Expression of human bone morphogenetic protein (BMP-2 and BMP-4) genes in transgenic bovine fibroblasts

Expressão dos genes bone morphogenetic protein (BMP-2 e BMP-4) em fibroblastos bovinos transgênicos

BMP; gene expression; fibroblast; oral and maxilofacial surgery; tissue reconstruction

BMP; expressão gênica; fibroblasto; cirurgia oral e maxilofacial; reconstrução de tecidos

COMMUNICATION

Expression of human bone morphogenetic protein (BMP-2 and BMP-4) genes in transgenic bovine fibroblasts

Expressão dos genes bone morphogenetic protein (BMP-2 e BMP-4) em fibroblastos bovinos transgênicos

C. Oleskovicz^{I, II}; S. Lisauskas^{III, IV}; F.J.L. Aragão^III,*

^IUniversidade Católica de Brasília - Campus II - Brasília, DF

^IIHospital de Base - Unidade de Cirurgia e Traumatologia Buco-Maxilo-Facial - Brasília, DF

^IIIEmbrapa Recursos Genéticos e Biotecnologia PqEB Final Av. W3 Norte 70770-900 - Brasília, DF

^IVUniversidade de Brasília - Campus Brasília, DF

Keywords: BMP, gene expression, fibroblast, oral and maxilofacial surgery, tissue reconstruction

RESUMO

cDNAs dos genes bone morphogenetic protein-2 (BMP-2) e bone morphogenetic protein-4 (BMP-4) foram sintetizados a partir de RNA total extraído de tecidos ósseos de pacientes que apresentavam trauma facial (fraturas do maxilar entre o 7º e o 10º dia pós-trauma) e clonados num vetor para expressão em células mamíferas, sob controle do promotor de citomegalovírus (CMV). Os vetores contendo os genes BMP-2 e o BMP-4 foram utilizados para a transfecção de fibroblastos bovinos. mRNAs foram indiretamente detectados por RT-PCR nas células transfectadas. As proteínas BMP-2 e BMP-4 foram detectadas mediante análises de Western blot. Os resultados demonstram a possibilidade de produção desses fatores de crescimento celular em fibroblastos bovinos. Essas células poderão ser utilizadas como fontes doadoras de material genético para a técnica de transferência nuclear na geração de animais transgênicos.

Palavras-chave: BMP, expressão gênica, fibroblasto, cirurgia oral e maxilofacial, reconstrução de tecidos.

Losses of bone tissues occur due to several factors, most importantly due to accidents. The bone tissue has an uncommon capacity to regenerate with the complete substitution of the harmed tissue by de novo formed tissue. However, there is a physiological limit in which the organism is capable to accomplish the task of self-reconstruction without surgical intervention. When the bone loss is larger than this critical limit, a surgical intervention is necessary in order to achieve tissue reparation. With the development of internal rigid fixation systems (use of metallic reconstruction plates and screws), the opened fracture reductions reached a satisfactory level. Nevertheless, the results achieved with the use of these techniques are still uncertain in cases of large tissue destruction in which the losses do not allow a correct juxtaposition of the bone fragments. In order to try to solve these limitations, surgeons have been searching and testing several types of therapeutic approaches to obtain aesthetic and functional restorations of the harmed anatomical area. Among the alternatives, the most used are autografts (bone transferred from one site to another in the same individual), alografts (bone transferred from one individual to another), xenografts (bone transferred from one specie to another) and aloplastics (synthetic materials). Although the autografts are successfully used, they have some disadvantages, such as the limited amount of bone tissue that can be obtained and necessity of a surgery in the donor area. Consequently, there is considerable interest in developing novel alternatives to de novo regenerate bone tissue. With the increase of the knowledge of the genetic osteogenic factors and genetic engineering, genetic therapy is becoming a viable alternative to obtain a satisfactory result in bone regeneration.

The in vivo repair of bone tissue is controlled by the temporary expression of growth factors genes (Bolander, 1992; Sandberg et al., 1993; Linkhart et al., 1996; Bostrom et al., 1998), trigged by the presence bone morphogenetic proteins (BMPs). BMP can be produced in animal cells in culture or in transgenic animals. Transfected bovine fibroblasts lines can be used as a source of donor nucleus to generate transgenic animals through nuclear transfer into enucleated bovine oocyte. These transgenic animals can further produce proteins with pharmaceutical relevance (Houdebine, 2000).

