Download AML1(7/7) embryos do not express certain hematopoiesis

Oncogene (1998) 17, 2287 ± 2293
ã 1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00
http://www.stockton-press.co.uk/onc
AML1(7/7) embryos do not express certain hematopoiesis-related gene
transcripts including those of the PU.1 gene
Hitoshi Okada1, Toshio Watanabe2, Masaru Niki2, Hiroshi Takano1, Natsuko Chiba2,
Nobuaki Yanai3, Kenzaburo Tani4, Hitoshi Hibino4, Shigetaka Asano4, Michael L Mucenski5,
Yoshiaki Ito6, Tetsuo Noda1 and Masanobu Satake2
1
Department of Cell Biology, Cancer Institute, Toshima-ku, Tokyo 170; 2Department of Molecular Immunology; 3Department of
Cell Biology, Institute of Development, Aging and Cancer, Tohoku University, Seiryo-machi, Aoba-ku, Sendai 980; 4Department of
Hematology-Oncology, The Institute of Medical Science, The University of Tokyo, Shirokanedai, Minato-ku, Tokyo 108, Japan;
5
Human Genome Sciences, Inc., 9410 Key West Avenue, Rockville, Maryland 20850, USA; 6Department of Viral Oncology,
Institute for Virus Research, Kyoto University, Shogoin, Sakyo-ku, Kyoto 606, Japan
The AML1 and PEBP2b/CBFb genes encode the DNAbinding and non-binding subunits, respectively, of the
heterodimeric transcription factor, PEBP2/CBF. Targeting each gene results in an almost identical phenotype,
namely the complete lack of de®nitive hematopoiesis in
the fetal liver on embryonic day 11.5 (E11.5). We
examined and compared the expression levels of various
hematopoiesis-related genes in wild type embryos and in
embryos mutated for AML1 or PEBP2b/CBFb. The
RNAs were prepared from the yolk sacs of E9.5
embryos, from the aorta-gonad- mesonephros regions of
E11.5 embryos and from the livers of E11.5 embryos and
RT ± PCR was performed to detect various gene
transcripts. Transcripts were detected for most of the
hematopoiesis-related genes that encode transcription
factors, cytokines and cytokine receptors, even in tissues
from homozygously targeted embryos. On the other
hand, PU.1 transcripts were never detected in any tissue
of AML1(7/7) or PEBP2b/CBFb(7/7) embryos. In
addition, transcripts for the Vav, ¯k-2/¯t-3, M-CSF
receptor, G-CSF receptor and c-Myb genes were not
detected in certain tissues of the (7/7) embryos. The
results suggest that the expression of a particular set of
hematopoiesis-related genes is closely correlated with the
PEBP2/CBF function.
Keywords: transcription factor; hematopoiesis; AML1;
PEBP2b/CBFb; PU.1; RT ± PCR; gene targeting
Introduction
The AML1 gene encodes the DNA binding subunit of
the heterodimeric transcription factor, PEBP2/CBF
(Bae et al., 1993, 1994; Kagoshima et al., 1993, 1996;
Meyers et al., 1993; Ogawa et al., 1993b). The gene,
also known as PEBP2aB or CBFA2, belongs to the
mammalian gene family, runt, all of which contain the
conserved Runt domain which is responsible for the
DNA binding activity of AML1. Several di€erent
approaches have been used to study the biological
signi®cance of the AML1 gene. Firstly, AML1 is
located at the chromosomal breakpoints associated
Correspondence: M Satake, Department of Molecular Immunology,
Institute of Development, Aging and Cancer, Tohoku University,
Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan
Received 14 April 1998; revised 22 May 1998; accepted 26 May 1998
with several subtypes of human acute myeloid or
lymphoblastic leukemia (Erickson et al., 1992; Golub et
al., 1995; Mitani et al., 1994; Miyoshi et al., 1991,
1993; Nucifora et al., 1993; Romana et al., 1995;
Shurtle€ et al., 1995). The chimeric genes generated
between AML1 and counterpart genes by chromosomal translocations are considered to be responsible for
leukemogenesis.
