-mail: jmheard
@pasteur.fr.
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.
© 2001 by The American Society of Hematology

BLOOD, 15 APRIL 2001 䡠 VOLUME 97, NUMBER 8 2213

 From www.bloodjournal.org by on March 15, 2011. For personal use only.
2214 SAMAKOGLU et al
theoretically result from various, possibly combined mechanisms
acting on ␤min-globin gene transcription, ␤min-globin or ␣-globin
mRNA stability, mRNA translation efficiencies, and globin chain
proteolysis. Moreover, additional indirect effects of Epo that would
participate in the appearance of an effective erythropoiesis cannot
be excluded as possible actions on membrane defects, oxidative
processes, or iron compartmentalization, because each of these
phenomena plays a crucial role in pathophysiology.19
We investigated these issues in the C57Bl/6Hbbth mouse model
of ␤ thalassemia.20 These animals have a complete deletion of the
mouse ␤major-globin gene. Because of the compensatory elevation
of ␤min-globin chains, which decreases the amount of unpaired ␣
chains, the mouse phenotype is more relevant to that of human ␤
thalassemia intermedia than to ␤ thalassemia major.21 The stimula-
tion of ␤min-globin expression in C57Bl/6Hbbth mice is considered
as a suitable model of the reactivation of fetal erythropoiesis in
humans.22-25 We have induced Epo oversecretion in these animals
by introducing an expression vector for murine Epo into skeletal
muscles. Gene transfer was performed by the intramuscular
injection of naked DNA associated with an electric shock. In
comparison with naked DNA injection alone, this method increases
gene expression level and persistence.26 Various parameters were
measured on blood samples taken from these animals before and
twice after treatment. Values were compared in individual animals
using statistical analysis. Investigations included a survey of globin
mRNA content in reticulocytes, as measured by real-time quantita-
tive polymerase chain reaction (PCR), a study of globin chain
synthesis, and the analysis of membrane proteins components and
iron compartments. Data indicate that the accumulation of ␤min-
globin mRNA accounts for the appearance of an effective erythro-
poiesis in response to Epo overproduction in ␤ thalassemic mice.
Materials and methods
Plasmid preparation and administration
Plasmid MCK3.3/rtTA2S-S2 contains the coding sequence for rtTA2S-S2,27
a variant of the original tetracycline-inducible transactivator rtTA.28 Expres-
sion is controlled by the muscle creatine kinase (MCK) 3.3-kilobase (kb)
promoter29 and a polyadenylation signal derived from the bovine growth
hormone gene.30 The murine Epo complementary DNA (cDNA) has been
inserted in the same construct downstream of heptamerized tetO sequences
associated with a minimal human cytomegalovirus promoter.31 Plasmid
DNA was prepared by standard double CsCl gradient purification and
resuspended in sterile saline solution. Avertine-anesthetized C57Bl/6Hbbth
mice, 3 to 4 months old (bred from animals obtained at the Jackson
Laboratory, Bar Harbor, ME), were injected in the quadriceps muscles with
100 ␮g plasmid DNA. Electrostimulation was performed immediately after
injection, as described previously.26 Electrical shocks consisted of 8 trains
of 103 pulses (45 V, 50 mA, 200 ␮s/phase). The electric field was applied
through a pulsar 6 bp-a/s stimulator (FHC, Bowdoinham, ME). Injected
muscles were resected at sacrifice (22 weeks) and high-molecular-weight
DNA was prepared. Vector DNA detection by PCR showed persistence in
all treated animals. Doxycycline-HCl (Sigma, Saint-Quentin Fallavier,
France) was dissolved in the drinking water to a final concentration of 200
␮g/mL with 5% sucrose.
Hematology and Epo detection
Hemoglobin (Hb), hematocrit (Hct), and red blood cell (RBC) counts were
determined by standard procedures. Reticulocytes were counted after
staining with brilliant cresyl blue. Serum Epo concentrations were mea-
sured by enzyme-linked immunosorbent assay (ELISA).32 The assay relies
on the capture of mouse Epo on microtitration plates coated with an
antihuman Epo monoclonal antibody (mAb; R&D Systems, Minneapolis,
BLOOD, 15 APRIL 2001 䡠 VOLUME 97, NUMBER 8
MN) and revelation with a horseradish-conjugated antihuman Epo mAb
(Roche Molecular Biochemicals, Mannheim, Germany). Quantification
with recombinant mouse Epo indicates a sensitivity of 1 mU/mL.
