J Bone Miner Metab (2004) 22:278–282
DOI 10.1007/s00774-003-0485-8

Short communication© Springer-Verlag 2004

Bone mass increase specific to the female in a line of transgenic miceoverexpressing human osteoblast stimulating factor-1
Tamotsu Hashimoto-Gotoh1,2, Hideo Ohnishi3, Atsushi Tsujimura1,2, Hiroaki Tsunezuka2, Kan Imai2,
Haruchika Masuda2, and Toshitaka Nakamura3
1

Genomic Medical Sciences, Graduate School of Medicine, Kyoto Prefectural University of Medicine, Kamigyo-ku, Kyoto 602-8566, Japan
Department of Biochemistry and Molecular Genetics, Research Institute for Neurological Diseases and Geriatrics, Kyoto Prefectural
University of Medicine, Kyoto, Japan3
Department of Orthopedics, University of Occupational and Environmental Health, Kitakyushu, Japan
2

Abstract We have reported that transgenic mice overexpressing human osteoblast stimulating factor-1 (osf1) under thecontrol of the human osteocalcin promoter have a significantly
higher bone mineral content and density than nontransgenic
littermates. Consequently, bone mass loss due to estrogen
deficiency was compensated for in ovariectomized female
mice. Here, we show that in this transgenic line, the bone mass
increase was evident in female, but not male, mice, as evaluated using the ash assay, double-emission X-ray analysis, andcalcein double-labeling to determine the bone formation
rate. To elucidate a possible influence on gene expression, we
analyzed genomic structures of the inserted transgene and its
flanking regions in mouse chromosomes. The results revealed
that the transgene was integrated in the mouse repetitive
sequences, 234-bp-long γ-satellite repeats, as inverted
multiple (5 ⫹ 8) copies. Twelve copies at most seemed to befunctional, but no direct evidence supporting female-specificmRNA synthesis of the transgene was obtained.
Key words OSF1 · BDF1 mouse · primary hybrid strain ·littermates · bone mass

Introduction
Osteoblast stimulating factor-1 (OSF1 [1], also named
HB-GAM [2] and PTN [3]), a heparin-binding Lys-rich
18-kDa protein, belongs to the family that also includes
midkine (MK) [4]. One of the important differences
between the osf1 and mk genes may be that the former
is not expressed in the ovary or oocytes and thus is not
expressed in early embryonic cells, whereas the latter istranscribed abundantly in those cells [5]. This finding
strongly suggests that OSF1 may be responsible forrather mature processes of biological functions. Fur-

Offprint requests to: T. Hashimoto-Gotoh(e-mail: thg@koto.kpu-m.ac.jp)
Received: August 5, 2003 / Accepted: September 24, 2003

thermore, the function of OSF1, but not MK, may be
specific to nerve and osteoblast cells, because the gene
expression is restricted to these cells in mature animals[2,5]. In osteoblasts, osf1 is expressed between the earlyand middle stages of osteoblastic differentiation. OSF1
stimulates osteoblasts not only in differentiation but
also in proliferation [6] and even recruits osteoblast
cells [7]. In fact, we have proposed that OSF1 may not
be relevant to embryonic bone morphogenesis but
rather may function in later stages of bone metabolism
such as bone remodeling [8]. This function is distinct
from the case of bone morphogenetic protein-2(BMP2), which works during early embryogenesis
for ventral patterning [9] and endochondral bonemorphogenesis [10].
Recently, Tare et al. have reported that our OSF1
transgenic mice increase bone mass only in males, but
not females [11,12]. However, they failed to use appropriate littermate controls indispensable for quantitative
experiments using transgenic mice, particularly thosethat are created in the primary hybrid strains. In thisreport, we show that bone mass increase in ourtransgenic mice is in fact more apparent in female thanin male mice as evaluated by the ash assay, doubleemission X-ray analysis (DXA), and an examinationof the bone formation rate (BFR) using calcein
double-labeling.

