Abstract
The growing use of mouse embryonic stem (ES) cells in research emphasizes their importance in studies of molecular mechanisms that maintain pluripotency and direct cellular differentiation. Although systems for regulatable transgene expression are essential for fine analysis of cellular processes at the molecular level, a strategy for the establishment of multiple ES cell lines carrying any of these systems has not yet been described. Here, we report our development of the ROSA-TET system, an effective system for the establishment of multiple ES cell lines carrying a tetracycline (Tc)-regulatable transgene at the Gt (ROSA)26asSor (ROSA26) locus. This system contains a knock-in step of a construct carrying both loxP and its mutant sequences into the ROSA26 locus, followed by a subsequent exchange step that introduces a cDNA to be Tc-regulated to the locus using the recombinase-mediated cassette exchange reaction. Both steps are demonstrated to give desired clones with high efficiency, suggesting that this system can be introduced readily into any ES cell lines, leading to the simultaneous establishment of multiple cell lines carrying different Tc-regulated cDNAs. We believe that use of this system will strongly accelerate molecular biological research using ES cells.