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Locus Control Region-Ay Transgenic Mice: A New Model for Studying the
Induction of Fetal Hemoglobin in the Adult
By P. Constantoulakis, 6. Josephson, L. Mangahas, T. Papayannopoulou, T. Enver,
F. Costantini, and G. Stamatoyannopoulos
All pharmacologic agents that induce fetal hemoglobin (Hb)
have been discovered with in vivo studies of humans,
macaques, and baboons. We tested whether transgenic mice
carrying human fetal (y) globin genes provide a model for
studying the pharmacologic induction of HbF in the adult. In
initial studies, phenylhydrazine-induced hemolytic anemia,
5-aracytidine, butyrate, or combinations of these treatments
failed to activate the human y-globin gene in a transgenic
mouse line carrying a 4.4-kb Gy globin gene construct that is
expressed only in the embryonic stage of mouse development. Subsequently, adult mice carrying the human Ay gene
linkedto the locus control region (LCR) regulatorysequences
and expressing heterocellularly HbF (about 25% y-positive
cells) were used. Treatments with erythropoietin, 5-azacytidine, hydroxyurea, or butyrate resultedin induction of y gene
expression as documented by measurement of F-reticulocytes, the y/y + p biosynthetic ratio and the level of steady
state y mRNA. Administration of erythropoietin or butyrate
to transgenic mice carrying a pLCR-p (human) globin construct, failed to increase human @-globinexpression. These
results suggest that the kLCR-Ay transgenic mice provide a
new model for studying the induction of fetal Hb in the adult,
0 1991 by The American Society of Hematology.
N HUMANS, FETAL hemoglobin (Hb) switches off
containing the locus control region (LCR)* were used. This
region is located 5 to 22 kb upstream of the human
embryonic €-globin gene and contains five developmentally
Transgenic mice
stable DNAse I hypersensitive
carrying constructs containing the human P-globin gene
linked to the LCR, display high level position and integration-site-independent human globin gene expression.“ Animals carrying a 3.3-kb fragment of the human Ay-globin
gene linked to an LCR casette express the human fetal gene
in the embryonic stage of development in a copy-numberdependent, position of integration independent fashion but
also continue to express small amount of Ay globin during
the adult life.”
In the experiments described here, we used nonLCR-G-y transgenic mice and LCR-Ay transgenic mice, to
ask whether administration of known in vivo HbF inducers
would also induce y gene expression in the adult transgenic
animals.
I
during the perinatal period, but some trace amount is
found in every person during adult life. A variety of
physiologic conditions and pharmacologic agents such as
erythropoietin (Epo), 5-azacytidine, arabinocytosine, hydroxyurea, and normal metabolites like butyric acid analogues, have been found to induce HbF production in the
AI1 studies on the in vivo induction of HbF have
been done either in primates or in humans, ie, species that
possess fetal Hb.In the studies described in this report we
tested whether transgenic mice can be used for studying the
pharmacologic induction of HbF in the adult.
In contrast to humans, who have three stages of Hb
development, embryonic, fetal, and adult, mice have only
two stages of Hb development, embryonic and adult.
Embryonic globins (ph’,e ) are produced during the embryonic stage of mouse development, while adult globins (pmaj,
p“”) are produced when the definitive stage of erythropoiesis starts in the fetal liver. Transgenic mice have been used
for studying the developmental control of human globin
genes. Thus, it was initially shown that mice carrying a
4.4-kb fragment containing the human Gy globin gene
display a low level y globin expression in the embryonic red
blood cells (RBCs) of the yolk sac but not in cells of the
definitive erythrop~iesis.‘~
In subsequent studies, constructs
~~
~
~
From the Division of Medical Genetics, Depattment of Medicine,
Universityof Washington, Seattle; and the Department of Genetics and
Development, College of Physicians and Surgeons, Columbia University, New York, Ny:
Submitted August 6, 1990; November 6, 1990.
Supported by Grants HL-20899 and DK-31232from the National
Institutes of Health.
