Measure of transient transfection efficiency using beta-galactosidase protein.
Although the control elements which regulate the transcriptional activity of promoter sequences are largely determined by the use of reporter plasmids in transient transfection analyses, controlling variability in these experiments can often be a vexing problem. Problems arise when the promoter of the internal control plasmid, used to correct for transfection efficiency, either affects test promoter strength or is itself regulated by trans-acting factors or inducing agents used to study the test promoter. Here we report the use of beta-galactosidase protein as an unbiased standard of transfection efficiency. beta-Galactosidase protein is readily internalized by adherent cell lines when incorporated into a calcium phosphate precipitate; significant enzyme activity can be recovered up to 72 h after transfection. Use of beta-galactosidase protein as a control obviates the concerns associated with promoter-dependent reporter plasmids as controls.
Howcroft
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TK
Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892-1360, USA.
Kirshner
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SL
Singer
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DS
D002130:Calcium Phosphates / Q000737:chemistry; D002460:Cell Line; D010957:Plasmids; D014162:Transfection / Q000379:methods*; D001616:beta-Galactosidase / Q000737:chemistry* / Q000235:genetics
D002130:Calcium Phosphates; C485828:alpha-tricalcium phosphate; C485830:tetracalcium phosphate; C485838:calcium phosphate, monobasic, anhydrous; C020243:calcium phosphate; D001616:beta-Galactosidase; C485829:calcium phosphate, dibasic, anhydrous