Secondly, recent gene targeting studies have shown
that the function of AML1 is indispensable for the
development of de®nitive hematopoiesis in the murine
fetal liver on embryonic day 11.5 (E11.5) (Okuda et al.,
1996; Wang et al., 1996a). In AML1(7/7) fetal livers,
no hematopoietic cells of any lineage are generated and
CFU-C activity cannot be detected. The defect caused
by homozygous disruption of AML1 is considered to
be intrinsic to multipotent hematopoietic progenitors.
This notion is based on the analysis of chimeric mice
using AML1 (7/7) embryonic stem (ES) cells.
Primitive hematopoiesis which originates in the yolk
sac on E7.5 is apparently una€ected by AML1
targeting. However, AML1(7/7) embryos start to
die on E12.5 due to massive hemorrhage of primitive
erythrocytes in the central nervous system by a
mechanism that remains to be elucidated. Interestingly, almost identical phenotypes have been reported
for targeting of the gene, PEBP2b/CBFb (Niki et al.,
1997; Sasaki et al., 1996; Wang et al., 1996b). PEBP2b/
CBFb encodes the non-DNA binding subunit of
PEBP2/CBF (Ogawa et al., 1993a; Wang et al.,
1993). Therefore, gene targeting studies have demonstrated that the two subunits of the transcription factor
PEBP2/CBF, namely AML1 and PEBP2b/CBFb,
function as one entity for the generation of hematopoietic progenitors of the de®nitive type.
There exist a number of reports of target genes
regulated by the transcription factor, PEBP2/CBF.
These include the GM-CSF receptor (Frank et al.,
1995; Takahashi et al., 1995), M-CSF receptor (Zent et
al., 1996; Zhang et al., 1994, 1996), IL-3 (Nimer et al.,
1996), myeloperoxidase, neutrophil elastase (Nuchprayoon et al., 1994) and T cell receptors genes
(Giese et al., 1995; Hernandez-Munain and Krangel,
1994, 1995; Sun et al., 1995; Wotton et al., 1994 and
see a review by Leiden, 1993). The above claims,
however, mostly rely on studies using cell lines
transfected by reporter plasmids whose transcription
is driven by the promoter of a suspected PEBP2/CBF
regulated gene. In this study we have taken an
PU.1 expression in AML1 targeted embryos
H Okada et al
2288
alternative approach by examining genes whose
expression is a€ected in embryonic tissues with an
AML1 (7/7) or PEBP2b/CBFb (7/7) background.
The expression of genes encoding transcription factors,
cytokines and cytokine receptors, which are all known
to be involved in hematopoiesis, was examined. The
RNAs used for RT ± PCR analysis were prepared from
the yolk sac, the embryonic aorta-gonad-mesonephros
(AGM) region and the fetal liver. The AGM region
has recently been shown to be the bona®de origin of
de®nitive hematopoiesis in the murine embryo
(Medvinsky and Dzierzak, 1996; Medvinsky et al.,
1993; MuÈller et al., 1994). Multipotent hematopoietic
progenitors develop ®rst in the AGM region and then
settle in the liver through circulation or by direct
migration. The transcripts whose expression was
a€ected by mutation of AML1 or PEBP2b/CBFb
include those of c-Myb, ¯k-2/¯t-3, M-CSF receptor, GCSF receptor and Vav. In particular, PU.1 transcripts
were never detected in any tissue of homozygously
targeted embryos. These ®ndings are discussed in
respect to gene regulation by PEBP2/CBF.
a
b
Figure 1 Targeting the AML1 gene by homologous recombination. (a) The targeting strategy used to delete the fourth exon of
AML1. In the targeting vector the transcriptional direction of neo is opposite to those of the AML1 and DTA genes. E3 and E4
indicate the third and fourth exons of AML1. Probes A, B, C and D whose locations are as indicated were used for Southern
hybridization as in panel (b). The sense primers a or c and the antisense primer b were used for PCR to genotype F2 embryos.