Address reprint requests to G . Stamatoyannopoulos, MD, Dr Sci,
Professor of Medicine, Medical Genetics, RG-25, University of Washington, Seattle, WA 98195.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section 1734 sole& to
indicate this fact.
0 1991 by TheAmerican Society ofHematofogy
0OO6-4971J91~7706-#30$3.
OOJO
1326
MATERIALS AND METHODS
Animals. Three types of transgenic mice were used: the G9 line
of Gy-mice, carrying the construct containing the 4.4-kb Bgl IIISph
I fragment with the Gy-globin gene and 1.5 kb of 5’ and 0.3 kb of 3’
flanking sequence^^^; the 1279 line of pLCR-Ay mice, carrying the
construct pBSpLCR-Ay, containing four hypersensitive sites from
the 5’ region of the @-globinlocus within a 2.5-kb fragment,”linked
to a 3.3-kb Hind111 DNA fragment containing a copy of the human
Ay-globin gene; and the pB17 line of pLCR-@mice, containing the
2.5-kb pLCR casette linked to a 4.8-kb Bgl 11-EcoRV fragment
containing the human @-globingene.2u
Drugs. Recombinant human Epo (rhuEpo; Genetics Institute,
Cambridge, MA), 5-azacytidine (Sigma Chemical Co, St Louis,
MO), and hydroxyurea (Sigma) were administered intraperitoneally in schedules and doses described below. a-amino-n-butyric
acid (Sigma) was dissolved in normal saline and administered
subcutaneously through a surgically implanted osmotic pump (Aka
Corporation, Palo Alto, CA) over a 5-day period according to the
manufacturers’ directions.
*The LCR was previously known as LAR (locus activation
region) or DCR (dominant control region). The new term, LCR,
was agreed upon by the participants of the Seventh Conference on
Hemoglobin Switching, Airlie House, Virginia, September 8-11,
1990.
B/uod, Vol77,No 6 (March 15). 1991: pp 1326-1333
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HeF STIMULATION IN TRANSGENIC MICE
1327
done as previously described?’ The probes used were human y
Hematologic studies. Twenty to thirty microliters of blood was
pTlAy,Z6
human p P S P ~ ~ ~ and
, , , ,mouse
~
ci pSP64”ci.”
drawn daily from the mouse tail and used for the analyses
For the polymerase chain reaction (PCR)-RNA analysis of the
described below. Proportions of reticulocytes were calculated by
human y-globin expression, mouse blood RNA was isolatedMand
counting 2,000 cells. The proportion of HbF containing reticuloreverse transcribed in the presence of the two y-primers
cytes (F-reticulocytes) was evaluated using a previously described
(Gi’:GAGGACAAGGCTACTATCACAand G::GTAT(JTGGAmethod2lZ; in brief, the reticulum in the reticulocytes was first
GGACAGGGCACT) that generate a 417-bp fragment from RNA
precipitated by incubating blood with 1% Brillant Cresyl Blue
transcripts. Following amplification (25 cycles, 94°C 6 minutes,
(BCB); and smears were prepared, fixed in methanol, and labeled
50°C 2 minutes, 72°C 2 minutes) we analyzed the PCR product with
with the anti-y-chain monoclonal antibody (MoAb) and antimouse
agarose gel electrophoresis.
F(ab’)2-FITC in the presence of 0.005% acridine orange. With this
method, the precipitated reticulum in F-reticulocytes appears
RESULTS
red-orange on a green background; reticulocytes that do not
Studies of nonLCR-Gy transgenic mice. It has been
contain HbF show the red-orange reticulum on a black backpreviously shown that transgenic mice carrying the 4.4-kb
ground. The preparations were viewed in a Zeiss Universal
Gy-globin gene fragment without LCR sequences express
Fluorescent microscope (Zeiss Inc, New York, NY)equipped with
the human gene only during the embryonic stage of developepiilluminationand a 200 Mercury/lSO Zenon power source.
