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 μM 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.
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Abbreviations
- EGFP:
-
Enhanced green fluorescent protein gene
- pMt:
-
Metallothionein promoter
- S2:
-
Schneider 2 Drosophila melanogaster cells
- S2MtEGFP:
-
S2 cells transfected with Mt inducible plasmid containing the EGFP gene
- NaBu:
-
Sodium butyrate
- CuSO4 :
-
Copper sulfate
- DES:
-
Drosophila expression system
- Hygro:
-
Hygromycin
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Acknowledgements
This work was supported in part by grants from the FAPESP (02/09482-3, 05/50565-8), CNPq and Fundação Butantan. We thank Dr. Bergmann Morais (UnB, Brasília, Brazil) for pGie1EGFP plasmid supplying and Dr. Jorge MC Ferreira Jr and Dr. Orlando Ribeiro, for flow cytometry analysis. C.A. Pereira is recipient of CNPq research fellowship. S.A.C. Jorge and M.A.G. Santos had scholarships from FAPESP (01/08914-4, 02/04003-0, 03/08978-8).
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Santos, M.G., Jorge, S.A.C., Brillet, K. et al. Improving heterologous protein expression in transfected Drosophila S2 cells as assessed by EGFP expression. Cytotechnology 54, 15–24 (2007). https://doi.org/10.1007/s10616-007-9060-9
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DOI: https://doi.org/10.1007/s10616-007-9060-9