Abbreviations used for the restriction sites were B for BamHI and X for XbaI. (b) Southern blot analysis of F2 embryos. Genomic
DNAs of E11.5 F2 embryos were digested by XbaI in the cases of probes A, C and D or by BamHI in the case of probe B and gel
electrophoresed. The blotted ®lters were hybridized with probes A, B, C or D as indicated. The genotypes of AML1 locus in each
embryo are indicated. The numbers alongside the ®lters indicate the size of the detected bands in kilobases
PU.1 expression in AML1 targeted embryos
H Okada et al
Results
Generation of mouse embryos homozygous for the
AML1 disruption
The fourth exon of the AML1 genome encodes the
central portion of the Runt domain in the AML1
polypeptide (Miyoshi et al., 1995). This was replaced
by the neo gene derived from the targeting vector
(Figure 1, panel a). By screening the ES cells
transfected with the targeting vector, 18 homologous
recombinants were obtained. Using two ES clones, we
obtained two lines of F1 mice which were heterozygous
for the AML1 mutation. Two lines of F2 o€springs
having the AML1(7/7) genotype displayed identical
phenotypes like those described in the Introduction
and remained alive until E12.5.
The results of Southern blot analysis of the
genotypes of the F2 embryos are presented in panel
b. Genomic DNA was prepared from E11.5 embryos.
Probe A was derived from the genomic sequence and
located 3' to the homologous sequence used in the
vector. In the case of the heterozygous (+/7)
genotype, both the 13.2 kb band of the wild type and
the 7.5 kb band from the mutated allele were detected
in XbaI-digested DNA. Only the 13.2 and 7.5 kb bands
were found for the (+/+) and (7/7) genotypes,
respectively. Probe B was located within the 5'
homologous sequence used in the vector. This probe
detected both the wild type 4.0 kb band and the
mutated 2.0 kb band in BamHI-digested DNA
prepared from the heterozygous (+/7) embryo.
Probes C and D are derived from sequences of the
AML1 fourth exon and neo gene, respectively. In XbaIdigested genomic DNA, probe C hybridized with the
13.2 kb band for the wild type (+/+) and heterozygous (+/7) embryos but not for the (7/7)
homozygote. On the other hand, the 6.9 kb band was
detected by probe D for the (+/7) heterozygote and
the (7/7) homozygote but not for the wild type (+/
+) embryo. Thus, the overall results of Southern blot
analysis con®rmed the expected pattern of homologous
recombination.
Histological examination of the yolk sac
The embryonic hematopoietic tissues were examined
histologically. Figure 2 shows sections of the yolk sacs
from E9.5 embryos. At this stage, numerous primitive
erythrocytes with nuclei were identi®ed for both the
heterozygously and homozygously mutated embryos.
This indicates that AML1 is dispensable for primitive
hematopoiesis.
Expression of hematopoiesis-related genes in
AML1-mutated embryos
Figure 2 Histological examination of the yolk sacs from
AML1(+/7) and (7/7) embryos. Sections of the yolk sacs
were prepared from E9.5 embryos in utero and stained with
hematoxylin and eosin. The genotypes of AML1 are (+/7) and
(7/7) as indicated
The E12.5 embryos of AML1 (7/7) were devoid of
hematopoietic cells of the de®nitive type in the liver
(data not shown). We next examined whether the
expression of hematopoiesis-related genes was a€ected
by the AML1 mutation. We selected three hematopoietic tissues, namely the yolk sac of an E9.5 embryo,
the AGM region of an E11.5 embryo and the liver of
an E11.5 embryo. RNAs were prepared from each
tissue and gene expression was examined by the RT ±
PCR method. Adequateness of PCR-ampli®cation was
checked by examining the size and restriction enzyme
digestion pattern of the PCR products. At least four
embryos obtained from two di€erent litters were used
for one genotype. In addition, we also examined
embryos mutated for PEBP2b/CBFb, whose generation we reported previously (Niki et al., 1997). Table 1
summarizes the results of RT ± PCR analysis which was
performed for each transcript, for each tissue and for
each genotype of AML1 or PEBP2b/CBFb. Since the
PCR method employed was not very quantitative, the
results are simply expressed as being positive or
negative. Therefore, even if a transcript was found be
present at a lower level in the tissue of one genotype
compared to that of same tissue of another genotype, it
was still considered to be positive.