Quantitative real-time PCR
The method is based on the detection of fluorescence generated by the
TaqMan probe degradation.33 The probe anneals between 2 amplification
primers and is degraded by the nucleolytic activity of the ampliTaq Gold
DNA polymerase at each polymerization step. Fluorescence is monitored in
real time. Intensity is related to the initial number of DNA copies, which can
be assessed be determining the threshold cycle (Ct).34
Total RNA extraction from blood samples and first-strand synthesis
were performed using standard procedures (RNeasy mini-kits, Qiagen,
Santa Clarita, CA; oligodeoxythymidine priming, Clontech, Palo Alto,
CA). Quantitative real-time PCR of cDNA was carried out with specific
double fluorescently labeled probes in an Abi Prism 7700 Sequence
Detector (Perkin-Elmer Applied Biosystems, Norwalk, CT). 6-Carboxyfluo-
rescein (FAM) was the 5⬘ fluorescent reporter and tetramethylrhodamine
(TAMRA) the 3⬘ end quencher. Probes were designed to span exon
junctions to prevent amplification of contaminating genomic DNA. The
following primers and probes were used: ␤minor-globin forward primer:
5⬘-ACCTGGGCAAGGATTTCACC-3⬘; ␤minor-globin reverse primer: 5⬘-
CCACTCCAGCCACCACCT-3⬘; ␤minor-globin probe: 5⬘-FAM-TGCTG-
CACAGGCTGCCTTCCAG-TAMRA-3⬘; ␣-globin forward primer: 5⬘-
AATATGGAGCTGAAGCCCTGG-3⬘; ␣-globin reverse primer: 5⬘-
AACATCAAAGTGAGGGAAGTAGGTCT-3⬘; ␣-globin probe: 5⬘-FAM-
AAGGATGTTTGCCAGCTTCCCCACTACT-TAMRA-3⬘.
Amplification reactions (25 ␮L) contained 10 ␮L sample cDNA or
standard DNA, 1 ⫻ TaqMan buffer, 5 mM MgCl2, 0.2 mM dA/dC/dGTP,
0.4 mM dUTP, 0.125 U AmpliTaq Gold, 0.2 mM primers (forward and
reverse), and 0.1 mM TaqMan probe. Amplification was performed by 50
cycles of 95°C, 15 seconds and 60°C, 1 minute. Standard amplification
curves were obtained by serial dilutions of the cDNA of interest, which
concentration was accurately determined.16 GAPDH mRNAs were used as
internal controls for extraction, reverse transcription, and amplification.
Data were edited using the Primer Express software (Perkin-Elmer, Applied
Biosystems, Foster City, CA).
Globin chain synthesis
Globin chain synthesis was analyzed by metabolic labeling as previously
described.9 Briefly, blood cells were washed and resuspended in methio-
nine- and cystein-free RPMI 1640 medium for 30 minutes at 37°C before a
20-minute labeling with 250 ␮Ci 35S/methionine/cystein (Amersham Life
Science, Arlington Heights, IL) and a 1-hour chase. Protein extracts (50 ␮g)
were analyzed on urea-Triton-polyacrylamide gel electrophoresis. Gels
were dried for quantification on a Phosphorimager (Molecular Dynamics,
Sunnyvale, CA). Erythrocyte ghost extracts35 and inside-out membranes
(IOMs)36 were prepared as described.
Membrane-bound immunoglobulin
Surface immunoglobulins were detected using a bead-rosette assay per-
formed on RBC suspension.37 Ten microliters of polystyrene beads
(Dynabeads M-450; Dynal, Great Neck, NY) coated with affinity-purified
goat antimouse IgG were incubated with 2.5 ⫻ 105 RBCs for 1 hour,
shaking gently at room temperature. The percentage of RBCs with attached
beads was determined using direct light microscopy. At least 400 RBCs
were counted in each experiment. Addition of soluble mouse immunoglobu-
lin to the suspension medium reduced the proportion of erythrocyte-bound
immunoglobulins in untreated ␤ thalassemic mice to that observed in
normal mice, indicating that the RBC-bead interaction was immunoglobu-
lin mediated.
Determination of free iron, nonheme iron, and heme iron
concentrations
Free iron (nonheme, nonferritin iron) was determined by reactivity with
ferrozine within 2 minutes of incubation. Freshly prepared ghosts or IOMs
 From www.bloodjournal.org by on March 15, 2011. For personal use only.
BLOOD, 15 APRIL 2001 䡠 VOLUME 97, NUMBER 8
were dissolved in 500 ␮L 0.6% sodium dodecyl sulfate (SDS) in 0.2 M
sodium acetate, pH 4.5, and incubated 5 minutes at room temperature in
0.35 M ascorbic acid, 10 M sodium metabisulfide in 0.2 M sodium acetate,
pH 4.5. The color developing solution (100 ␮L, 200 mg ferrozine and
1.25 g thiourea dissolved in 50 mL water) was added for 2 minutes and the
absorbance was measured at 562 nm. Each measurement was made in
duplicate. Concentration of total nonheme iron (membrane iron plus any
other iron such as ferritin iron) was determined after a 24-hour incubation,
after which no further reaction occurred. A millimolar extinction coefficient
of 27.9 was used for determining iron concentration. Total heme iron
content (including free heme, hemoglobin, and hemichrome) was measured
by absorbance at 398 nm of either a known amount of membrane
preparations or of IOMs dissolved in formic acid. A millimolar extinction
coefficient of 83.5 ⫾ 1.8 at 398 nm was used for determining heme iron
concentration. All reagents were rendered iron-free by treatment with a
chelating resin (Sigma Chemical, St Louis, MO).
