Conditional gene expression systems have developed into essential tools for the study of gene functions. However, their utility is often limited by the difficulty of identifying clonal cell lines, in which transgene control can be realized to its full potential. Here, we describe HeLa cell lines, in which we have identified-by functional analysis-genomic loci, from which the expression of transgenes can be tightly controlled via tetracycline-regulated expression. These loci can be re-targeted by recombinase-mediated cassette exchange. Upon exchange of the gene of interest, the resulting cell line exhibits the qualitative and quantitative properties of controlled transgene expression characteristic for the parent cell line. Moreover, by using an appropriate promoter, these cell lines express the tetracycline controlled transcription activator rtTA2-M2 uniformly throughout the entire cell population. The potential of this approach for functional genomics is highlighted by utilizing one of our master cell lines for the efficient microRNA-mediated knockdown of the endogenous human lamin A/C gene.