Materials and methods
Animals
The line of osf1 transgenic mice expressing human osf1
under the control of human osteocalcin promoter(HPoc-Hosf1) has been constructed in a primary hybrid
strain, BDF1, between two congenic strains, female
C57BL/6 and male DBA/2 [8]. The homozygoustransgenic progeny were maintained by repeated inbred

T. Hashimoto-Gotoh et al.: Bone mass increase in female OSF1 micecrossing, and therefore their genetic background isbiased from that of BDF1. Therefore, it is most likelythat one may obtain artificial observations if they arecompared directly with BDF1 mice as controls. Thus,
before experiments, they were crossed back three times
to BDF1 mice purchased from Nihon CLEA (Tokyo,
Japan). The resulting heterozygous transgenic parents
were then crossed to produce transgene negative, heterozygous, and homozygous littermates. Mice were fed
as described previously [8].
Measurements of bone mass and bone formation rate
Bone mineral content and density were evaluated withthe ash assay using bilateral femora and by doubleemission X-ray analysis (DXA) using vertebrae (L2–L4)
as described previously [8]. The bone formation rate wascalculated using vertebra (L3) and tibia dually labeledwith calcein at 7-day intervals as previously described[13]. Histomorphometry was performed with a semiautomatic image-analyzing system linked to a light microscope. For each undecalcified vertebral body, the areaof secondary spongiosa was measured, but the region
within 0.3 mm from the growth plate–metaphyseal junction was not examined to exclude primary spongiosa. In
the right tibia section, the metaphyseal cancellous bone
area located within 0.2 mm distal to the growth plate–
metaphyseal junction was analyzed. As parameters ofbone formation, the followings were measured and calculated: mineralizing bone surface (MS/BS, %), mineralapposition rate (MAR, µm/day) calculated as the distance between double labels divided by interval labelingtime and multiplied by p/4, and bone formation rate
(BFR/BS, µm3/µm2/day).

279

Results
Transgene negative, heterozygous, and homozygous
littermates were bred under the same conditions fromparental mating to death at 30 weeks of age and examined to determine their bone mineral content anddensity as described in Materials and methods. It was
revealed that, in females, the bone mineral content ofbilateral femurs was significantly higher in homozygoustransgenic mice than in nontransgenic littermates.
However, in males, no significant differences were observed among negative, heterozygous, and homozygous
littermates (Fig. 1A). Essentially the same results wereobtained by DXA using vertebrae (Fig. 1B). Moreover,in homozygous mice, the bone mass of females was
significantly higher than that of males by either method,
ash assay or DXA (Fig. 1A,B).
The MAR values of the third lumbar vertebra andright tibia were higher in homozygous transgenics thanin nontransgenic littermates only in females (Fig. 2A).
Similarly, the BFR/BS values of lumbar and tibia werehigher in homozygous transgenics than in nontransgenic
littermates only in females (Fig. 2B). The MS/BS values

Genomic analysis of the transgene and flanking regions
Mouse genomic DNA was prepared from the liver anddigested partially by Sau3AI, filled with Klenow enzyme in the presence of dGTP and dATP, and fractionated by sucrose gradient density centrifugation for
isolation of DNA fragments of approximately 40 kbp.
Then, the isolated DNAs were used for cloning into
cosmid vector, pHSG429 [14], digested with XhoIand partially filled in the presence of TTP and dCTP,
and introduced into Escherichia coli STBL4 after invitro packaging (Invitrogen, Carlsbad, CA, USA).
The library was screened by colony hybridization withhuman osf1 cDNA labeled with [32P]dCTP. For cloning
the entire transgene repeats located between HindIII
and AccI sites, pHC79 [15] was used. The cloned
DNAs were subcloned into pKF4 [16] for sequencing
analysis.

