Upon integration of a gene of interest into pUHD10-3, the resulting vector should be transfered into the tTA or rtTA-positive cell lines by cotransfection together with a second selection marker. Selection markers or other constructs having proven or suspected enhancer activity, should be avoided. Since the cotransfected DNAs will frequently cointegrate and the minimal promoter may serve as an enhancer trap. Therefore, enhancerless selection markers are recommended to obtain cells with a low basal expression level. This should, however, be a less critical concern when the goal is only a conditional overexpression.