It was isolated the human bone morphogenetic proteins cDNAs (BMP-2 and BMP-4) that were positioned under control of a mammalian constitutive promoter. The vectors were used to transfect bovine fibroblasts and express the BMP-2 and BMP-4 genes.

Total RNAs were isolated from bone tissue collected from patients with facial trauma (jaw fractures between the 7^th and 10^th day pos-trauma). All samples were harvested to get the best fracture reduction. All surgical procedures were carried out in the Unidade de Cirurgia e Traumatologia Buco-Maxilo-Facial, Hospital de Base de Brasília in accordance with the ethical standards of the institutional committee and the Helsinki Declaration.

RNA was isolated using Trizol¹ and the cDNA synthesis was conducted with the Superscript II kit¹ according to the manufacturer's instructions.

The BMP genes were amplified by PCR using the primer pairs BMP2forw (gtgcttcttagacggactgc) / BMP2rev (gtactagcgacacccacaac) to amplify the 1,233 bp BMP-2 gene, and BMP4forw (agccattccgtagtgccatc) / BMP4rev (aaggactgcctgatctcagc) to amplify the 1,373bp BMP-4 gene. Each PCR reaction was carried out in a 25µl mixture containing 60mM Tris-SO₄ (pH 8.9), 18mM ammonium sulfate, 2mM MgSO₄, 200µM of each dNTP, 200nM of each primer, 1U of Taq Platinum high fidelity polymerase¹. The mixture was overlaid with mineral oil, denatured for 2min at 94ºC in a MJ Research thermal cycler² and amplified for 30 cycles (94ºC for 15s, 55ºC for 30s, 68ºC for 2min). The products were run on a 1% agarose gel, stained with ethidium bromide and visualized with UV light. The amplified DNA sequences were cloned into the pGEM-T easy vector³ and sequenced. The genes were then cloned in the NotI restriction site from the pCMV-b vetor, replacing the b-galactosidase gene⁴>, under control of the cytomegalovirus (CMV) promoter for expression in mammalian cells to generate the vectors pCMV-BMP2 and pCMV-BMP4.

Bovine fibroblasts were transfected with the vectors pCMV-BMP2 and pCMV-BMP4 using the LipofectAmine Plus¹ according to the manufacturer's instructions. In order to detect BMP-2 and BMP-4 genes expression (mRNA) in the transgenic fibroblasts cells, RT-PCR was carried out. Total RNA isolation and PCR were conducted as previously described. Fig. 1 shows the RT-PCR amplification of the 1,233 bp (BMP-2) and 1,373 bp (BMP-4) DNA sequences in 3 independent fibroblasts lines. RT-PCR with total RNA without cDNA synthesis revealed no amplification attesting no contaminations with DNA (Fig. 1, lane 6). RT-PCR analyses with non-transfected fibroblasts lines (negative control) revealed no DNA amplification (Fig. 1, lane 5). Western blot analysis, carried out as described (Ramoshebi et al., 1999; Viñals et al., 2002) using polyclonal antibodies⁵ revealed that both BMP-2 and BMP-4 protein is produced in the transfected fibroblasts (Fig. 2).

In this report it was able to express human BMP-2 and BMP-4 genes in bovine transgenic fibroblasts. It suggested that the BMPs could be produced in cell in culture. In addition, the BMP genes can be introduced and expressed in vivo (Rech et al., 1996), in order to trigger the de novo osteogenesis process.

ACKNOWLEDGMENTS

The authors thank Robert G.N. Miller and Soraya C.M. Leal-Bertioli for critical reading of the manuscripts.

Recebido para publicação em 18 de junho de 2003

Recebido para publicação, após modificações, em 19 de dezembro de 2003

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1

Invitrogen, Carlsbad, CA, USA.

2

Waltham, MA, USA.

3

Promega, Madison, WI, USA.

4

Clontec, Palo Alto, CA, USA.

5

Creative BioMolecules, Hopkinton, MA, USA.

Publication Dates

• Publication in this collection
  29 Oct 2004
• Date of issue
  Aug 2004

History

• Accepted
  19 Dec 2003
• Received
  18 June 2003