Erythropoiesis in the Rps19 disrupted mouse: Analysis of erythropoietin
response and biochemical markers for Diamond-Blackfan anemia
H. Matssona, E.J. Daveya, A.S. Fröjmarka, K. Miyakeb, T. Utsugisawab, J. Flygareb, E. Zahouc,
I. Bymanc, B. Landinc, G. Ronquistd, S. Karlssonb, N. Dahla,⁎
aDepartment of Genetics and Pathology, The Rudbeck Laboratory, Uppsala University, 751 85 Uppsala, Sweden
bMolecular Medicine and Gene Therapy, The Strategic Center for Stem Cell Biology and Cell Therapy, Lund University, Lund, Sweden
cUnit of Clinical Chemistry, Karolinska Hospital, Huddinge, Sweden
dDepartment of Medical Sciences, Clinical Chemistry, Uppsala Akademiska Hospital, Uppsala, Sweden
Submitted 29 November 2005
Available online 7 February 2006
(Communicated by A. de la Chapelle, M.D., Ph.D., 1 December 2005)
The human ribosomal protein S19 gene (RPS19) is mutated in approximately 20% of patients with Diamond-Blackfan anemia (DBA), a
congenital disease with a specific defect in erythropoiesis. The clinical expression of DBA is highly variable, and subclinical phenotypes may be
revealed by elevated erythrocyte deaminase (eADA) activity only. In mice, complete loss of Rps19 results in early embryonic lethality whereas
Rps19+/−mice are viable and without major abnormalities including the hematopoietic system. We have performed a detailed analysis of the
Rps19+/−mice. We estimated the Rps19 levels in hematopoietic tissues and we analyzed erythrocyte deaminase activity and globin isoforms which
are used as markers for DBA. The effect of a disrupted Rps19 allele on a different genetic background was investigated as well as the response to
erythropoietin (EPO). From our results, we argue that the loss of one Rps19 allele in mice is fully compensated for at the transcriptional level with
preservation of erythropoiesis.
© 2005 Elsevier Inc. All rights reserved.
Keywords: Rps19; Mouse model; Diamond-Blackfan anemia; Erythropoiesis
The ribosomal protein S19 (RPS19) is part of the small
subunit of the ribosome. The gene for RPS19 is mutated in
approximately 20% of patients with Diamond-Blackfan anemia,
DBA [1–5], a congenital anemia characterized by absence or
decreased numbers of erythroid precursors in bone marrow
[6,7]. Approximately 30% of DBA patients also present with
additional abnormalities [7–9]. Most RPS19 mutations are
predicted to result in truncations of the protein and it has been
suggested that haploinsufficiency of RPS19 is a disease
mechanism in DBA [10,11]. The strong association between
RPS19 mutations and erythroblastopenia, which can partially be
rescued in vitro , suggests that RPS19 is an important
regulator of erythropoiesis and necessary for normal prolifer-
ation and differentiation of erythroid cells in bone marrow. The
relation between RPS19 and other factors involved in
erythropoiesis is unclear and patients with DBA do not respond
to erythropoietin (EPO) which is a key regulator of erythropoi-
esis [6,13]. The phenotypic spectrum of DBA is broad ranging
from the classical presentation of red cell aplasia in early
infancy to an isolated increase in erythrocyte adenosine
deaminase (eADA) activity . Other findings associated
with atypical or subclinical forms include elevated mean
corpuscular volume (MCV) and elevated fetal hemoglobin
(fHb) levels [9,14–17] which are also observed in individuals
with the transient form erythroblastopenia .
The Rps19+/−mouse model was previously reported as
hematologically normal . We hypothesized that analysis of
eADA, fHb and MCV which are used as diagnostic markers for
DBA may reveal a silent phenotype also in the Rps19+/−mouse.
We also hypothesized that the apparently normal phenotype in
the C57BL/6J Rps19+/−mice may be explained by a strain-
Blood Cells, Molecules, and Diseases 36 (2006) 259–264
⁎Corresponding author. Fax: +46 18 554025.
E-mail address: email@example.com (N. Dahl).