Globin chain anaZysk. Blood aliquots were incubated in the
ment.” Adult animals of line G9 containing this construct
presence of ’[HI-leucine (specific activity 60 Cimmol), in leucinewere examined for y globin expression after staining of
free Iscove’s modified Dulbecco’s medium (IMDM)and 10% fetal
blood with fluorescent anti-y-globin antibodies, or by
calf serum (FCS) at 37°C for 12 to 16 hours. At the end of
isoelectric focusing of radiolabeled globin chains or by
incubation, the samples were washed with Na, K, Mg (NKM)saline
y-globin mRNA PCR. No y globin was detected in the
and either analyzed immediately or stored in liquid nitrogen for
blood of these animals by these techniques. To test whether
later analysis.Cell lysates were subjected to globin chain isoelectric
the y-globin transgene is activated by acute erythropoietic
focusing followed by fluorography as previously described.= The
stress, acute hemolytic anemia was induced by phenylhydrarelative proportion of synthesized globin chains was determined by
zine
and the appearance of F-reticulocytes was monitored
automated densitometry of the resulting fluorograms (Gelman,
by
immunofluorescence
labeling. Despite an increase in
Automatic Computing Densitometer, Ann Arbor, MI).
reticulocytes from 2%-3% to 30%-35%, no F-reticulocytes
RNA undysk. Whole blood was used for slot blot cytoplasmic
globin could be
could be detected. To test whether
hybridization analysis of globin RNA as follows. The cells were
counted (1 to 5 x 106cells) and pelleted by centrifugation.The cell
pellet was washed twice in phosphate-bufferedsaline (PBS), it was
resuspended in cold Tris-EDTA (TE) buffer (10 mmoVL Tris, 0.1
mmol/L EDTA) and 20 U of RNAse inhibitor were added
(RNasin; Promega, Madison, WI). The cells were then lysed with
5% “-40 solution and, after centrifugation, the cytoplasm was
transferred in tubes containing SSC (0.15 mol/L NaCl, 0.015 m o w
Trisodium Citrate) and formaldehyde (6X SSC, and 8% formaldehyde final). After denaturing at 65°C for 15 minutes, the samples
were used for “spot blotting” immediately, or they were stored at
-70°C for later analysis. The “spot blotting” technique was
performed using the Schleicher& Schuell (Keene, NH) Minifold I1
apparatus, as previously described.” In brief, the samples were
diluted to final volume of 300 FL with 15x SSC and loaded on
prewet (in 15x SSC) S and S (Schleicher and Schull) nitrocellulose
paper under vacuum. After drying, the nitrocellulosewas baked in
a vacuum oven at 80°C for 2 hours. After 3 hours of prehybridization in a 65°C water bath, the blots were hybridized with 50 ng of in
vitro transcribed radioactive (a-”P-UTP) RNA probes. For detection of mouse globin RNA, the mouse 3’ p-major globinz specific
probe was used. For the detection of the human y-globin mRNA
the 0.6-kb EcoRIIHindIII fragment of the 3’ end of the human
Gy-globin gene was used. Hybridization was allowed to take place
between the homologous RNA sequences for at least 16 hours, e - :
4
0
4
8
12
18
followed by extensive washing under stringent conditions (2x SSC
Daysofbeabnent
with 0.1% sodium dodecyl sulfate [SDS] at 65°C six times, followed
by 0.1 x SSC with 0.1% SDS in 50% formamide at 65°C two times).
Fig 1. Epo stimulates y globin expression in transgenic mice. Two
kLCR-Ay transgenic mice were injectedwith 3.000 U of Epo per kg of
After exposing the blot in a Kodak X-OMAT AR film (Eastman
body weight every 12 hours for 1 day (left) and for 3 days (right). The
Kodak, Rochester, NY) at -7O”C, the relative amounts of y- and
number of treatment days is indicated by the arrows in each panel.
p-globin mRNAs were calculated by both scanning in a densitomeTop panels: number of total reticulocytes during the treatment
ter and by directly measuring the radioactivity (counts per minute)
period. Middle panels: proportionof fetal Hb expressing reticulocytes
of every slot in a liquid scintillation f3-counter.