RBC survival
Survival of RBCs was measured using a nonradioactive method.38 Mice
were injected intravenously 3 times over a 24-hour period with 1 mg
NHS-X-Biotin (Calbiochem, San Diego, CA). For analysis, 2.5 ␮L
capillary blood obtained by tail puncture was washed 3 times in phosphate-
buffered saline (PBS) with 0.5% bovine serum albumin (BSA) and
incubated 30 minutes at 4°C with 5 ␮L ALEXA-conjugated streptavidin
(Pierce Chemical, Rockford, IL). Samples were analyzed using a FACScan
(Becton Dickinson, Mountain View, CA). Labeled cells were expressed as a
percentage of total cell count (5000 per sample). Routinely, more than 94%
of circulating RBCs stained positive 24 hours after the last injection.
Statistical analysis
Statistics were performed using the SPSS software (SPSS, Chicago, IL).
Results
The effect of a transient Epo secretion from muscles was investi-
gated in 9 C57Bl/6Hbbth homozygous ␤ thalassemic mice. Animals
were studied regarding several parameters relevant to erythropoi-
esis at 3 time points: 1 week before treatment, and 4 and 11 weeks
after treatment. Data collected in individual animals before and
after treatment were analyzed using the nonparametric Wilcoxon
2–related samples test to search for relationships between the
various explored parameters. Treatment consisted of a single
intramuscular injection of DNA encoding a tetracycline-inducible
expression vector for murine Epo. Electrostimulation was per-
formed immediately after injection. We expected that inducible
expression would allow choice of various levels of Epo secretion,
as we previously observed,26 thus facilitating the analysis of
relationships between parameters. Doxycycline was given in
drinking water (200 ␮g/mL), starting 1 day after DNA injection.
Because this single dosage induced a large range of effects
depending on the individual animal, it was continuously provided
until sacrifice at 22 weeks.
Animals suffered severe anemia before treatment (Hct,
32.9% ⫾ 1.06%; Hb, 90 ⫾ 4.7 g/L; RBC count, 9.7 ⫾ 1.6 ⫻ 109/
mL) with characteristic hypochromia (mean corpuscular hemoglo-
bin concentration [MCHC], 27.2 ⫾ 1.5 pg/dL), anisocytosis, and
microcytosis (mean corpuscular volume [MCV], 35 ⫾ 1.2 fL)
(Table 1 and Figure 1A). Serum Epo concentrations, which were
below background detection level in normal mice, ranged from 18
to 61 (mean 34 ⫾ 16) mU/mL in ␤ thalassemic mice (Table 1 and
Figure 1B).
Epo STIMULATES ␤MINOR-GLOBIN mRNA ACCUMULATION 2215
Epo delivery from electroinjected muscles
All treated animals showed increased serum Epo concentrations 4
weeks after injection (200 ⫾ 75 mU/mL). Individual values were
distributed over a large range (132.6-325.4 mU/mL) (Table 1 and
Figure 1B). Concentrations decreased to 150 ⫾ 47 mU/mL be-
tween weeks 4 and 11. They were below the detection level in sera
of mice 1, 3, 5, and 8 at week 22. Thus, plasmid DNA electroinjec-
tion induced a transient Epo secretion in ␤ thalassemic mice, with
peak values variable between animals. When given to normal mice,
a similar treatment plan resulted in a more stable Epo secretion.26
Improvement of erythropoiesis
The Hct, Hb concentration, and RBC counts increased in all treated
mice (Table 1). Values for serum Epo concentrations were distrib-
uted over a large range. Some animals became polycythemic, but
others were normocythemic and yet others remained anemic.
Wilcoxon tests showed significant relationships between Hct, Hb,
RBC values, and serum Epo concentrations (P ⫽ .0077 at 4 weeks
and P ⫽ .0117 at 11 weeks). These data confirmed that increasing
serum Epo concentration improves erythropoiesis in ␤ thalassemic
mice.6,9,39,40
In mouse 1, which was polycythemic at 4 weeks, erythropoietic
stimulation was associated with a correction of MCHC and MCV
(33.4 g/dL and 41.3 fL, respectively). This mouse died of polycythe-
mia 5 weeks after gene transfer. In mice 2 through 7, in which Hct
values ranged between 45.3% and 55.6% (mice 3-7) and 70.3%
(mouse 2) at 4 weeks, improvement of hypochromia and microcy-
tosis was partial. In mice 8 and 9, in which serum Epo concentra-
tions were only slightly increased, Hct values remained below 40%
and hypochromia and microcytosis were almost unchanged. These
results show that the improvement of hypochromia and microcyto-
sis was limited unless Hct reached high values.