Fig. 1. Bone mineral content (BMC) in femur (A) and bonemineral density (BMD) in vertebra (B). BMC and BMD were
evaluated by ash assay and double-emission X-ray analysis(DXA), respectively, as described in Materials and methods.
Means of groups were compared by ANOVA, and thesignificance of differences was determined with the post hoc
test using Bonferroni–Dunn’s method. A n ⫽ 9 (NE) and n ⫽4 (HO) in male; n ⫽ 5 (NE) and n ⫽ 9 (HO) in female. B n ⫽7 (NE) and n ⫽ 7 (HO) in male; n ⫽ 8 (NE) and n ⫽ 8 (HO)in female. NE, HE, and HO indicate transgenic negative,
heterozygous, and homozygous littermates, respectively

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T. Hashimoto-Gotoh et al.: Bone mass increase in female OSF1 mice
A

B

Fig. 2. Mineral appositional rates (MAR) and bone formation
rates per bone surface (BFR/BS) in vertebrae and in tibiae.
MAR and BFR/BS values were determined in vertebrae (Aand B, respectively) and tibiae (C and D, respectively),as described in Materials and methods. The p values were
calculated with Student’s t test: n ⫽ 9 (NE) and n ⫽ 4 (HO) inmales; n ⫽ 4 (NE) and n ⫽ 9 (HO) in females

were also higher in homozygous transgenics than in
nontransgenic littermates only in females but not inmales (data not shown).
We hypothesized that the chromosomal location of
the transgene might have an influence on its gene expression. For example, the transgene might be inserted
close to some gene controlled by enhancer sequencessuch as the estrogen-responsive element (ERE). Therefore, we analyzed the genomic structure of transgene
copies as well as flanking regions. We constructed a
cosmid library, and obtained two positive clones, E3C5
and A2F7, containing the left and right flanking regions,
respectively (Fig. 3A). By using the sequence information obtained from these clones, we then obtained asingle clone, H3A1, covering all the transgene copies䉴transgene copies are repeated 13 times as shown on the left by
B1 to B13. The top band indicated by “Full size” in lane [c]represents the entire H3A1 DNA singly cut by HindIII. Notethat DB9 is migrating slightly faster than expected in theladderFig. 3. Schematic structure of the human osf1 transgene
copies and flanking regions (A) and Southern blot analysis ofmultiple insertions of the transgene (B). A Cosmid clones,
E3C5 and A2F7, were obtained in pHSG429 as described inMaterials and methods. Another clone, H3A1, was obtainedby cloning DNA fragments (~40 kbp in size) prepared by
HindIII and AccI (filled in) digestion between HindIII and
BamHI (filled in) on pHC79 [15]. DNA sequences of theseclones were determined after subcloning, as described inMaterials and methods. Open arrows with small closed boxes,
representing the human osf1 open reading frame, indicate
the orientation and approximate size (2.65 kbp) of respective
transgene expression units. Two open boxes shown by D1 andD2 indicate the regions deleted in the transgene copies. The
solid line on the top indicates the approximate size (~100 kbp)
and regions covered by the three cosmid clones. The flanking
regions consisting of γ-satellite repeated sequences are shownby hatched boxes. Note that the γ-satellite repeats are inverted
together with transgene copies, as shown by shadows in theboxes as well as by arrows on the top. Nucleotide sequencesat the inversion point are . . . GGCTAA/AAGTAC . . .
linked between . . . GGCTAA[TAAAGG . . .] and [. . .
TAGAGA]AAGTAC . . . , in which the sequences in [ ] aredeleted as shown by D1. The nucleotide positions of A/A at
the junction correspond to A at the 2243rd and T at the1184th nucleotide positions in the transgene (accession no.
AB004306). A 248-bp-long deletion between nucleotide positions 894 and 1141, D[CTTCAG . . . AACGAA], contained inthe 3rd copy from the left, is indicated by D2. The nucleotide
sequences of the left and right flanking regions will appear
in the DDBJ databases under accession nos. AB105218 andAB105219, respectively. B Because HindIII does not cut at all
and BamHI cuts only once in the transgene DNA, the H3A1
DNA was digested completely with HindIII and completely
with an excess amount of BamHI (lane [a]) and partiallywith two different dilutions of BamHI (lanes [b] and [c]), thenseparated on a 0.6% agarose gel and probed with the 32Plabeled HincII–PstI fragment (2.2 kbp in size) of pHC79.
Size marker positions are indicated on the left (bp). The osf1

