Improvement of Transfection Efficiency of Epithelioma Papulosum Cyprini Carp Cells by Modification of Cell Cycle and Use of an Optimal Promoter

Improvement of Transfection Efficiency of Epithelioma Papulosum Cyprini Carp Cells by... Several methods to improve transfection of epithelioma papulosum cyprini (EPC) carp cells have been tested and are reported here. By modifying the cell cycle state of EPC cell monolayers and selecting the best promoter for the plasmid to be transfected, we increased transfection efficiency from 12.8% to 55.1% and decreased the coefficient of variation among different experiments from 54.1% to 11.8%. Thus 2- to 3-fold higher transfection efficiencies were obtained when the EPC monolayers were treated with colchicine or thymidine before transfection. In addition, the plasmids pMOKβgal and its shorter derivative pMVC1.4βgal, both containing 218 bp of additional sequences upstream of the cytomegalovirus promoter contained in plasmid pCMVβ, consistently produced higher transfection efficiencies than pCMVβ. Combination of the two methods resulted in an improvement of both efficiency and reproducibility. These results should facilitate transfection of EPC cells to use as a model to obtain transgenics, to conduct quantitative transfected-cell fusion assays, to improve DNA-immersion-vaccination methods, or to obtain infectious cDNA from fish RNA viruses. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Marine Biotechnology Springer Journals

Improvement of Transfection Efficiency of Epithelioma Papulosum Cyprini Carp Cells by Modification of Cell Cycle and Use of an Optimal Promoter

Marine Biotechnology, Volume 6 (5) – Nov 4, 2004

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Publisher
Springer Journals
Copyright
Copyright © 2004 by Springer-Verlag
Subject
Life Sciences; Freshwater & Marine Ecology; Microbiology; Zoology; Engineering, general
ISSN
1436-2228
eISSN
1436-2236
D.O.I.
10.1007/s10126-003-0008-6
Publisher site
See Article on Publisher Site

Abstract

Several methods to improve transfection of epithelioma papulosum cyprini (EPC) carp cells have been tested and are reported here. By modifying the cell cycle state of EPC cell monolayers and selecting the best promoter for the plasmid to be transfected, we increased transfection efficiency from 12.8% to 55.1% and decreased the coefficient of variation among different experiments from 54.1% to 11.8%. Thus 2- to 3-fold higher transfection efficiencies were obtained when the EPC monolayers were treated with colchicine or thymidine before transfection. In addition, the plasmids pMOKβgal and its shorter derivative pMVC1.4βgal, both containing 218 bp of additional sequences upstream of the cytomegalovirus promoter contained in plasmid pCMVβ, consistently produced higher transfection efficiencies than pCMVβ. Combination of the two methods resulted in an improvement of both efficiency and reproducibility. These results should facilitate transfection of EPC cells to use as a model to obtain transgenics, to conduct quantitative transfected-cell fusion assays, to improve DNA-immersion-vaccination methods, or to obtain infectious cDNA from fish RNA viruses.

Journal

Marine BiotechnologySpringer Journals

Published: Nov 4, 2004

References

  • Enhanced plasmid DNA transfection with lysosomotropic agents in cultured fibroblasts.
    Ciftci, K.; Levy, R.J.
  • Critical assessment of the nuclear import of plasmid during cationic lipid-mediated gene transfer.
    Escriou, V.; Carriere, M.; Bussone, F.; Wils, P.; Scherman, D.
  • Induced fusion of VHSV persistently infected fish cells.
    Fernandez-Alonso, M.; Coll, J.M.

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