The AML1 transcript was not detected in any tissue
of AML1(7/7) embryos but was detected in the liver
of PEBP2b/CBFb(7/7) embryos. On the contrary,
the PEBP2b/CBFb transcript was not detected in the
liver of PEBP2b/CBFb(7/7) embryos but was
detected in all the tissues of AML1(7/7) embryos.
2289
PU.1 expression in AML1 targeted embryos
H Okada et al
2290
were prepared from the livers of E11.5 embryos and
hybridized with the radiolabeled antisense RNA probe.
After digestion by RNase, the samples were run on a
sequencing gel (Figure 4). The probe used was a mixture
of antisense RNAs which allowed simultaneous
detection of both PU.1 and glyceraldehyde -3-phosphate
dehydrogenase (GAPDH) transcripts. GAPDH transcripts were detected to a similar extent irrespective of
the AML1 genotype. On the other hand, PU.1
transcripts were only evident for the AML1(+/+)
and (+/7) genotypes but not for the (7/7) genotype.
Each lane in Figure 4 represents an RNA sample
prepared from an individual embryo. Thus, the
combined results of Figures 3 and 4 con®rm that there
was, indeed, no PU.1 transcript expressed in the tissues
of AML1(7/7) or PEBP2b/CBFb(7/7) embryos.
Absence of PU.1 transcript in the AML1(7/7)
embryos
The PCR products generated from the PU.1 transcripts
were processed for Southern blot analysis (Figure 3).
The radiolabeled probe used corresponds to the
oligonucleotide sequence located between the sense
and antisense primers. No apparent band was detected
for the yolk sac, the AGM region or the liver of
AML1(7/7) embryos, whereas discrete, positive
signals were detected for the tissues of AML1(+/+)
and (+/7) embryos (see the upper panel). It is
particularly noteworthy that no PU.1 transcripts were
detected in the E9.5 yolk sac of AML1(7/7) embryos
that contained numerous primitive erythrocytes. From
the RNA samples described in the upper panel, similar
amounts of PCR products were generated when the
primers were set for the hypoxanthine phosphoribosyl
transferase (HPRT) cDNA. The absence of the PU.1
transcript in the liver RNA of PEBP2b/CBFb(7/7)
embryos was also con®rmed by Southern blot analysis
of the RT ± PCR products (data not shown).
The expression of the PU.1 transcript was also
examined by an RNase protection assay. The RNAs
Other genes whose expression was a€ected by the AML1
mutation
In addition to PU.1, several other hematopoiesisrelated genes are a€ected in their expression by
mutation of AML1 and PEBP2b/CBFb (Table 1).
The transcripts of the M-CSF receptor and Vav were
Table 1. Expression of various genes in AML1-, PEBP2b/CBFb- or c-Myb- mutated embryos as examined by RT±PCR
Genotype of AML1
Genotype of PEBP2 b/
Genotype of c-Myb
CBF b
E9.5 Yolk sac
E11.5 AGM
E11.5, Liver
E11.5, Liver
E11.5, Liver
(+/+) (+/±) (±/±) (+/+) (+/±) (±/±) (+/+) (+/±) (±/±) (+/+) (+/±) (±/±) (+/+) (+/±) (±/±)
AML1
PEBP2 b/CBF b
PEBP2 aA
GATA1
GATA2
Ikaros
Scl/tal1
PU.1
NFE2
c-Myb
¯k-1
¯k-2/¯t-3
¯t-1
SCF
c-Kit
Epo
Epo receptor
M-CSF
M-CSF receptor
G-CSF
G-CSF receptor
Vav
b-actin
HPRT
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Figure 3 RT ± PCR analysis of PU.1 and HPRT transcripts. PCR was performed on the cDNA templates prepared from the E9.5yolk sacs, the E11.5-AGM regions and the E11.5-livers, using the appropriate primers to detect PU.1 or HPRT transcripts. The
PCR products were gel electrophoresed and the blotted ®lters were processed for Southern hybridization. 32P-labeled probes used in
hybridization are described in Material and methods. Each lane represents a sample from an individual embryo and the AML1
genotypes of each embryo are as indicated
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PU.1 expression in AML1 targeted embryos
H Okada et al
to the paucity of hematopoietic cells in the AML1 (7/
7) or PEBP2b/CBFb (7/7) embryos.