High reticulocyte cell counts and percentages in the blood of ␤
thalassemic mice reflected chronic erythropoietic stimulation. A
decreased proportion of circulating reticulocytes was observed in
all treated mice (Table 1). This indicated that the appearance of a
more effective erythropoiesis was accompanied by a reduction of
erythroid cell proliferation, as previously reported.9 The reduction
largely varied depending on animals (eg, from 28% to 9.6%, 30.7%
to 20.5%, and 32.2% to 26% in mice 1, 5, and 8, at 4 weeks,
respectively). Wilcoxon tests showed a significant relationship
between reticulocyte proportions in the blood and serum Epo
concentrations (P ⫽ .0077 at 4 weeks; P ⫽ .0117 at 11 weeks).
However, reticulocyte counts remained elevated with respect to the
increased blood mass (between 18.7 ⫻ 105/␮L and 29.4 ⫻ 105 /␮L
at 4 weeks versus 3.1 ⫾ 0.4 ⫻ 105/␮L in normal mice), indicating
that erythropoiesis was still accelerated.
Reduced RBC survival is a hallmark of ␤ thalassemia. We
measured this parameter by labeling erythrocytes in vivo with
NHS-X-biotin.37 The half-life of normal erythrocytes measured by
this method was 18.9 ⫾ 1.9 days (n ⫽ 3), a value consistent with
that obtained using radiolabeling.37,38 Survival of erythrocytes in
untreated ␤ thalassemic mice was 9.2 ⫾ 1.1 days. Survival of
erythrocytes was 16.9 days when measured 14 weeks after
treatment in mouse 2, whose Hct value was 55.6% at that time. This
result showed that improved erythropoiesis was associated with
increased RBC survival.
Increased ␤min-globin mRNA amounts in reticulocytes
Beneficial effects of Epo on ␤ thalassemic erythropoiesis have been
previously reported in mice,6,9,39,40 and to a lesser extent in
 From www.bloodjournal.org by on March 15, 2011. For personal use only.

2216 SAMAKOGLU et al BLOOD, 15 APRIL 2001 䡠 VOLUME 97, NUMBER 8

Table 1. Hematology, globin mRNA amounts, globin chain synthesis, and red cell membrane proteins, at various time points in treated ␤-thalassemic mice
Treated mice
Wk M1 M2 M3 M4 M5 M6 M7 M8 M9

Serum Epo concentration (mU/mL) ⫺1 44.7 21.4 25.4 60.8 33.6 23.5 21.2 18.1 54.6
4 244.8 309.4 325.4 193 163 167 138.5 132.6 164
11 — 295.6 194.1 157.8 80.7 117 138 142.7 145.5
Hematocrit (%) ⫺1 33.1 33.3 32.5 32.4 34.2 33.3 32.4 30.7 34.1
4 80.5 70.3 55.6 53.5 49.6 46.7 45.3 39.8 38.2
11 — 63.8 46.6 41 37.5 38.1 34.2 34.1 33.3
Hemoglobin (g/dL) ⫺1 9.2 9 8.7 9.1 9.6 8.5 8.9 8.4 9.8
4 26.8 21.4 16 16.2 14.3 12.5 12.1 10.7 11.2
11 — 18.6 13.1 11.6 10.4 9.9 8.9 9.2 9.7
Red blood cell count (109 cells/mL) ⫺1 9.8 9.2 9.6 8.9 9.4 9.5 9.7 8.8 9.9
4 19.5 17.4 13.3 13.6 13.3 12.2 13.7 10.7 10.6
11 — 16.3 12.2 11 10.2 9.2 10.4 9.3 9.8
Reticulocytes (%) ⫺1 28 31.1 27.3 27 30.7 30.3 29.2 32.2 26.8
4 9.6 13.3 14.8 15 20.5 19.6 21.5 26 22.6
11 — 13.4 18.1 19.2 28.3 25.5 26.3 29 24
␤minor mRNA copy number ⫺1 — 18.3 18.1 18.1 17.3 15.3 15.3 16.3 14.4
4 — 48.3 44.7 35 32.1 26.9 28.7 17.5 15.7
11 — 51.2 33.5 25.6 18.7 16.2 22.4 16.4 14.8
␣-globin mRNA copy number ⫺1 — 57.8 58 58 55.4 48.6 48.4 54.3 44.6
4 — 57.5 58.3 58.5 55 48.5 48.6 54.5 45
11 — 57.7 58.1 58.2 55.6 48.9 48.5 54 44.2
␤minor/␣-globin chain synthesis ratio ⫺1 0.72 0.71 0.68 0.74 0.69 0.64 0.68 0.63 0.71
4 0.98 0.96 0.90 0.88 0.81 0.80 0.75 0.65 0.76
11 — 0.95 0.87 0.82 0.72 0.76 0.73 0.68 0.74
␣-chain content in erythrocyte membrane (% of ⫺/⫺) ⫺1 100 100 100 100 100 100 100 100 100
4 1 37.9 70.5 69.8 77.5 79.2 45.5 92 98
11 — 14.8 38.3 68.2 86.9 69.1 98.4 94.1 90.2
Spectrin/band 3-globin ratio ⫺1 0.59 0.69 0.62 0.63 0.71 0.72 0.64 0.59 0.55
4 1.35 1.36 1.26 1.25 1.02 1 1.11 1.08 0.98
11 — 1.37 1.31 1.22 1 1.2 0.86 0.83 0.76
Band 3-globin area (% of total proteins) ⫺1 32.5 25.5 26 26.8 25.8 23.7 29.8 30.6 33.9
4 20 16.1 17.8 19.3 20.7 21.7 24.9 23 25.5
11 — 15.5 17 20 21.1 20.1 27.1 28.2 27.3
Spectrin ␣ ⫹ ␤ chains (% of total proteins) ⫺1 19.3 17.6 16.1 16.9 18.3 17.1 19.1 18 18.6
4 27 21.9 22.5 24.1 21.1 21.7 26.7 24.9 24.9
11 — 21.2 22.4 24.5 21.1 24.2 23.3 23.3 20.8
Membrane-bound Ig (% positive cells) ⫺1 12 11 11 11 10 9 10 12 13
4 2 3 6 5 6 4 7 8 10
11 — 2 5 6 7 4 8 10 11

Epo indicates erythropoietin; Hb, hemoglobin; Ig, immunoglobulin.

