Construction and expression of a bicistronic vector containing human bone morphogenetic protein 2 and vascular endothelial growth factor-165 genes in vitro.

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Chinese Medical Journal 2009;122(4):471-473
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Brief report

Construction and expression of a bicistronic vector containing
human bone morphogenetic protein 2 and vascular endothelial
growth factor-165 genes in vitro

TIAN Xiao-bin, SUN Li, ZHANG Yu-kun, GAO Yong, FU De-hao and YANG Shu-hua

Keywords: bone morphogenetic protein 2; vascular endothelial growth factor-165; co-expression; bicistronic vector

t has been well documented that bone morphogenetic
proteins (BMPs), a group of proteins belonging to the
transforming growth factor-β (TGFβ) superfamily, can
induce bone formation, both in vivo and in vitro. Bone
morphogenetic protein-2 (BMP2) is a potent
osteoinductive factor and is being evaluated as a bone
growth inducer for orthopedic applications.1 Vascular
endothelial growth factor (VEGF), the best-characterized
angiogenic factor, plays an important role in bone growth
via the endochondral ossification pathway. Here, we
described the construction of a bicistronic vector
encoding human BMP2 and VEGF165. This vector will
aid in the investigation of angiogenic and osteogenic
factor interaction in bone formation and bone healing.

METHODS

Construction of the pIRES-BMP2 expression vector
The XhoI-BMP2-MluI sequence from the vector
pcDNA3.1-BMP22 was amplified by polymerase chain
reaction (PCR). The product was digested with XhoI and
MluI, separated by electrophoresis on a 1% agarose gel
and isolated using an Agarose Gel DNA Extraction Kit
(BioAsia, Shanghai, China). The purified product was
then ligated with an XhoI and MluI digested pIRES vector
(BD Biosciences, CA, USA) for 16 hours at 16°C in the
presence of T4 ligase (TaKaRa, Japan). Competent E. coli
DH5α were then transformed with the ligation mixture.
Transformed bacteria were spread on LB agar plates and
incubated overnight at 37°C. Single clones were then
picked and grown in LB medium supplemented with
ampicillin. Plasmid DNA from individual clones was
isolated using a Vector Miniprep Kit (BioAsia) and
analyzed by digestion with restriction endonucleases.
Finally, the pIRES-BMP2 vector was sequenced.

Construction of the pIRES-BMP2-VEGF165
expression vector
An XbaI-VEGF165-NotI sequence, containing the human
VEGF165 gene, was amplified by PCR from the vector
pUC-CAGGS-VEGF165 and then subcloned into the XbaI
and NotI sites of pIRES-BMP2 (The pUC-CAGGS-
VEGF165 vector was a gift from Dr. LIU Xiao-bing of
Tongji Medical College of Huazhong Science and
Technology University, Wuhan, China). The engineered
vector was propagated, isolated, analyzed and sequenced
as described above.

Cell culture and transfection
Mouse marrow stromal cells (MSCs) were cultured in
Dulbecco′s modified Eagle′s medium (DMEM, Gibco,
Germany) supplemented with 10% fetal bovine serum
and 0.1 mg/ml penicillin + streptomycin at 37°C in an
atmosphere of 5% CO2. The pIRES-BMP2-VEGF165
vector was transfected into MSCs by lipotransfection
according to the manufacturer′s protocol (Invitrogen,
USA). After 48 hours, the transfected cells were analyzed
directly for VEGF165 and BMP2 mRNA expression.
Empty pIRES vector was used as a control.

BMP2 and VEGF165 mRNA detection
BMP2 and VEGF165 mRNA were detected in transfected
cells by reverse transcription-PCR (RT-PCR). Total RNA
was isolated from harvested cells using Trizol (BioAsia).
First strand cDNA synthesis was performed using the
First Strand cDNA Synthesis Kit (Fermentas, Lithuania).
PCRs were performed using sense and antisense primers
of BMP2 and VEGF165.

Western blotting
Total 107 transfected MSCs were washed twice in
ice-cold phosphate buffered saline (PBS) and then lysed
with 200 µl lysis buffer. After centrifugation (20 000 × g
for 5 minutes), supernatants were analyzed by Western
blotting. BMP2 and VEGF165 proteins were detected
using anti-BMP2 antibody (Boster, Wuhan, China) at a
1:500 dilution and anti-VEGF165 antibody (Boster) at a
1:500 dilution, respectively. Proteins were detected using
I
DOI: 10.3760/cma.j.issn.0366-6999.2009.04.0021
Department of Orthopedics, People′s Hospital of Guizhou
Province, Guiyang, Guizhou 550002, China (Tian XB and Sun L)
Department of Orthopedics, Union Hospital, Tongji Medical
College of Huazhong Science and Technology University,
Wuhan, Hubei 430022, China (Zhang YK, Gao Y, Fu DH and
Yang SH)
Correspondence to: Dr. TIAN Xiao-bin, Department of
Orthopedics, People′s Hospital of Guizhou Province, Guiyang,
Guizhou 550002, China (Tel:
86-851-5924943. Email: txb6@vip.163.com)
This work was supported by the grants from the Science and
Technology Foundation of Guizhou Province (No. [2005]2053),
and the Special Foundation of Guizhou Province (No. 200514).
86-851-5937194. Fax:

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Chin Med J 2009;122(4):471-473
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an ECL kit (Boster).