(F-reticulocytes) during the treatment. Bottom panels: y/y + p”‘
For the RNAse protection assays, 100 FL of tail blood was
globin chain biosynthetic ratios, as obtained by isoelectricfocusing of
drawn, total RNA was isolated? and 200 ng was used in each assay,
peripheralblood reticulocytes during the treatment.
u
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1328
CONSTANTOUIAKiS ET AI.
induced by 5-azacytidine,the animalswere treated intraperitoneally with 10 mgkg of 5-azacytidine per day for 4 days.
This treatment resulted in cytoreduction followed by an
increase in reticulocytes up to 25% but no appearance of
F-reticulocytes. Because previous studies in baboons"' have
suggested that 5-azacytidine activates fetal globin production when it acts on an expanding erythropoicsis, mice were
trcatcd first with phenylhydrazine(10 mg/kg/d for 4 days) to
induce erythropoietic expansion and subscqucntly with
5-azacytidine for either 2 days or 4 days (4 mg pcr kg of
body weight per injcction). This manipulation also failed to
induce y globin expression as judged by the F-reticulocyte
assay or the lack of appearancc of radioactive y-globin
chains on isoelectric focusing gels.
It has bccn previously shown that the combination of
5-azacytidinewith butyrate reactivates the embryonicglobin
gene in chickens," and induces F-reticulocyte production in
baboons."." Transgenic animals of the Gy line were treated
with a schedule (previously used in baboons)'' consisting of
administration of 5-azacytidineon day 1 (4 mgkg, followed
12 hours later by 12 mgkg) and of 1 g/kg sodium butyrate or
250 mgkg a-amino-n-butyric acid on days 2 to 6.Thcrc was
no dctcctable y globin expression by the F-reticulocyte
assay or globin chain biosynthesis or y-globin RNA PCR.
Induction of human y-globin gene in GCR-Ay tmtqenic
mice tmared with E p . The FLCR-A~transgenic mice
were the offspring of a founder mousc containing three
A
slot
-Blot
- Hybrldlzatfon
~ _ _ -
copies of the human y-globin gene.'9 Adult heterozygous
animals having 23% to 32% y-positive cells by immunofluorescence and 0.03 to 0.09 yhu""/amRNA ratio by
RNA% protection were used for these studics.
To test whether Epo can induce y-globin gene expression
in the kLCR-Ay transgenic mice, animals were trcated for
1 day, or for 3 days, with doses of Epo previously shown to
induce HhF in baboons.'' Thus, Epo was administered as
intraperitoneal bolus injections of 3,000 U pcr kg of body
weight every 12 hours. As shown in Fig 1, this manipulation
induced erythroid regeneration, as indicated by the sharp
increase in reticulocytes (from 2% to 3% to 10% to 12% in
the 1-day treatment and to 20% to 22% in the 3-day
treatment). In parallel, there was induction of y globin
expression as indicated by the increase in F-reticulocytes
(Fig 1). The dcgrcc of erythroid regeneration and the
increase in F-reticulocytes were dependent on the dose of
Epo used (compare, in Fig 1, the rcsults of the 1-day
trcatment, to thc results of the 3-day treatment). A transient increase was obscrved in the y/y + f3"' globin chain
ratio by globin chain biosynthesis (Fig 1). in the y/y + f3"l
mRNA ratio as measured by cytoplasmic hybridization (Fig
2). and in the $a""- globin mRNA ratio as measured by
RNA% protection (Fig 2).
Induction of y gene q m s s i o n pLLR-Ay mice mated with
5-azucytidine. Animals were treated with a schedule, previously used in baboons,'' consisting of intraperitoneal
RNAse-PIotectlon
_ _ __
Increment of y/y$ mRNA Ratlo
day 0 day 4
Days of treatment
I) @ a-mouse
dm
-a
4
d'b.
--
vmw*
- -
B-
FiO 2 7 (11mRNA in "g.nk mk. t r o d
with Epo. Mouw ratkulotyh. (rLCR-Ay) wore anawed for globin RNA expression with cytoplasmic
slot blot hybrldlzation, andlor with R N h proteetion, using specific human (7). mouw (p), and mouw
(a)globin RNA probes (uta Materials and Methodr).