humans.11-15 However, little is known about the mechanisms by
which Epo induces the appearance of effective erythropoiesis. We
took advantage of the large distribution of serum Epo values and
hematologic parameters in treated animals to investigate possible
relationships between the amounts of ␤min-globin mRNA present
in reticulocytes at different time points and various parameters
relevant to the appearance of an effective erythropoiesis.
We followed globin mRNA amounts in the circulating reticulo-
cytes of living animals by quantitative real-time PCR. Total RNA
was extracted from blood samples of ␤ thalassemic mice before
treatment and at weeks 4 and 11 after DNA injection. cDNA was
synthesized by reverse transcription. Primers and probes were
designed for the amplification of both murine ␤min- and ␣-globin
cDNA. The mRNA copy numbers were estimated from cDNA
amounts, which were quantified by the amplification of a reference
DNA. These values represent an operational quantification in-
tended to allow comparisons between time points and animals.
Equivalent ␣-globin mRNA levels were measured in untreated
and treated mice, indicating that Epo did not affect the expression
of the ␣-globin gene (Table 1 and Figure 1C). Estimated copy
numbers ranged between 45 and 58 mRNA molecules per reticulo-
cyte. The estimated copy number of ␤min-globin mRNAs was
16.4 ⫾ 1.6/reticulocyte in untreated mice. Values were 2- to 3-fold
more abundant 4 weeks after DNA injection in mice 2 through 7
(Table 1 and Figure 1D). They further decreased between weeks 4
and 11, but remained higher than before treatment in mice 2, 3, 4,
and 7. A Wilcoxon test showed a strong relationship between
estimated ␤min-globin mRNA copy numbers and serum Epo
concentrations (P ⫽ .0117 at weeks 4 and 11). Thus, Epo induced
either the accumulation of ␤min-globin mRNAs or the production
of erythroid cells in which ␤min-globin mRNAs accumulate. The
␤min-globin mRNA copy numbers were also significantly related
to Hct values (P ⫽ .0117 at weeks 4 and 11). They remained
unchanged in mice 8 and 9, which showed little increase of serum
Epo concentration and did not improve erythropoiesis.
In normal mice, the ␤min plus ␤maj/␣-globin mRNA ratio is
close to 1. Before treatment, the ␤min/␣-globin mRNA ratio in our
␤ thalassemic mice was 0.31 ⫾ 0.01. Values rose above 0.55 in
mice 2 through 7 after treatment, reaching up to 0.9 in mouse 2
(Figure 1E). However, values never reached 1, indicating that
imbalance persisted at the mRNA level despite the accumulation of
␤min-globin mRNA induced by Epo.
 From www.bloodjournal.org by on March 15, 2011. For personal use only.
BLOOD, 15 APRIL 2001 䡠 VOLUME 97, NUMBER 8
Figure 1. Globin chain mRNA levels and globin chain synthesis in treated ␤
thalassemic mice. Blood samples were taken from 9 C57Bl/6Hbbth mice (M1-9) 1
week before treatment (⫺1), then 4 weeks (4) and 11 weeks (11) after naked DNA
electrotransfer into muscles. Hematocrits (A) are shown as well as serum Epo
concentration measured by ELISA (B). Copy numbers of ␣-globin (C) and ␤min-
globin (D) mRNA and ␤min/␣-globin mRNA copy number ratios (E) were determined
by quantitative real-time PCR. Globin chain synthesis levels were measured by
metabolic labeling and ␤min/␣-globin chain synthesis ratios are shown (F).
Increased ␤min-globin chain synthesis
Globin chain imbalance is a characteristic feature of ␤ thalassemia.
We examined whether the accumulation of ␤min-globin mRNAs in
treated mice would translate into a more efficient synthesis of
␤min-globin chains.
