Transient cotransfection assay

BOX 29.1. TRANSIENT COTRANSFECTION ASSAYS CAN BE USED TO STUDY TRANSCRIPTION IN VIVO... 

The biological activity of the synthesized compounds was evaluated by means of a transient transactivation assay. The cotransfection assay was conducted in mouse fibroblast NIH3T3 cells, using an expression plasmid for h AR and reporter plasmids, ARE/Luci (firefly luciferase), and pRL/ CMV (Renilla luciferase). 

Calcium-phosphate transfection has been used transiently to introduce plasmids containing the luciferase gene into animal cells (15) and electroporation has proved useful for plant and Dictyostelium cells, (16,26). For monkey CV-1 cells,10 yg of DNA was optimal for high levels of expression and the activity dropped sharply when DNA below or above this amount was used. In contrast, for electroporation of carrot cells (32) the signal increased almost linearly up to the highest amount of DNA used (50 yg/ml). It would be advisable to determine this optimal amount of DNA for other cell lines or transfection protocols. Extracts for luciferase activity are prepared and assayed as described (15,16). The Instruments used to measure activity in extracts must be calibrated periodically with purified luciferase of known specific activity to obtain an idea of the absolute amount of the enzyme being produced. For experiments in which luciferase and CAT genes are cotransfected into cells, the extracts should be made in the luciferase extraction buffer because CAT is stable in this buffer, whereas luciferase exhibits lower activity in the CAT extraction buffer. Furthermore, in such cotransfection experiments, the relative levels of CAT and luciferase expression have been found to depend upon the amounts of the two plasmids used and there is also the possibility of competition between the promoters on the two plasmids. Hence it is advisable to optimize the absolute amounts as well as the ratios of the two plasmids in such experiments. 

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