1079-9796/$ - see front matter © 2005 Elsevier Inc. All rights reserved.
specific genetic background and that erythroid stress in these
Rps19+/−mice would induce a subnormal erythroid response.
The previous report describing the Rps19+/−mouse model
showed mRNA levels for Rps19 in spleen cells and we now
aimed at measurements of the protein levels for Rps19 in both
spleen and bone marrow cells. To address these questions, we
produced an antiserum in rabbit against a peptide derived from
human RPS19 identical to the murine Rps19 sequence. We also
bred C57BL/6J Rps19+/−mice with FVB/NJ mice in order to
introduce the targeted allele on a different genetic background.
Human recombinant erythropoietin (hrEPO) has previously
been reported to efficiently stimulate erythropoiesis in mouse
[20,21] and we used hrEPO to test the effect on erythroid cell
formation in mice with a disrupted Rps19 allele. We present
herein the results from these investigations of Rps19+/−mice
together with the analysis of different diagnostic parameters
used in DBA.
Material and methods
We analyzed blood and bone marrow from Rps19+/−mice as
described previously . C57BL/6J Rps19+/−mice were bred
with FVB/NJ mice (Jackson Laboratory) for three (F3) to four
generations (F4) to produce Rps19+/−offspring with a high
proportion of the FVB/NJ genetic background. The analysis of
our mouse model is approved by the Uppsala Animal Research
Hematological analysis of FVB/NJ mice
Analysis of hemoglobin concentration, red- and white blood
cell count, hematocrit, mean erythroid corpuscular volume and
mean erythroid hemoglobin concentration was performed using
300 μl blood from F3 and F4 generations FVB/NJ Rps19+/−and
Rps19+/+mice (Unit of Clinical Chemistry, National Veterinary
Institute, Uppsala, Sweden).
Generation of Rps19 antiserum and Rps19 quantification
A synthetic peptide with amino acid sequence KLAKHKE-
LAPYDEN (N-terminal to C-terminal) identical to amino acids
38–51 of human RPS19 protein was immunized in rabbits. The
peptide sequence is 100% identical to murine Rps19. Total IgG
from immunized rabbits was affinity purified using Protein-G
Sepharose 4 Fast flow (Amersham Biosciences). Mouse bone
marrow (BM) cells were collected from femurs by flushing the
bones with filtered 1× PBS. Fresh mouse spleens were reduced
to pulp and single-cell suspensions were made in filtered 1×
PBS. A ribosome-enriched protein extract (RS) from K562 cells
and a His-tagged recombinant RPS19 protein served as control
for the specificity of the RPS19 antiserum and were used for
molecular weight determination of the native RPS19 protein.
We boiled 200,000 BM and spleen cells, respectively, for 10
min in 1× LPS loading buffer (Invitrogen) containing 50 mM
dithiotreitol and protein was separated on 12% Bis-Tris PAGE
gels (Invitrogen) at 200 V for 55 min and transferred to PVDF
filters (Amersham Biosciences) at 30 V for 80 min. A 1:1500
antibody dilution of the rabbit anti-RPS19, 1:1500 of the goat
anti-mouse Lamin B (Santa Cruz Biotechnology), 1:10,000
dilution of the HRP conjugated anti-rabbit IgG (Amersham
Biosciences) and 40 ng/mL of the HRP conjugated bovine anti-
goat IgG (Santa Cruz Biotechnology) was used for Rps19
quantification. Hybridized filters were developed using ECL
kits and Hyperfilm ECL films (Amersham Biosciences).