Discussion
Figure 4 Expression of PU.1 and GAPDH transcripts as assayed
by RNase protection. The cytoplasmic RNAs prepared from the
livers of E11.5 embryos were hybridized with 32P-labeled RNA
probes harboring the antisense sequences of PU.1 or GAPDH.
After digestion with RNase, the samples were gel electrophoresed
and processed for image analysis. The locations of undigested
RNA are indicated by bars. The genotypes of AML1 in each
embryos are (+/+), (+/7) or (7/7), as indicated
neither detected in the AGM nor in the liver of AML1
(7/7) embryos, but were detected in the yolk sac. The
expression of c-Myb and the G-CSF receptor was
undetectable in the liver of these embryos but was
detectable in the AGM and yolk sac.
The AML1 (7/7) embryos did not express ¯k-2/¯t-3
transcripts in the AGM or liver. As for the yolk sac, the
e€ect of the AML1 mutation on ¯k-2/¯t-3 expression
cannot be evaluated, since corresponding transcripts
were not detected in the yolk sac, even in the case of
wild type embryos. This result, however, con®rms that
the expression of ¯k-2/¯t-3 is strictly correlated with the
development of de®nitive type hematopoiesis (Matthews
et al., 1991).
It should be noted that homozygous mutations of
AML1 or PEBP2b/CBFb did not necessarily cause
complete shut down of expression of the hematopoiesis-related genes other than mentioned above.
Comparison with the c-Myb targeted embryos
Homozygous mutations of ¯k-2/¯t-3 (Mackarehtschian
et al., 1995), c-Myb (Mucenski et al., 1991) or PU.1
(McKercher et al., 1996; Scott et al., 1994), all impair
hematopoiesis of the de®nitive type, although only in
certain cell lineages and to di€erent extents. The
phenotype that best resembles the case of AML1 (7/
7) or PEBP2b/CBFb (7/7) is that associated with
gene targeting of c-Myb. In the liver of c-Myb (7/7)
embryos, no hematopoietic cells with the exception of
megakaryocytes can be observed and no CFU-C
activities can be detected. Therefore, we examined in
parallel what kind of genes are a€ected in their
expression in the c-Myb (7/7) embryonic liver.
Interestingly, transcripts of the PU.1, ¯k-2/¯t-3, Vav,
G-CSF receptor and M-CSF receptor genes were
detected by the RT ± PCR method (Table 1). This
indicates that the absence of de®nitive hematopoiesis in
the c-Myb (7/7) embryos did not result in the
complete lack of expression of the above mentioned
transcripts. Thus, the failure to detect a particular
species of transcript does not appear to be simply due
Of particular interest in the present study is the
observation that AML1 (7/7) and PEBP2b/CBFb
(7/7) embryos displayed identical loss of certain
transcripts of the liver. This is in good agreement with
the notion that deletion of even one of the genes of the
heterodimeric transcription factor PEBP2/CBF is
sucient to abolish its function. It is therefore likely
that the absence of certain transcripts in the
homozygously mutated embryos is somehow related
to the loss of PEBP2/CBF function.