Immunohistochemical analysis
Transfected MSCs were cultured in conditioned media for
7 days. The cells were fixed in ice-cold methanol and
incubated with anti-BMP2 antibody (Boster) at a 1:200
dilution or with anti-VEGF165 antibody (Boster) at a
1:500 dilution. Goat-anti-rabbit IgG antibody (Boster)
was used as secondary antibody.

RESULTS

Cloning and validation of the bicistronic
pIRES-BMP2-VEGF165 vector
Restriction digestion mapping and DNA sequencing
confirmed the correct engineering of BMP2 and VEGF165
cDNAs into the pIRES vector (Figure 1). The sequences
of BMP-2 and VEGF165 within the pIRES-BMP2-
VEGF165 vector were in agreement with the BMP2
(NM-001200) and VEGF165 (X 62568) sequences in the
GenBank database.



Figure 1. Restriction enzyme digestion analysis of recombinant
vector pIRES-BMP2-VEGF165. Lane 1: Marker. Lane 2:
recombinant vector pIRES-BMP2-VEGF165.
pIRES-BMP2-VEGF165 digested with XhoI/MluI. Lane 4:
pIRES-BMP2-VEGF165 digested with XbaI/NotI.
Lane 3:

Expression of BMP2 and VEGF165 mRNA in vitro
RT-PCR generated 1.2 kb and 592 bp products from
pIRES-BMP2-VEGF165 transfected cells. Cells transfected
with pIRES produced no RT-PCR products (Figure 2).



Figure 2. Co-expression of BMP2 and VEGF165 in mMSCs
detected by RT-PCR. Lane 1: Marker. Lane 2: BMP2
amplification of mMSCs/pIRES-BMP2-VEGF165. Lane 3:
BMP2 amplification of mMSCs/pIRES. Lane 4: VEGF165
amplification of mMSCs/pIRES-BMP2-VEGF165. Lane 5:
VEGF165 amplification of mMSCs/pIRES.
Expression of BMP2 and VEGF165 proteins
BMP2 and VEGF165 proteins were detected by Western
blotting analysis (Figures 3 and 4) and immunohisto-
chemistry (Figure 5) using anti-BMP2 and anti-VEGF165
antibodies, respectively.
pIRES-BMP2-VEGF165 were capable of BMP2 and
VEGF165 protein expression.

MSCs transfected with


Figure 3. Western blotting analysis of the BMP2 protein in
transfected mMSCs. Lane 1: Cells transfected with bicistronic
pIRES-BMP2-VEGF165 vector. Lane 2: Cells transfected with
empty pIRES vector. Lane 3: Non-transfected cells.



Figure 4. Western blotting analysis of the VEGF165 protein in
transfected mMSCs. Lane 1: Cells transfected with bicistronic
pIRES-BMP2-VEGF165 vector. Lane 2: Cells transfected with
empty pIRES vector. Lane 3: Non-transfected cells.



Figure 5. Immunohistochemistry analyses of the BMP2 (A) and
VEGF165 (B) proteins expressed in transfected MSCs (Original
magnification ×200).

DISCUSSION

BMPs were originally discovered in osteoinductive
extracts of bone matrix. BMP2 is a potent osteoinductive
factor, which has been shown to induce osteogenic
differentiation of mesenchymal cells. BMP2 can
stimulate differentiation of MSCs toward an osteoblastic
lineage, thereby increasing the number of differentiated
osteoblasts capable of forming bone.1 Recombinant
human BMP2 (rhBMP2) is manufactured by a
recombinant DNA biotechnology process and has been
evaluated preclinically and clinically for use in the
treatment of bone defects.3,4

Although BMP2 alone is sufficient to induce bone
formation,5 bone regeneration is a multistep process
involving many physiological and pathological

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Chinese Medical Journal 2009;122(4):471-473
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phenomena. Angiogenesis is postulated to be a crucial
step for new bone
best-characterized angiogenic factor, can enhance the
formation of new vessels
ossification and bone healing.7,8 Blocking VEGF leads to
a decrease in trabecular bone formation at the growth
plate, secondary to suppression of blood vessel invasion
and impairment of cartilage resorption.5 VEGF can
up-regulate BMP2 mRNA and protein levels in
endothelial cells.9,10 On the other hand, BMP2 can display
angiogenic activity during
upregulating VEGF expression.11,12 These studies indicate
that co-expression of BMP2
synergistically enhance bone formation and healing.13,14