Erythropoietin administration and flow cytometry
C57BL6J Rps19+/−and Rps19+/+mice were injected
subcutaneously for two consecutive days with sterile PBS
(controls) or 200 IE hrEPO (Epoetin beta, Roche) dissolved in
100 μl sterile PBS. A total of 16 Rps19+/−and 14 Rps19+/+mice
were injected with hrEPO whereas 7 Rps19+/−and 7 Rps19+/+
mice were used as controls. At day 5 after the initial injection,
BM cells were harvested from mouse femurs with cold sterile
PBS. BM cells were treated with a 1/100 dilution of 5 μM
Hoechst 33342 dye (Sigma-Aldrich) at 37°C for 1 h to label
nucleated cells. Cells were washed in PBS/0.5% FCS (Sigma-
Aldrich) and incubated with 1 μg of each phycoerythrin-
conjugated anti-mouse Ter119 (PharMingen) and fluorescein
isothiocyanate-conjugated anti-mouse CD71 (PharMingen) on
ice for 30 min. We used propidium iodide to label and eliminate
dead cells in the analysis. For the enumeration of Ter119
positive cells in BM, we collected data from 100,000 nucleated
cells. All samples were analyzed on a FACS Vantage flow
cytometer (Becton Dickinson).
eADA measurements and fetal globin isoforms
A total of 12 Rps19+/+and 6 Rps19+/−adult (20–24 weeks of
age) mice were tested for eADA activity. For each individual,
500 μl blood was centrifuged at 1000 × g for 5 min at 4°C.
Erythrocytes were resuspended in 3 volumes of buffer contain-
ing 0.9% sodium chloride and 10 mM glucose, incubated on ice
for 10 min and centrifuged as above. The eADA activity was
determined as previously described  assuming a mean
corpuscular hemoglobin of 30 pg and expressed in attokatal
(1 × 10−18)/erythrocyte. Mouse globin isoforms were deter-
mined from blood samples diluted 50 times in de-ionized sterile
water followed by a 10 fold dilution in 50% aqueous methanol
containing 0.2% formic acid. Samples were desalted using C18
zip tips (Millipore) according to protocol and analyzed with
mass spectrometry as previously described [23,24] using a
Quattro Micro (Waters) instrument controlled by MassLynx
software (Waters). The cut-offfor detection was set to 3% of the
β1 globin signal strength.
Analysis of blood parameters in FVB/NJ Rps19+/−mice
Blood samples from C57BL/6J Rps19+/−mice were
previously characterized . We now introduced this
260H. Matsson et al. / Blood Cells, Molecules, and Diseases 36 (2006) 259–264
genotype on the FVB/NJ strain and the corresponding Rps19+/+
and Rps19+/−mice were analyzed for hematological para-
meters. We obtained data including hemoglobin concentration,
hematocrit, MCVand red- and white blood cell numbers using
procedures previously reported . The parameters were
similar in Rps19+/+and Rps19+/−mice with no significant
differences (Student's t test, data not shown) when analyzed at
3 and 7 weeks of age (Table 1). In combination, our previous
findings and the results presented here argue against a strain-
specific compensatory mechanism for the loss of one Rps19
Normal Rps19 levels in C57BL/6J Rps19+/−mice
The relative amount of Rps19 protein were measured in cell
lysates from total bone marrow (Fig. 1A) and spleen cells (Fig.
1B) using a rabbit polyclonal antibody against an N-terminal
peptide within RPS19. A polyclonal antibody against murine
Lamin B was used as a standard. The specific signals of
approximately 18 kDa detected in extracts from spleen and BM
corresponded to the size of the RPS19 protein in ribosome
enriched sample (RS) from K562 cells. The signals were
quantified relative to the Lamin B signals by densitometry.
After quantification, the ratio of Rps19 in Rps19+/+mice with
respect to Rps19+/−mice were 1.1 for total BM and 0.7 for
spleen cells, respectively. The result suggests similar levels of
Rps19 in BM and spleen of Rps19+/−mice and wild type
Erythropoietin response in C57BL/6J Rps19+/−bone marrow
We investigated the effect on erythropoiesis after stimulation
by human recombinant erythropoietin (hrEPO) in 16 C57BL/6J
Rps19+/−mice and 14 wild type mice. The response to hrEPO is
measured as the fraction of nucleated cells in BM with positive
staining for the erythroid cell surface marker Ter119 which is
expressed on cells of the proerythroblast stage to mature
erythrocytes. Our results show a distinct increase in the relative
number of Ter119 positive BM cells in hrEPO treated mice
when compared to controls injected with PBS. Rps19+/−mice
show a slightly lower number of Ter119 positive cells in
response to hrEPO when compared to Rps19+/+mice (Fig. 2).