Recently, the culture system of AGM in which
hematopoietic cells can be generated in vitro has been
developed (Mukouyama et al., 1998). We subjected the
AGM regions of the AML1 (7/7), c-Myb (7/7) as
well as wild type E11.5 embryos to this culture,
according to the procedure described. Neither from
the AML1 (7/7) or from the c-Myb (7/7) culture,
hematopoietic cells were produced at all (details of these
cultures will be described elsewhere). Interestingly,
transcripts of PU.1 and Vav were not detected in the
AML1 (7/7) culture but detected in the c-Myb (7/7)
as well as the wild type cultures by the RT ± PCR
method (unpublished results). This indicates that AML1
is indeed indispensable for the expression of PU.1 and
Vav irrespective of the presence or absence of
hematopoiesis, at least in the in vitro culture of AGM.
The expression of M-CSF receptor has been reported
to be regulated by AML1 (Zhang et al., 1996). The
AML1 and C/EBP transcription factors can associate
with each other, bind to their adjacent sites together on
the promoter of the M-CSF receptor and transactivate
it synergistically. This was revealed by transfection
studies using various cell lines. Thus, it is noteworthy
that the present study employing gene targeted
embryos identi®ed the M-CSF receptor as a candidate
gene whose expression is regulated by PEBP2/CBF.
The transcripts which behaved similarly to the M-CSF
receptor in the targeted embryos include those of PU.1,
¯k-2/¯t-3 and Vav. None of these transcripts were
detected in the AGM or liver of AML1 (7/7) or
PEBP2b/CBFb (7/7) embryos. The possibility that
the promoters of PU.1 and Vav are likewise regulated
by PEBP2/CBF remains to be elucidated, since survey
of their promoter regions (7330 nt through to +1 nt
of the PU.1 promoter, Chen et al., 1995 and 7870 nt
through to +1 nt of the Vav promoter, Ogilvy et al.,
1998) revealed multiple candidate sequences therein,
though not typical, for the PEBP2/CBF binding
(unpublished observation). In the case of the PU.1
promoter, several transcription factors such as Oct1/2,
Spl and PU.1 itself have been reported to be important
for its transactivation in myeloid and B lymphoid cell
lines (Chen et al., 1995, 1996).
Overall, the present study has shown that a
particular set of hematopoiesis-related genes are
signi®cantly a€ected in their expression by mutations
of AML1 and PEBP2b/CBFb. These observations
should help clarify the relationship between the gene
expression and the PEBP2/CBF transcription factor in
exact molecular and cellular terms.
2291
PU.1 expression in AML1 targeted embryos
H Okada et al
2292
Materials and methods
Targeting vector and homologous recombination
The targeting vector for AML1 replaces a 0.6-kb region
that encompasses the fourth exon of AML1 with the neo
cassette containing the neo gene regulated by the promoter
and the poly(A)+ addition sequence of the murine
phosphoglycerol kinase gene (Figure 1a). The neo gene
was ¯anked by a 6.3 kb SmaI-EcoRI fragment and a 1.3 kb
SmaI-BamHI fragment, each corresponding to 5' and 3'
genomic sequences of AML1 surrounding the fourth exon.
In addition, the loxP sequences were added to both ends of
the neo cassette. The gene for the diphtheria toxin A
subunit (DTA) was incorporated into the 3' end of the
vector. The vector was linearized with NotI and electroporated into J1 ES cells. Two hundred and thirty G418
resistant ES cell colonies were picked up, expanded as
described previously (Nakai et al., 1995) and screened for
homologous recombination by Southern blotting using
probe A depicted in Figure 1a. Eighteen ES cell clones
were identi®ed as heterozygous for the AML1 disruption
and two clones were injected into blastocysts derived from
C57BL/6J mice. Chimeric mice were bred against C57BL/
6J female mice. Southern blot analysis of tail DNA of F1
mice showed germline transmission of the mutant AML1
allele. F1 mice heterozygous for the mutation were
intercrossed to generate F2 o€spring. For genotyping F2
mouse embryos routinely, PCR was performed on genomic
DNA as a template. The locations of primer a, 5'CAGGTTTGTCGGGCGGAGCGGTAGA-3', primer b,
5'-CCAAGTCCATCACAGCTGTGCGTT-3' and primer
c,
5'-GCTAAAGCGCATGCTCCAGACTGCCTTG-3',
are depicted in Figure 1a. PCR ampli®cation (PerkinElmer) was performed for 35 cycles (948C, 1 min; 608C,
2 min; 728C, 2 min). The size of the PCR products were
211 bp from the wild type allele and 340 bp from the
mutated allele. All the presented data was obtained using
F2 mouse embryos. Targeting the PEBP2b/CBFb and cMyb genes was described previously (Mucenski et al., 1991;
Niki et al., 1997).