The pIRES vector contains the internal ribosome entry
site (IRES) of the encephalomyocarditis virus (ECMV).
This sequence permits two genes of interest (in our case,
BMP2 and VEGF165) to be separately translated from a
single bicistronic mRNA. Additional advantages of
bicistronic vectors compared
monocistronic vectors are lower costs of preparation and
lower levels of endotoxins produced during vector
preparation. In the present work we have constructed a
bicistronic vector encoding BMP2 and VEGF165. This
vector has the potential to be transfected in vivo into cells
of restricted, localized sites to co-express the
osteoinductive and angiogenic factors. The co-expression
will enable the interaction between angiogenic and
osteogenic factors in bone formation and bone healing to
be investigated.

REFERENCES

1.
Khan SN, Bostrom MP, Lane JM. Bone growth factors.
Orthop Clin North Am 2000; 31: 375-388.
2.
Tian XB, Sun L, Yang SH. Cloning and construction of bone
morphogenetic protein 2 eukaryotic expression vector. Chin J
Reparat Reconstr Surg (Chin) 2006; 20: 112-115.
3.
Yoneda M, Terai H, Imai Y, Okada T, Nozaki K, Inoue H, et al.
Repair of an intercalated long bone defect with a synthetic
biodegradable bone-inducing implant. Biomaterials 2005; 26:
5145-5152.
4.
Chen FM, Wu ZF, Wang QT, Wu H, Zhang YJ, Nie X, et al.
Preparation of recombinant human bone morphogenetic
protein-2 loaded dextran-based microspheres and their
formation.6 VEGF, the
during endochondral
bone formation by
and VEGF may
with using two
characteristics. Acta Pharmacol Sin 2005; 26: 1093-1103.
Tian XB, Sun L, Yang SH, Zhang YK, Hu RY, Fu DH.
Osteogenic potential of the human bone morphogenetic
protein 2 gene activated nanobone putty. Chin Med J 2008;
121: 745-751.
Geris L, Gerisch A, Sloten JV, Weiner R, Oosterwyck HV.
Angiogenesis in bone fracture healing: a bioregulatory model.
J Theor Biol 2008; 251: 137-158.
Kaigler D, Wang Z, Horger K, Mooney DJ, Krebsbach PH.
VEGF scaffolds enhance angiogenesis and bone regeneration
in irradiated osseous defects. J Bone Miner Res 2006; 21:
735-744.
Gerber HP, Vu TH, Ryan AM, Kowalski J, Werb Z, Ferrara N.
VEGF couples hypertrophic cartilage remodeling, ossification
and angiogenesis during endochondral bone formation. Nat
Med 1999; 5: 623-628.
Bouletreau PJ, Warren SM, Spector JA, Peled ZM, Gerrets RP,
Greenwald JA, et al. Hypoxia and VEGF up-regulate BMP2
mRNA and protein expression in microvascular endothelial
cells: implications for fracture healing. Plast Reconstr Surg
2002; 109: 2384-2397.
10. Peng H, Usas A, Olshanski A, Ho AM, Gearhart B, Cooper
GM, et al. VEGF improves, whereas sFlt1 inhibits,
BMP2-induced bone formation and bone healing through
modulation of angiogenesis. J Bone Miner Res 2005; 20:
2017-2027.
11. Deckers MM, van Bezooijen RL, van der Horst G,
Hoogendam J, van Der Bent C, Papapoulos SE, et al. Bone
morphogenetic proteins stimulate angiogenesis through
osteoblast-derived vascular endothelial growth factor A.
Endocrinology 2002; 143: 1545-1553.
12. Raida M, Clement JH, Leek RD, Ameri K, Bicknell R,
Niederwieser D, et al. Bone morphogenetic protein 2 (BMP-2)
and induction of tumor angiogenesis. J Cancer Res Clin Oncol
2005; 131: 741-750.
13. Samee M, Kasugai S, Kondo H, Ohya K, Shimokawa H,
Kuroda S. Bone morphogenetic protein-2 (BMP-2) and
vascular endothelial growth factor (VEGF) transfection to
human periosteal cells enhances osteoblast differentiation and
bone formation. J Pharmacol Sci 2008; 108: 18-31.
14. Kanczler JM, Oreffo RO. Osteogenesis and angiogenesis: the
potential for engineering bone. Eur Cell Mater 2008; 15:
100-114.

(Received November 5, 2008)
5.
6.
7.
8.
9.
Edited by HAO Xiu-yuan