Genetic heterogeneity of stably transfected cell lines revealed by expression profiling with oligonucleotide microarrays

Large-scale gene expression measurements with oligonucleotide microarrays have contributed tremendously to biological research. However, to distinguish between relevant expression changes and falsely identified positives, the source and magnitude of errors must be understood. Here, we report a source of biological variability in microarray experiments with stably transfected cell lines. Mouse embryonic fibroblast (MEF/3T3) and rat schwannoma (RT4) cell lines were generated to provide regulatable schwannomin expression. The expression levels of 29 samples from five different mouse embryonic fibroblast clonal cell lines and 18 samples from 3 RT4 cell lines were monitored with oligonucleotide microarrays. Using hierarchical clustering, we determined that the changes in gene expression induced by schwannomin overexpression were subtle when compared with those detected as a consequence of clonal selection during generation of the cell lines. The hierarchical clustering implies that significant alterations of gene expression were introduced during the transfection and selection processes. A total of 28 genes were identified by Kruskal-Wallis rank test that showed significant variation between clonal lines. Most of them were related to cytoskeletal function and signaling pathways. Based on these analyses, we recommend that replications of experiments with several selected cell lines are necessary to assess biological effects of induced gene expression.