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To assay eukaryotic DNA sequences for their transcriptional regulatory activity, these sequences are routinely linked to a reporter gene (e.g., CAT gene coding for chloramphenicol acetyltransferase) transfected into cells and transient expression of the reporter gene is monitored (1). For comparison cotransfection with a second reporter gene, e.g. the gene coding for /3-galactosidase is frequently done (2). Expression of the cotransfected control plasmid, however, is likely to be modulated by transacting enhancer or silencer DNA elements. This potential artefact was reported recently (3). Here, we show that the level of CAT activity can be normalized to the amount of CAT plasmid DNA introduced into recipient cells by DNA transfection. Cells of a human B-cell line were transfected with various recombinant CAT DNA constructs using DOTAP reagent (Boehringer Mannheim). Two days after transfection, cells were extensively washed and equal numbers of cells were lysed. To determine the amount of CAT enzyme synthesized, 135 fd of the crude cell lysate (150 /J) were subjected to CAT ELJSA (Boehringer Mannheim). For monitoring the level of plasmid DNA actually transferred into recipient cells, 15 yl of the lysate were incubated with RNase A (100 /tg/ml), and subsequently with Proteinase K (100 /xg/ml), each for 30 min at 37°C. Two volumes of 20xSSC were added, the DNA was dotted in a series of dilutions onto Hybond N membrane (Amersham Buchler), and hybridized to ^P-labeled pCAT DNA as probe (Figure 1). Intensities of the hybridization signals indicate the amount of transferred plasmid DNA present in crude cell extract compared to a standard. The level of CAT enzyme (determined by CAT ELJSA) or of CAT activity (monitored by thin layer chromatography of reaction products) is now normalized to the relative amount of transferred CAT DNA (Table 1). Small amounts of CAT plasmid DNA in the transfection mixture result in low amounts of DNA introduced into the cells (Figure 1, lanes 4—6). However, the quotient of the CAT enzyme level present in the cell extract relative to the amount of CAT plasmid DNA transferred into the cells is constant under these conditions. Obviously, this procedure is done under the condition that cells with similar growth parameters and cultures with the same cell densities are transfected. On the other hand, this protocol does not need cotransfection of a second plasmid and, therefore, does not exhibit the potential artefact in determination of transacting enhancer or silencer activities.

A procedure to standardize CAT reporter gene assay