Globin chain synthesis was investigated by metabolic labeling
of blood cells (Table 1 and Figure 1F). The ␤min/␣-globin chain
synthesis ratio was 0.69 ⫾ 0.036 before treatment. Values ranged
between 0.74 and 0.99 in mice 1 through 7 at 4 weeks after DNA
injection and were still higher than before treatment in mice 2
through 4 at 11 weeks. Wilcoxon tests showed that ␤min/␣-globin
chain synthesis ratios were related to the ␤min/␣-globin mRNA
ratio (P ⫽ .0117 at weeks 4 and 11), to serum Epo concentrations
(P ⫽ .0077 at week 4, P ⫽ .0117 at week 11), and to Hct
(P ⫽ .0077 at week 4, P ⫽ .0117 at week 11). As for mRNA levels,
the ratio never reached 1, indicating that unpaired ␣ chains were
still produced. These results showed that the accumulation of
Epo STIMULATES ␤MINOR-GLOBIN mRNA ACCUMULATION 2217
␤min-globin mRNA in response to Epo stimulation subsequently
stimulates the synthesis of ␤min-globin chains.
Production of qualitatively improved RBCs
Unpaired ␣-globin chains affect erythrocyte quality by liberating
iron, binding to membranes, and altering lipids and proteins
through oxidative mechanisms. These phenomena result in prema-
ture cell destruction. We examined whether increased ␤min-globin
synthesis subsequent to Epo stimulation allowed for a correction of
these defects.
The ␣-globin chain content was measured in erythrocyte ghosts.
Results showed a dramatic reduction at 4 weeks in mice 2 through 7
and a complete disappearance in mouse 1 (Table 1). Changes were
minimal in mice 8 and 9. Improvement persisted at week 11 for
mice 1 through 3, whereas phenotype reversed to accumulation in
other mice. Wilcoxon tests showed a significant relationship
between the reduction of ␣-globin chain content in erythrocyte
ghosts and serum Epo concentrations (P ⫽ .0077 at 4 weeks,
P ⫽ .0173 at 11 weeks), ␤min/␣-globin mRNA ratio (P ⫽ .0117 at
4 and 11 weeks) and ␤min/␣-globin synthesis ratio (P ⫽ .0109 at 4
weeks, P ⫽ .0117 at 11 weeks).
Spectrin and band 3 are 2 functionally and quantitatively
important proteins of erythrocyte membranes. In normal erythro-
cytes, spectrin ␣ plus ␤ chains and band 3-globin area represent
30.6% ⫾ 5.9% and 22.1% ⫾ 3.5% of total membrane proteins,
respectively. Spectrin was decreased in ␤ thalassemic erythrocytes
before treatment, whereas the band 3-globin area was slightly
increased, so that the ratio of spectrin to band 3 was 0.63 ⫾ 0.06 in
␤ thalassemic mice compared to 1.39 ⫾ 0.14 in heterozygotes.
After treatment, the proportions of spectrin ␣ plus ␤ chains
increased in mice 2 through 7 and the amount of proteins found in
the band 3-globin area decreased (Table 1). This was associated
with the disappearance of many protein fractions in the low-
molecular-weight range, indicating a general improvement of
membrane protein profiles (not shown). Improvement was related
to the disappearance of ␣-globin chains in membranes (P ⫽ .0109
at 4 weeks, P ⫽ .0117 at 11 weeks) and to the increased amount of
␤min-globin mRNA (P ⫽ .0117 at 4 and 11 weeks).
A relationship has been described between the presence of
surface immunoglobulins and the abnormal arrangement of band 3
proteins in ␤ thalassemic cells.41,42 The proportion of erythrocytes
carrying surface immunoglobulin detectable by bead-rosette anti-
immunoglubulin assay was determined before and after treatment.
Results showed a significant reduction of membrane-bound immu-
noglobulins in treated mice (Table 1). Reduction was related to that
of proteins found in the band 3-globin area (P ⫽ .0077 at 4 weeks,
P ⫽ .0117 at 11 weeks), to the spectrin/band 3-globin ratio
(P ⫽ .0077 at 4 weeks, P ⫽ .0117 at 11 weeks), to the ␣-globin
chain content in membranes (P ⫽ .0109 at 4 weeks, P ⫽ .0117 at
11 weeks) and to ␤min-globin mRNAs levels (P ⫽ .0117 at 4 and
11 weeks).
Altogether, these data indicated that the accumulation of
␤min-globin mRNA and the increased synthesis of ␤min-globin
chains in response to Epo stimulation led to the production of
qualitatively improved erythrocytes.
Iron decompartmentalization in erythrocytes
Iron decompartmentalization promotes the targeting of auto-
oxidative damages to the cell membrane and thereby contributes to
␤ thalassemia pathophysiology.43
 From www.bloodjournal.org by on March 15, 2011. For personal use only.
