Mouse Mix is activated early during ES cell differentiation and is conditionally expressed in i-Mix ES cell lines. (A) RT-PCR analysis of mMix expression in EBs (E14 line). Controls (-RT, -DNA) were routinely performed. NT indicates minus template control. (B) Strategy for conditional expression of mMix in ES cells. The vector plox-Mix1, encoding amino-terminal FLAG-tagged mMix, was inserted into the X chromosome using Cre-mediated recombination.42 In the presence of DOX, rtTA protein expressed from the ROSA26 locus on chromosome 6 (chr6) can bind to a tetracycline operator (tetOP), and expression of mMix is activated. (C) Western blot analysis of 1 of the 3 i-mMix ES cell lines (clone 2) cultured at different concentrations of DOX. Actin was used as a loading control. (D) Nuclear expression of FLAG-tagged mMix protein in cells from induced i-Mix EBs. Magnification, × 40.