The average fold increase of the number of Ter119 positive cells
in the Rps19+/−mice is 1.69 ± 0.39 times the corresponding
number in PBS controls 5 days after the initial hrEPO injection.
The corresponding average cell number was 1.83 ± 0.42 in the
wild type mice. This difference in hrEPO response between the
Rps19+/−and Rps19+/+mice is not significant (P = 0.51,
Student's t test).
eADA activity and globin isoforms in C57BL/6J Rps19+/−mice
Erythrocyte ADA activity was measured from freshly
prepared red cells of adult mice (Table 2). The eADA activity
was similar in Rps19+/−mice when compared to normal
controls (P = 0.70, Student's t test). We also determined the type
Measurements of peripheral blood parameters for Rps19+/−and Rps19+/+mice at 3 and 7 weeks of age
(no. of mice)
126.0 ± 4.0
126.6 ± 7.9
146.4 ± 6.5
146.8 ± 4.9
7.7 ± 0.3
7.7 ± 0.5
9.3 ± 0.5
9.5 ± 0.4
6.1 ± 1.3
6.5 ± 0.8
8.4 ± 2.5
7.5 ± 2.2
40.8 ± 2.2
40.9 ± 2.9
47.0 ± 2.7
47.2 ± 2.7
53.0 ± 2.3
52.8 ± 1.8
50.8 ± 1.8
49.7 ± 1.4
310.5 ± 9.5
311.1 ± 7.0
312.2 ± 8.6
313.4 ± 9.2
The table presents mean values including standard deviation for all measurements. B-Hb, blood hemoglobin; RBC, Red Blood Cell count; WBC, White Blood Cell
count; MCV, Mean Corpuscular Volume; MCHC, Mean Corpuscular Hemoglobin Concentration.
Fig. 1. Western blot analysis presenting Rps19 protein levels in Rps19+/−bone
marrow and spleen cells of Rps19+/+and Rps19+/−mice. Ribosome enriched
ultra-centrifuged cell extract from K562 cells (RS) and 100 ng recombinant His-
tagged human RPS19 (R-RPS19) protein are used as controls for the specificity
of the anti-RPS19 antibody. A rabbit polyclonal antibody against an N-terminal
peptide sequence of human RPS19 detects the murine Rps19 protein of
approximately 18 kDa in BM and RS samples (A) as well as in spleen cells (B).
Signals detected by an antibody against the nuclear envelope protein Lamin B
are used as references for quantification. Selected molecular weight in kDa of a
protein ladder is shown to the left in the figure.
261 H. Matsson et al. / Blood Cells, Molecules, and Diseases 36 (2006) 259–264
of globin forms present in blood of Rps19+/−and Rps19+/+mice
by mass spectrometry (MS). One α and β-globin chain with
masses 14,981.02 Da and 15,616.89 Da were detected
corresponding to the A allele of Hba-a1 and the S allele of
Hbb-b1 (Table 3), respectively, in both adult and newly born
Rps19+/−and Rps19+/+mice. No expression of the early
embryonic zeta (Hbz), epsilon (Hbb-y) or beta H0 (Hbb-bh0)
chains, the embryonic beta type (Hbb-bh1) or the adult beta 2
chains (Hbb-b2) was detected.