(A) Left panel: changes in the y / y + pw ratio during
the course of treatment (pretreatment y / y + p"* ratio
Is taken as 1). obtained by cytopiasmk slot blot
hybridization. Right panel: results of R N h proteetion analysis pedormed at days 0 and 4 of the
treatment with Epo. (E) Autoradiogram from the slot
blot hybridization experiment described in (A) (Epo
treatment 3,000U/kg x 6). The column "wlis" shows
the number of RBCs applied to each slot. do to d12
correspond to the days of treatment. y"" and pindicate the probes used for hybridization with each
row of filters.
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1329
HBF STIMULATION IN TRANSGENIC MICE
bolus injection of 4 mg per kg of body weight, followed 12
hours later by an injection of 12 m a g . As in the studies of
primates:' an initial decline and a subsequent increase in
the number of reticulocytes was observed in response to
5-azacytidine treatment (Fig 3). During the rebound of
reticulocytes, an increase in F-reticulocytes (from 30% to
32% to 65% to 67%) also took place (Fig 3). There was a
fivefold to sevenfold increase in the y/y + pmaiglobin chain
I
20 r
biosynthetic ratio (Fig 3), a sixfold to sevenfold increase of
the y/y + pmaJmRNA ratio by the slot blot hybridization
assay (Fig 4), and a fourfold to fivefold increase in the
,/amause
mRNA ratio by the RNAse protection assay (Fig 4).
pLCR-Ay transgenic mice treated with hydroxyurea. TO
test whether hydroxyurea can induce y globin expression in
the kLCR-Ay transgenic mice, we administered intraperitonealy 200 mg of hydroxyurea per kg of body weight daily
for 5 days each week for total of 5 weeks. This dose was
previously shown by Alter and Wagner3jto induce p-minor
globin gene expression in adult mice. The treatment resulted in very sharp changes in the production of reticulocytes (Fig 5) consistent with sequential cycles of cytoreduction and erythroid regeneration. At the same time there
was a gradual increase in the F-reticulocyte production
from 28% to 30% to 64% to 66% (Fig 5).
Induction of pLCR-Ay expression by butyrate. Butyric
acid analogues induce erythroid differentiation in erythroleukemia lines34335
and activate fetal globin genes in erythroid cell c u l t ~ r e . 'In
~ ' vivo
~ ~ studies have shown inhibition of
the y to p switch in sheep fetuses treated with butyrate,"
reactivation of embryonic globin gene expression in adult
chickens treated with butyrate and 5-azacytidine? and
stimulation of fetal Hb production in butyrate-treated adult
baboon^."^'^
t
lot
1
2o
0.06
n
i t
0
4
0
4
8
12
16
Days of treatment
Fig 3. 5-azacytidine stimulates y globin expression in transgenic
mice. Means of two experiments in which pLCR-Ay transgenic mice
were treated with an injectionof 4 mg of 5-azacytidine per kg of body
weight, followed 12 hours later by a 12-mg/kg injection. Vertical
arrows show the days of 5-azacytidine administration. Top panel:
number of total reticulocytes during the treatment period. Middle
panel: proportion of HbF-expressing reticulocytes during the treatment. Bottom panel: changes in the y/y + @""' ratio during the course
of treatment.
To test whether butyrate induces y-globin gene expression in transgenic mice, we administered 200 mg of a-aminon-butyric acid per kg of body weight per day for 5 days,
through a subcutaneous osmotic pump, a treatment schedule that has been previously shown to increase HbF
production in bab00ns.l~While this manipulation produced
only a small increase of reticulocytes, it substantially increased F-reticulocyte production (from 28% to 30% to
54% to 56%, Fig 6). The induction of y globin expression
was also apparent in the increase of y/y + pmaJglobin
biosynthetic ratio (Fig 6) and the increase in y globin
mRNA (Fig 7).