Histological examination
The E9.5 embryos in utero were ®xed in formaldehyde
solution for 6 h. The yolk sacs were then removed and
embedded in paran. Transverse sections with a 10 mm
thickness were mounted on slide glasses and stained with
hematoxylin and eosin.
RT ± PCR analysis
E9.5 and E11.5 live embryos were selected on the basis of
the presence of a heart beat. Total RNA samples were
prepared from the yolk sacs, the AGM regions or the livers
of embryos using ISOGEN (Nippon Gene). RNA (2 mg)
was reverse transcribed at 428C for 60 min using a cDNA
cycle kit (Invitrogen) and the products were dissolved in
10 ml of distilled water. PCR ampli®cation of the cDNAs
(0.2 ml each for one reaction) was performed for 35 cycles.
One cycle was 948C, 1 min; 608C, 2 min and 728C, 2 min.
The PCR products were analysed by 2% (wt/vol) agarose
gel electrophoresis. All the primer sets for the genes listed
in Table 1 were as described previously (Keller et al., 1993;
Koopman et al., 1989; McClanahan et al., 1993; Takahashi
et al., 1997; Weiss et al., 1994). For the PCR products of
PU.1, c-Myb and HPRT, Southern blot analysis was also
carried out. The blotted ®lters were hybridized with 32Pend-labeled oligonucleotide harboring the sequences of
PU.1, 5'-CTATCGCCACATGGAGCTGGAACAG-3', cMyb, 5'-TCTGGGGAACAGATGGGCAGAGAT C - 3'
or HPRT, 5'-TACGAGGAGTCCTGTTGATGTTGCC-3'.
The hybridization was done at 428C for 12 h in 66SSPE,
56Denhardt's solution, 1% (wt/vol) SDS and 0.1 mg/ml
of denatured salmon sperm DNA. The blotted ®lters were
washed twice at 608C for 15 min in 0.26SSC and 0.1%
(wt/vol) SDS and processed for autoradiography.
RNase protection analysis
The antisense RNA probes were prepared as follows. The
cDNA sequence of PU.1 (nt 20 through to nt 571 in the
coding region) was subcloned into the PstI-SacI sites of
pBluescript. Two mg of the plasmid DNA was linearized by
digestion with HindIII and used as a template. Transcription was performed by T7 RNA polymerase and [a-32P]UTP was included in the reaction. Total radioactivity
incorporated into RNA was 26107 c.p.m. The antisense
RNA probe for GAPDH was prepared in a similar way.
The 32P-labeled RNA probes (16105 c.p.m. for PU.1 and
16103 c.p.m. for GAPDH) were mixed with 10 mg of the
cytoplasmic RNA prepared from the livers of E11.5
embryos. Hybridization and RNase digestion were performed using a RPAII Ribonuclease Assay Kit (Ambion
Inc.). The ®nal sample was run on an 8% (wt/vol)
polyacrylamide-8M urea sequencing gel. The dried gel was
processed for image analysis.
Acknowledgements
We thank I Imamura for secretarial assistance. This work
was supported by research grants from the Ministry of
Education, Science, Sports and Culture of Japan, The
Asahi Glass Foundation, The Mochida Memorial Foundation for Medical and Pharmaceutical Research and
Intelligent Cosmos Academic Foundation.
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