| Full text | |
| Author(s): |
Santos, Mariza G.
;
Jorge, Soraia A. C.
;
Brillet, Karl
;
Pereira, Carlos A.
[4]
Total Authors: 4
|
| Document type: | Journal article |
| Source: | Cytotechnology; v. 54, n. 1, p. 15-24, May 2007. |
| Field of knowledge: | Biological Sciences - Biochemistry |
| Abstract | |
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) | |
| FAPESP's process: | 02/09482-3 - Heterologus gene expression in dipteran cells: molecular biology and bioprocess engineering |
| Grantee: | Carlos Augusto Pereira |
| Support Opportunities: | Research Projects - Thematic Grants |
| FAPESP's process: | 01/08914-4 - Expressão dos genes do vírus da hepatite B (HBsAg) e do vírus da raiva (GPV/PV) em células de drosófilas |
| Grantee: | Soraia Attie Calil Jorge |
| Support Opportunities: | Scholarships in Brazil - Post-Doctoral |
| FAPESP's process: | 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. |
| Grantee: | Carlos Augusto Pereira |
| Support Opportunities: | Regular Research Grants |
| FAPESP's process: | 02/04003-0 - Estudo de metodos de transfeccao de dna plasmidial em celulas de "drosophila melanogaster". |
| Grantee: | Mariza Augusta Gerdulo dos Santos |
| Support Opportunities: | Scholarships in Brazil - Scientific Initiation |
| FAPESP's process: | 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 |
| Grantee: | Mariza Augusta Gerdulo dos Santos |
| Support Opportunities: | Scholarships in Brazil - Master |