T. Hashimoto-Gotoh et al.: Bone mass increase in female OSF1 miceand both the flanking regions, namely mouse γ-satellite
repeat sequences [17]. Southern blot analysis of H3A1
DNA indicated that the transgene was inserted as
multiple copies, most likely 13 repeats (Fig. 3B). Apparently, the 9th band from the bottom migrated slightly
faster than expected, suggesting some deletion(s) in thetransgene.
Furthermore, DNA sequencing analysis revealed
that 5 and 8 copies of the transgene were invertedly
repeated, and that the 8th copy from the right and the
5th copy from the left (Fig. 3A) fused as a tail-totail inversion with a deletion in the middle. Anotherdeletion was also found in the 5⬘-untranslated region of
the 3rd copy from the left (Fig. 3A). Therefore, thetransgenic mice contain at most 12 functional transgene
copies per haploid, twice more than the initial estimation [8]. Concerning the flanking regions, it was revealed that the transgene copies were located in the
middle of the mouse repeated sequences, called γsatellite repeats, and no signs of any enhancer-like sequences such as an ERE were obtained, at least within~50 kbp either side of the center of the transgene copies.

Discussion
We have reported that bone mass loss resulting from
estrogen deficiency was compensated for in ovariectomized female mice in our transgenic line, which
overexpresses human osf1 under the control of a regulatory promoter containing the vitamin D response element (VDRE) for the human osteocalcin gene [8]. In
this study, we compared the bone mass increase in thetransgenic mice between males and females and foundthat it occurred only in females as judged by three criteria: bone mineral content (BMC), bone mineral density(BMD), and bone formation rate (BFR) (see Figs. 1, 2).
In principle, there may be three possible explanations
for the female-specific phenotype derived from the osf1
transgene. First, the mRNA synthesis could be more
efficient in females, probably due to an ERE which islocated somewhere close to the transgene (positional
effect). Second, it may be due to the VDRE containedin human osteocaolcin promoter used, which was multiply repeated in the mouse chromosome and thus could
partially be cross-reactive to estrogen (repetition
effect). Third, OSF1 protein synthesis or function may
be more active in the presence of some other protein(s),whose gene(s) is (are) under the control of an ERE.
The first possibility is unlikely because we found no
ERE sequences close to the transgene (within ~50 kbp).
The second possibility would be that the humanosteocalcin promoter with VDRE inserted repeatedly
would cross-react with the estrogen receptor, and thus
the HPoc-Hosf1 transgene may be expressed more281

efficently. In fact, the VDRE in the osteocalcin promoter contains features in common with that of the
ERE [18]. Concerning the third hypothesis, we have no
confirming evidence so far.
Recently, Tare et al. [11,12] reported, using ourtransgenic mice, that bone mass increase was observed
only in males but not in females. However, they failed
to employ appropriate control mice or littermate miceindispensable for such experiments [7,8,19]. Instead,they used directly BDF1 mice purchased from animal
breeders. Although our transgenic mice were createdin BDF1 embryos, they have been maintained astransgenic homozygotes by inbred crossing and must
therefore be genetically biased from the original strain.
It is well known that, in mouse experiments, one mutation can have markedly different phenotypes whenplaced on different genetic backgrounds [20]. The variation is caused by different alleles at modifying loci invarious mouse inbred strains. This consideration isparticularly important in the case of BDF1, because it isgenetically uniform because of the nature of a primary
hybrid strain between two congenic strains, from which
their descendents are largely biased after repeatedinbred crossings.
The transgenic mice used in this work can be obtained as “bone-rich mice” from the Center for Animal
Resources and Development, Kumamoto University,
Kumamoto 860-0811, Japan (http://card.medic.
kumamoto-u.ac.jp).
Acknowledgments. We thank Dr. T. Shiroishi for his
advice on mouse genetics, and Drs. G. Schütz and M.
Sato for critical reading of this manuscript.

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