The ribosomal proteins are well characterized as components
in the protein synthesis machinery. However, the association of
heterozygous RPS19 mutations with DBA in 20% of patients
has led to the suggestion of an extra-ribosomal function for
RPS19 in erythropoiesis. Recent findings support the idea that
haploinsufficiency in patients carrying nonsense RPS19 muta-
tions may be a mechanism in the development of erythroblas-
topenia. In two independent studies, reduced RPS19 expression
generated by siRNA against the RPS19 transcript resulted in
erythroid defects correlating to the RPS19 levels in CD34+cells
[10,25]. Furthermore, mutations producing premature stop
codons in RPS19 are shown to reduce the amount of RPS19
mRNA and RPS19 protein in CD34+BM cells of DBA patients
. Interestingly, this down-regulation was not detected in
blood mononuclear cells (MNC) from the same patients. It has
been shown that the need for RPS19 varies with different stages
of erythropoiesis and the amount of RPS19 decreases during
erythroid maturation . It is possible that the regulation of
RPS19 levels is different in MNC compared to erythroid cells
or, that MNC cells require lower amount of RPS19. Recently,
two studies present novel implications on the effects of several
previously reported RPS19 mutations. Firstly, a study presents a
maturation defect of pre-ribosomes upon depletion of the
RPS19 homologues in yeast . Deletion of both yeast RPS19
genes is lethal which is consistent with the absence of
blastocysts homozygous for Rps19 disruption in mouse .
When common RPS19 mutations present in DBA patients were
introduced into yeast, a similar effect was obtained as with
under-expression of RPS19 with accumulation of pre-40 S
particles and failure of ribosome assembly in the nucleus. This
finding present the first indication of a ribosome dysfunction,
caused by an impaired function of RPS19, that could affect
cellular growth in highly proliferative tissue such as erythroid
cells. Secondly, the Serine–Threonine kinase PIM-1 was
identified as a binding partner to RPS19 in a yeast two-hybrid
screen using a human liver c-DNA library . PIM-1 is
expressed in erythroid cells and is regulated by several growth
factors, thereby erythropoietin. RPS19 is phosphorylated by
PIM-1 in vitro and associates with ribosomes in human 293 T
cells suggesting a role in translational control. Also, the
introduction of three RPS19 mutations present in DBA patients
altered the binding efficiency of the PIM-1 and RPS19 proteins.
Further studies of the specific cellular function of the
association between PIM-1 and RPS19 are needed and presence
of this interaction in primary cells remains to be determined.
From our measurements, we conclude that the RPS19
protein levels are similar in MNC from bone marrow and spleen
of Rps19+/−and Rps19+/+mice. This is analogous to the normal
Rps19 transcript levels previously detected in Rps19+/−spleen
cells  which support the hypothesis of a transcriptional up-
regulation of Rps19 mRNA levels in Rps19+/−mice. In our
analysis, we have used total BM cells and a specific reduction in
Rps19 expression in the sub-population of early erythroid
progenitor cells of Rps19+/−mice cannot be excluded. On the
Erythrocyte adenosine deaminase (ADA) activity in red blood cells of adult
Genotype (no. of mice)Average eADA activity
(attokatal/erythrocyte ± SD)
0.45 ± 0.03⁎
0.46 ± 0.06
SD, standard deviation.
⁎P = 0.70 (Student's t test).
Globin isoforms present in bloodof adult andnewly bornRps19+/−andRps19+/+
(no. of mice)
Detected globin isoformsGlobin allele,
3 daysRps19+/+(3)Hba-α2, Hbb-β2
Rps19+/−(4) Hba-α2, Hbb-β2
aAge interval = 17–20 weeks.
Fig. 2. The response to human recombinant erythropoietin (hrEPO) in bone
marrow of Rps19+/+and Rps19+/−mice. Cells positive for Ter119 expression
were detected with FACS 5 days after the initial administration of hrEPO. The
average increase of Ter119 positive cells in bone marrow after hrEPO
stimulation is compared to the background level of Ter119 positive cells in
PBS injected mice. Data from a total of 14 Rps19+/+and 16 Rps19+/−hrEPO
injected mice together with 14 PBS injected mice are pooled and presented as
the fold increase in Ter119 positive cell number after hrEPO stimulation with
262H. Matsson et al. / Blood Cells, Molecules, and Diseases 36 (2006) 259–264
other hand, an effect on erythropoiesis in the Rps19+/−mice is
not supported by our previous and present analysis of blood
parameters such as Hb, hematocrit and MCV .
We also present the results of the mutant Rps19 allele on a
different genetic background. Blood was found normal in 3 and
7 week-old FVB/NJ Rps19+/−mice compared to age matched
controls. The absence of a detectable blood phenotype on two
different mouse strains argues against the genetic background
and modifier genes as important factors for the normal
hematopoietic phenotype in C57BL/6J Rps19+/−mice.
