Improvement of transfection efficiency of epithelioma papulosum cyprini carp cells by modification of cell cycle and use of an optimal promoter.
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 pMOKbetagal and its shorter derivative pMVC1.4betagal, both containing 218 bp of additional sequences upstream of the cytomegalovirus promoter contained in plasmid pCMVbeta, consistently produced higher transfection efficiencies than pCMVbeta. 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.
Rocha
A
A
SGIT, INIA, Biotecnología. Crt. La Coruña Km. 7, 28040 Madrid, Spain.
Ruiz
S
S
Coll
J M
JM
D000818:Animals; D002347:Carps; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D003078:Colchicine / Q000494:pharmacology; D003587:Cytomegalovirus / Q000235:genetics; D005697:Galactosides; D007211:Indoles; D010957:Plasmids / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D013936:Thymidine / Q000494:pharmacology; D014162:Transfection / Q000379:methods*; D001616:beta-Galactosidase / Q000378:metabolism
D005697:Galactosides; D007211:Indoles; D001616:beta-Galactosidase; D003078:Colchicine; C044888:5-bromo-4-chloro-3-indolyl beta-galactoside; D013936:Thymidine