2218 SAMAKOGLU et al
We measured the size of various erythrocyte iron compart-
ments. Pretreatment values illustrated iron overload in ␤ thalasse-
mic cells as compared to heterozygotes. Data were consistent with
previous observations made in mouse37 and human44 ␤ thalassemic
cells. Values were not significantly different after treatment. In
ghost erythrocyte membranes, the accumulation of free iron
represented 5.26 ⫾ 0.85 nmoL/mg protein before treatment and
4.92 ⫾ 1.22 at 4 weeks (Figure 2A). Nonheme iron, including both
free iron and other nonheme iron deposits such as ferritin iron, was
12.2 ⫾ 4.27 nmol/mg before treatment and 12.13 ⫾ 4.43 at 4
weeks (Figure 2B). Heme iron, including hemoglobin, hemichrome,
and free heme, was measured in erythrocyte ghosts and in IOMs.
Values found for heterozygotes, untreated, and treated mice (4
weeks) were 1.73 ⫾ 0.09, 2.75 ⫾ 0.72, 2.29 ⫾ 0.63 nmol/mg,
respectively, in ghosts, and 0.75 ⫾ 0.06, 1.19 ⫾ 0.36, 0.99 ⫾ 0.35
nmol/mg, respectively, in IOMs (Figure 2C).
Figure 2. Iron decompartimentalization in treated ␤ thalassemic mice. Erythro-
cyte ghosts and inside-out membranes (IOMs) were prepared from normal mice (z),
heterozygous C57Bl/6Hbbth mice (s), and ␤ thalassemic C57Bl/6Hbbth mice before
treatment (䡺), and at 4 (f) and 11 (u) weeks after naked DNA injection. Free iron (A)
and nonheme iron (B) were measured in ghosts by spectrophotometry. Heme iron
was measured in IOMs via ferrozine reactivity (C).
BLOOD, 15 APRIL 2001 䡠 VOLUME 97, NUMBER 8
Discussion
This study was conducted of exploring the mechanisms governing
the effects of Epo overproduction in ␤ thalassemia. Transient Epo
secretion was induced in ␤ thalassemic mice by naked DNA
electrotransfer into muscles. Globin mRNA content in reticulo-
cytes, globin chain synthesis, and erythrocyte membrane defects
were analyzed. As we previously observed, high serum Epo
concentrations were strongly related to the appearance of an
effective erythropoiesis that improved the ␤ thalassemic phenotype.9
Real-time quantitative PCR allowed a precise measurement of
globin mRNAs in circulating reticulocytes of living animals. The
amount of ␣-globin mRNA was independent of serum Epo
concentration, indicating that Epo affects neither the synthesis nor
the stability of these transcripts. In contrast, a significant relation-
ship existed between serum Epo concentrations and ␤min-globin
mRNA content in reticulocytes. This suggests that a consequence
of high serum Epo concentrations would be to stimulate either the
accumulation of ␤min-globin transcripts in erythroid progenitors or
the proliferation of erythroid progenitors committed to high
␤min-globin gene expression.
The ratio of ␤min/␣-globin mRNA was directly related to that
of globin chain synthesis, both being proportional to serum Epo
concentrations. However, ␤min-globin mRNA amounts increased
more rapidly with serum Epo concentrations than ␤min-globin
chain synthesis. Thus, translation of ␤min-globin mRNA was not
facilitated by high serum Epo concentration and the effect of Epo
on globin chain synthesis can be accounted for solely by the
accumulation of ␤min-globin mRNAs. It is therefore unlikely that
Epo modulates the translational efficiency of globin mRNAs.
The amount of membrane-bound ␣-globin chains was inversely
proportional to serum Epo concentrations. The ␣-globin-mRNA
amounts, ␣-globin chain synthesis, and ␣-globin content (not
shown) in erythrocytes did not vary with serum Epo levels. In
contrast, membrane-bound ␣-globin chain levels were inversely
related to ␤min-globin mRNA copy numbers and to ␤min/␣-globin
chain synthesis. Thus, the amount of unpaired ␣ chains seemed to
be solely determined by the available amount of ␤min-globin
chains. Accelerated synthesis alone can account for increased
amounts of ␤min-globin chains. It may also be combined with
reduced proteolysis, although there is no direct evidence for such
an effect of Epo. The observation that the spectrin/band 3 ratio and
membrane-bound immunoglobulins were significantly linked to
␣-chain contents in erythrocyte membranes and thus to ␤min-
globin chain synthesis levels indicates that Epo effects on mem-
brane protein components were mediated by its action on the
accumulation of ␤min-globin mRNA.
Taken together, these results indicate that the mechanisms by
which Epo overproduction induced an effective erythropoiesis in ␤
thalassemic mice was primarily by favoring the accumulation of
␤min-globin mRNA in immature erythroid cells. Consistently,
accumulation of ␤C globin mRNA in response to intense Epo
stimulation was previously described in anemic sheep5 and in
sheep bone marrow cultures.17 Drugs that reactivate fetal erythro-
poiesis in human erythroid cell cultures, such as hydroxyurea,
5-azacytydine and butyrate, increase ␥-globin mRNA amounts.16
In contrast, it has been shown that the compensatory increase of
␤min-globin chain synthesis in untreated ␤ thalassemic mice
affects mRNA translation rather than accumulation.18 We assume
that this mechanism was hidden by the accumulation of ␤min-globin
mRNA in ␤ thalassemic mice secreting high amounts of Epo.