The finding of a normal erythropoiesis in Rps19+/−mice led
to the hypothesis that loss of an Rps19 allele may be rate-
limiting in erythroid precursor cells under proliferative stress.
An increased demand on erythropoiesis may result in a relative
deficiency of erythroid cells. We also considered previous
investigations showing that erythroid formation in DBA
patients does not respond to erythropoietin (EPO) which is a
major regulator of erythropoiesis [12,13,27]. To test the
sensitivity to EPO and, the capacity of erythroid formation in
Rps19+/−mice, we analyzed the response to EPO in Rps19+/−
and wild type mice. We demonstrate a similar response to
hrEPO in BM of Rps19+/−mice when compared to Rps19+/+
controls. Our results suggest a normal sensitivity to EPO as well
as a maintained capacity in the erythroid lineage under stress in
mice with one disrupted Rps19 allele.
Patients with DBA frequently show persistent levels of fetal
hemoglobin and we hypothesized that a proportion of
hemoglobin in the Rps19+/−mice after birth may consist of
persistent embryonic hemoglobin. In mouse, the site for
erythroid formation as well as the type of hemoglobin produced
changes during development. The primitive erythropoiesis in
yolk sac produces mainly embryonic hemoglobin and three
globin tetramers are described, E1 (x2,y2), E2 (α2,y2) and E3
(α2,z2) . Later stages of erythropoiesis take place in fetal
liver, spleen and BM and definite erythrocytes contain only the
adult hemoglobin forms (α2, β2)majand (α2, β2)min. The
mRNA expression of embryonic and adult globins during
development has previously been analyzed in mouse  and
expression of the embryonic gamma globin has been detected
up to day 15.5 after gestation. We determined the types of
globin isoforms present in blood on the protein level by MS and
we show that only the adult α and β-chains, corresponding to
the A allele for Hba-a1 and the S allele for Hbb-b1, are present
in blood of both wild type and Rps19+/−mice. It cannot be
excluded that embryonic globin is present in Rps19+/−mice at
levels below the limit of detection using MS but our results do
not imply any altered globin pattern.
Finally, we also investigated the eADA activity in the
Rps19+/−mice. The eADA activity is found increased in a
majority of patients with DBA and this may be the only marker
for subclinical forms of the disease. In our analysis, the eADA
activity was found almost identical when comparing adult
Rps19+/−mice and wild type mice.
We have performed a detailed biochemical analysis of the
mouse model with a disrupted Rps19 gene in order to identify
abnormalities similar to those associated with RPS19 mutations
in humans. We have investigated the mouse model for DBA
associated markers such as eADA activity, immature forms of
hemoglobin, and Rps19 levels in spleen and BM cells. We also
investigated the Rps19+/−mice for EPO sensitivity, response to
erythropoietic stress and variation with different genetic
background. We did not detect any abnormalities in the
Rps19+/−mice. From our combined results, we conclude that
a yet unknown mechanism, most likely at the transcriptional
level, may compensate for the lack of one functional Rps19
allele in mouse. More detailed knowledge about the regulation
of the Rps19 gene and its transcript are needed to understand the
possible role for Rps19 in murine erythropoiesis. Moreover,
increasing knowledge of mechanisms by which RPS19 acts in
human will pave the way for further investigations of this mouse
The authors would like to acknowledge Dr. Karpova at the
Novosibirsk Institute of Bioorganic Chemistry for providing the
vector for recombinant RPS19 expression, Jan Grawé at the
department of Genetics and Pathology for help with FACS
analysis and Gunilla Frenne at the Uppsala Academical
Hospital for eADA measurements.
This work was supported by grants from the National Heart
Lung and Blood Institute (grant no. 5 R01 HL079567-02 to N.
D.), the Children's Cancer Foundation of Sweden, the Swedish
Medical Research Council, the Swedish Cancer Society, the
DBA Foundation Inc., Ronald McDonald's fund, Torsten and
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