Studies of pLCR-p transgenic mice. The induction of the
human y-globin gene in the pLCR-Ay transgenic mice
could be mediated by an action on sequences of the Ay
globin gene or on sequences of the kLCR cassette, or on
sequences of both the wLCR and the Ay gene. To investigate the role of the sequences of the pLCR cassette we
examined whether Epo or butyrate can induce p globin
expression in pLCR-P transgenic mice. Fifteen percent to
20% of the P-like globin chains of the transgenic animals we
used are human p globin chains.
One animal was treated for 3 days with the doses of Epo
shown to stimulate y gene expression in the LCR-Ay
transgenic animals (Fig 1). Levels of steady state p mRNA
were analyzed with cytoplasmic slot blot hybridization and
with RNAse protection assays. Despite the Epo-induced
erythroid regeneration, there was no increase in the phuman/
phuman
+ pmaJmRNA ratio as measured by cytoplasmic RNA
hybridization (Fig 8), or the phuman/amoure
mRNA ratio as
measured by RNAse protection (Fig 9). Indeed, the phuman/
phuman
+ pmaimRNA ratio slightly decreased during the
treatment period (Fig 8). Similar results were obtained
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CONSTANTOUIAKIS ET AL
1330
day 0 day 6
Increment of
Y/*s mRNA Ratio
0
ry-human
-6
6.
5.
9 4-
2 3-
I
a-mouse
Days of treatment
following treatment with a-amino-n-butyric acid (200 mgl
kg/d for 5 days).
DISCUSSION
The results presented here show that several compounds
that induce HbF in humans and baboons also induce y
globin in the pLCR-Ay transgenic mice. Our observations
suggest that these transgenic mice may offer a convenicnt in
vivo system for testing induction of HbF by various pharmacologic agents. Transgenic mice carrying LCR-globin constructs may also provide opportunitics to delincatc the cis
sequences that are involved in y-globin gene inducibility.
Previous studies have shown that administration of high
doses of Epo stimulate HbF production in bahoons.by
inducing rapid erythroid rcgeneration." The induction of
HbF by Epo and cytotoxic drugs has been attributed to
premature commitment of early erythroid cells. Presumably
such cclls contain fmns-acting elements that induce fetal
globin expression and these dements are retained in the
tcrminal cells when premature commitment occurs. In the
studies described herc, we find that Epo can induce human
-
4 O
-6.
FIg 5. Hydro.tkrmkh.7 gkMn e r p "
In tnnsgenk
mk.. Two anlmah (A and B) WII. treated wkh a .eh.dule of
th.vrrtk.1 a n ~ m
in the "h)
c o d h u d r ~ r y ~1indk.t.d
m
ing of daily lntraperitocnalInjmkms of200mg per kg of body weight,
for 5 days each week, for a total of 5 weeks. Top panels: changes in
ruticuloqte production during the treatment course. Bottom paneh:
proportion of F-retkulocytes during the &week treatment period;
note that F-rstkuloqtes increased several weeks after the onset of
treatment.
FIg4. 7gbbInmRNAintnmgenic mk. treated with Suacytidine. During the treatment of
the pLCR-Ay transgenic mke
with kzacytldine, described in
Fig 3, we analyzed the globin
RNA oxprasion of the mticulocytes with slot blot hybridization
and R N A n protection assays.
LStt panel: results of slot blot
hybridizationanalysis during the
treatment. Right panel: RNAW
protection analysis at days 0 and
6 of the treatment.
4
0
4
8.
l P l 8
I
Butymterthnul.h.7pkMne~in~mk..
two (rlCR-&tnnrg.nk
m k . (A and B) were treated wkh 200 mglkg
ol a-amlno-n-butyric acid per day for 5 days through a continuousRow osmotk pump. The box- insIda the pawls indicate the days of
butyrate admlnistrmtbn. Top panels: number of total m t k u l m
during the treatment pe&. Middle panels: proportion of F-retkulocytes during the treatment period. Bottom panels: changes in the
y / y + p"* ratio during the course of treatmont. Not. the incroaw in
F-reticulocytesand y / y + p- globin chain biosynthetk ratio desplte
the small increase in ntkuloqtes.
