How were the parental ES cell lines developed?
MC1 mouse ES cells derived from 129S6/SvEvTac (Olson et al. Cancer Res., 2003) were transfected with linearized pMWROSATcH (Masui et al. Nucleic Acids Res., 2005 ) to generate the ROSA-TET locus by homologous recombination. Cells were selected in presence of hygromycin B and in absence of doxycycline (Dox). The expression of hygromycin B resistant gene is activated by the hCMV*-1 promoter via binding of tTA that is expressed by the endogenous ROSA26 promoter in a promoter-trapping manner. The homologous recombination was confirmed by southern blotting using 5′ external probe and 3′ internal probe.
