Improving heterologous protein expression in transfected Drosophila S2 cells as assessed by EGFP expression

Autor(es)

Santos, Mariza G. ; Jorge, Soraia A. C. ; Brillet, Karl ; Pereira, Carlos A. ^[4]

• Tipo de documento: Artigo Científico
• Fonte: Cytotechnology; v. 54, n. 1, p. 15-24, May 2007.
• Área do conhecimento: Ciências Biológicas - Bioquímica
• Assunto(s): Expressão gênica Insetos Drosophila melanogaster

Resumo

Drosophila melanogaster S2 cells were co-transfected with plasmid vectors containing the enhanced green fluorescent protein gene (EGFP), under the control of metallothionein promoter (pMt), and the hygromycin selection gene, in view of establishing parameters for optimized gene expression. A protocol of transfection was worked out, leading after hygromycin selection, to ¡­90% of S2MtEGFP fluorescent cells at day 5 after copper sulfate (CuSO4) induction. As analyzed by confocal microscopy, S2MtEGFP cell cultures were shown to be quite heterogeneous regarding the intensity and cell localization of fluorescence among the EGFP expressing cells. Spectrofluorimetry kinetic studies of CuSO4 induced S2MtEGFP cells showed the EGFP expression at 510 nm as soon as 5 h after induction, the fluorescence increasing progressively from this time to attain values of 4.6 ¡¿ 105 counts/s after 72 h of induction. Induction with 700 ¥ìM of CuSO4 performed at the exponential phase of the S2MtEGFP culture (106 cells/mL) led to a better performance in terms of cell growth, percent of fluorescent cells and culture intensity of fluorescence. Sodium butyrate (NaBu) treatment of CuSO4 induced S2MtEGFP cell cultures, although leading to a loss of cell culture viability, increased the percent of EGFP expressing cells and sharply enhanced the cell culture fluorescence intensity. The present study established parameters for improving heterologous protein expression in stably transfected Drosophila S2 cells, as assessed by the EGFP expression. (AU)

• Processo FAPESP: 02/09482-3 - Expressão de genes heterólogos em células de dípteros: biologia molecular e engenharia de processos
• Beneficiário: Carlos Augusto Pereira
• Modalidade de apoio: Auxílio à Pesquisa - Temático
• Processo FAPESP: 01/08914-4 - Expressão dos genes do vírus da hepatite B (HBsAg) e do vírus da raiva (GPV/PV) em células de drosófilas
• Beneficiário: Soraia Attie Calil Jorge
• Modalidade de apoio: Bolsas no Brasil - Pós-Doutorado
• Processo FAPESP: 05/50565-8 - Construcao de vetores e estrategias para expressao dos genes do virus da hepatite b (hbsag) e do virus da raiva (gpv/pv) em celulas de drosofila.
• Beneficiário: Carlos Augusto Pereira
• Modalidade de apoio: Auxílio à Pesquisa - Regular
• Processo FAPESP: 02/04003-0 - Estudo de métodos de transfecção de DNA plasmidial em células de Drosophila melanogaster
• Beneficiário: Mariza Augusta Gerdulo dos Santos
• Modalidade de apoio: Bolsas no Brasil - Iniciação Científica
• Processo FAPESP: 03/08978-8 - Co-expressão de genes virais (GPV - raiva e HBsAg-hepatite B) com o gene da Green Fluorescent Protein (EGFP) em células de Drosophila melanogaster como instrumento de optimização de bioprocesso para produção de antígenos virais
• Beneficiário: Mariza Augusta Gerdulo dos Santos
• Modalidade de apoio: Bolsas no Brasil - Mestrado