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HeF STIMULATION IN TRANSGENIC MICE
1331
y-human
-7.
ygkMnmRNAlnbw
wt"dtnnrg.nk
mko.
Globin RNA analysis dudng th.
butyrate treatment of the
pLCR-Ay transgenic mice described in Fig 6. Left panel: wsub of dot blot hybridhatlon
during the treatment period.
Right panel: RNAw protection
analysis of mouse blood RNA on
days 0 and 5 of butyrate treat-
Days of treatment
a-mouse
for 6 day8
ment.
y-globin gene expression in the cells of mice. This result
suggests that the "factors" of fast regeneration which
activate y globin expression are not specks specific.
The failure to detect an induction of y gene of the
non-LCR-y transgenic mice could be explaincd by one of
0.08 -
I
111
I
0.04
0.02
4 la-
0 4
0.1 I
-?-,i'"
ma
i-ot-rlCRg-k-withEpo.Om
& C R + t m w k " o w a InJoeredwith 3,000U d Epo per kg of
body weight every 12 hours for 3 days. Treatment days a n indicated
by arrows in the panoh. Top panel: total roticuloqte numbw. Bottom
panel: changes in the p-/p+ p- mRNA rrti0 obtained by
cvtoplmk slot Mot hybridhation onalysh.
1-1
I
I
O4t
owe-
Fig 9. Hu" @ gbbln "A In &cft-gtmmgank mkr. One
animol (M)
wms tro.t.d with Epo for 3 days 13.000 U/kg evwy 12
hours). and anoth.r (right) with a-amlnon-buyrk acid (200 mglkgldl
for 5 dovs. In selected days, globin mRNA wn analyzed by RNA..
protection. Top left: levels of protected human p- and "ea-globin
mRNAs, W o r e Epo treatment, ond 4 days from the onset of treatment. Top right: levels of human 8- and mouse a-globin mRNAs,
b d o butyrate
~
treatment, and 4 days from the 0M.t of tmatment.
Bottom panels: mRNA ratios (p-/p+ (IC....)obtained from th.
RNA.. protoctbn analysis after autoradiography scanning (m) or
liquid scintillation counting (C).
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
CONSTANTOULAKIS ET AL
1332
three possibilities. First, it is possible that induction of Gy
transgene has occurred but y globin accumulation was
below the threshold of detection by the methods we
applied. The sensitivity of the PCR technique argues
against this possibility. Second, the compounds we used
may induce y gene expression by acting on sequences of the
LCR. This possibility is less likely in the case of butyrate
and Epo, because these compounds failed to induce p gene
expression in the p,LCR-p transgenic mice. Third, the
inducers we used may augment y expression only when the
y-globin genes are already transcriptionally active. Thus, it
can be argued that there was stimulation of y expression in
the pLCR-Ay transgenic mice because the genes are
dysregulated, ie, already “on”, in the cells of the adult
animals. Studies of transgenic mice carrying LCR-globin
constructs in which both the y and p genes are submitted to
developmental controlZowill offer useful insights in the
induction of fetal Hb in the adult.
REFERENCES
1. Stamatoyannopoulos G, Nienhuis AW: Hemoglobin switching, in Stamatoyannopoulos G, Nienhuis AW, Leder P, Majerus
PW (eds): The Molecular Basis of Blood Diseases. Philadelphia,
PA, Saunders, 1987, p 66
2. DeSimone J, Heller P, Hall L, Zwiers D: 5-Azacytidine
stimulates fetal hemoglobin synthesis in anemic baboons. Proc Natl
Acad Sci USA 79:4428,1982
3. Ley TJ, DeSimone J, Anagnou NP, Keller GH, Humphries
RK, Turner PH, Young NS, Heller P, Nienhuis AW: 5-Azacytidine
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1991 77: 1326-1333
Locus control region-A gamma transgenic mice: a new model for
studying the induction of fetal hemoglobin in the adult
P Constantoulakis, B Josephson, L Mangahas, T Papayannopoulou, T Enver, F Costantini and G
Stamatoyannopoulos
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