Homogeneous tetracycline-regulatable gene expression in mammalian fibroblasts.
The expression of transfected genes in mammalian cells is rapidly repressed by epigenetic mechanisms such that, within a matter of weeks, only a fraction of the cells in most clonal populations still exhibit detectable expression. This problem can become prohibitive when one wants to express two ectopically introduced genes, as is necessary to establish cell lines that harbor genes regulated by the tetracycline-controlled transactivators. We describe an approach to establish Chinese hamster ovary (CHO) cell lines that stably induce a tet-responsive reporter gene in all cells of a transfected clonal population. Screening of more than 100 colonies resulting from a standard co-transfection of the tetracycline transactivator (tTA) with a green fluorescent protein (GFP) reporter plasmid failed to identify a single colony that could induce GFP in more than 20% of cells. The presence of chromatin insulator sequences, previously shown to protect some transfected genes from epigenetic silencing, moderately improved stability but was not sufficient to produce homogeneous transformants. However, when cell lines were first established in which selection could be maintained either for the expression of tTA activity (co-transfection with a tTA-responsive selectable marker) or the presence of tTA mRNA (bicistronic message encoding a selectable marker), these cell lines could be subsequently transfected with the GFP reporter construct, and nearly 10% of the resulting colonies exhibited stable homogeneous tet-responsive GFP expression in 100% of the expanded clonal cell population.
Izumi
M
M
Biodesign Research Group, Institute of Physical and Chemical Research Wako, Saitama 351-0198, Japan.
Gilbert
D M
DM
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013752:Tetracycline / Q000494:pharmacology*; D014162:Transfection
D008164:Luminescent Proteins; D012333:RNA, Messenger; D049452:Green Fluorescent Proteins; D013752:Tetracycline