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The β-galactosidase reporter gene is commonly used as a control for transfection efficiency in the promoter reporter assay system. While investigating vasoactive intestinal peptide response elements in the promoter of the prolactin gene, we found that primary pituitary cells from turkey hens highly expressed endogenous β-galactosidase. Therefore, we developed a new protocol for determining transfection efficiency using the β-lactamase gene, which is present on many expression vectors. Transcript levels of β-lactamase were measured by RT-PCR after transfection of different amounts of the pGL3-basic and pGL3-control vectors. A high correlation was observed between the amount of plasmid transfected and β-lactamase mRNA levels. Although no eukaryotic promoter was present, there was apparently leaky expression of the β-lactamase gene. Expression of β-lactamase was independent of expression from the simian virus 40 or turkey prolactin promoters cloned upstream of the luciferase gene.