Correct evaluation of reporter assays in different cell lines by direct determination of the introduced plasmid amount.
Transfection efficiency in reporter gene assays is usually determined by cotransfection of a reference reporter gene under the control of a constitutively active strong promoter and determination of the reference enzyme activity. The SV40 promoter-driven beta-galactosidase reporter plasmid is frequently used as the reference reporter plasmid. Here we show that the beta-galactosidase expression in different cell lines does not correctly reflect the amount of plasmid taken up by cells and thus is not an accurate measure of transfection efficiency. The direct determination of introduced plasmid concentration in lysates of transfected cells is suitable for monitoring the transfection efficiency in reporter gene assays even if different cell lines are compared.
Siedow
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A
Medizinische Klinik I, Gastroenterologie und Infektiologie, Universitätsklinikum Benjamin Franklin der Freien Universität Berlin, Germany. muccias@zedat.fuberlin.de
Gratchev
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A
Hanski
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C
D001681:Biological Assay / Q000379:methods*; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D006801:Humans; D010957:Plasmids; D014407:Tumor Cells, Cultured