 From www.bloodjournal.org by on March 15, 2011. For personal use only.

BLOOD, 15 APRIL 2001 䡠 VOLUME 97, NUMBER 8 Epo STIMULATES ␤MINOR-GLOBIN mRNA ACCUMULATION 2219

Several lines of evidence suggest that the accumulation of
␤min-globin mRNA in C57Bl/6Hbbth mouse reticulocytes is subse-
quent to the expansion of an erythroid progenitor compartment in
which ␤min-globin transcripts are abundant. A similar mechanism
could possibly be recruited for a reactivation of fetal erythropoiesis
in humans, although effectiveness may require higher Epo serum
concentration. In vitro cultures of mouse45 and human10 erythroid
progenitors have shown that Epo stimulates the expansion of a cell
compartment with a potential for ␤min- or ␥-globin synthesis.
Consistent results were obtained during a short stimulation with
rhuEpo in normal or anemic baboons.7 A model has been proposed
based on the observation that (1) the distribution of HbF is
heterocellular in normal human adult bone marrow with a small
percentage of maturing progenitors (F cells) containing HbF46,47
and (2) the most primitive burst-forming unit-erythrocyte, when
triggered prematurely to making erythroblasts, propagates an
increased proportion of F cells among their progeny.48 The model
states that increasing HbF synthesis during stress, thus possibly in
response to intense Epo stimulation, involves the accelerated
maturation of progenitors, leading to reprogramming or to recruit-
ing erythroid progenitors that will give rise to F cells.49 Investigat-
ing whether this model may account for anemia correction in ␤
thalassemic mice requires that erythropoiesis is maintained at a
steady state, preferentially normocythemia. Indeed, exploration
during acute Epo stimulation would not allow us to distinguish the
effects observed in ␤ thalassemic mice from those previously
described in normal animals.
We obtained robust Epo secretion in ␤ thalassemic mice by the
intramuscular injection of naked DNA followed by an electric
shock, as previously reported in normal mice.26 Despite a slow
decline of Epo serum concentrations over time, this method had a
sustained effect on erythropoiesis in normal mice and in rats,
resulting in a persistent polycythemia.50 Although serum Epo
concentration decreased with a similar kinetics in ␤ thalassemic
mice, the effects on erythropoiesis were much more limited in time.
Serum Epo concentrations above which effects on erythropoiesis
were visible were higher in ␤ thalassemic mice than in normal
animals. Whereas threshold values varied depending on ␤ thalasse-
mic animals, they appeared higher than 150 mU/mL. In contrast,
normal animals with 32 mU/mL were already polycythemic.26 A
likely explanation is that different mechanisms are involved.
Whereas induction of polycythemia in normal animals is the
consequence of the stimulation of cells committed to adult
erythropoiesis, improvement of erythropoiesis in ␤ thalassemic
mice supposes the recruitment of cells committed to the accumula-
tion of ␤min-RNA. Because effects on erythropoiesis were not
maintained over time, we could not attempt to obtain steady-state
normocythemia by modulating Epo secretion levels through the
tetracycline-dependent expression cassette present in the vector.
This would be better performed by using gene transfer vector
derived from type 2 adeno-associated virus, which allows sustained
Epo delivery levels.9
It is noticeable that disease correction remained partial even at
the peak Epo secretion. Microcytosis and hypochromia persisted,
except in animals with intense polycythemia. Iron overload was not
corrected in erythrocytes. Persistent dyserythropoiesis, as illus-
trated by high reticulocyte counts, probably accounted for incom-
plete phenotypic reversion. We presume that therapeutic strategies
for ␤ thalassemia based on Epo delivery will have to find a
compromise between the induction of effective erythropoiesis
providing some clinical benefit and the persistence of an ineffective
erythropoiesis, the complete reversion of which would require the
induction of polycythemia.
Despite this intrinsic limitation, the strategy could be of interest
for improving erythropoiesis in ␤ thalassemic patients. Electrotrans-
fer of naked DNA is a safe, noninvasive, and low-cost method that
could be proposed, possibly in combination with other treatments,
to large populations. Although tolerance to high Epo dosages has
been documented, transient secretion is certainly advantageous in
terms of security, whereas readministration would ensure a sustained
effect, if desired. Moreover, control of dose delivery would probably be
feasible by modulating doxycycline dosage, if required.
Acknowledgments
We especially acknowledge Dr W. Hillen and Dr H. Bujard for
providing us with the transactivator rtTA2s-S2 and for their
authorization to use it prior to publication. We are grateful to Dr Y.
Beuzard and to Dr G. Ciliberto for helpful discussions during the
origin of the work. We also thank Dr O. Schwartz for useful
comments on the manuscript.

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