Analysis of ES cells transduced with LVs carrying a hygromycin resistance gene.

<p>(<b>A</b>) Construct of LV carrying additional hygromycin resistance gene driven by PGK promoter and cDNA of TK under control of EOS-S4-promoter (STPH). (<b>B</b>) ES cells were transduced with STPH (1.5 copy numbers per genome in average) or not transduced (WT) and treated with (+) or without (-) 20 µM GCV for 72 hours after pre-selection with (+Hygro) or without hygromycin (-Hygro). Representative brightfield images are shown (n=3). (<b>C</b>) (<b>D</b>) and (<b>E</b>) Relative cell survival of untransduced ES cells (WT) (C), STPH-transduced ES cells (1.5 copy numbers per genome in average) without pre-selection (D) and STPH-transduced ES cells (1.5 copy numbers per genome in average) with pre-selection (E) with (+) or without (-) 20 µM GCV treatment (n=3, Mean±SEM; ***P<0.001 compared to without GCV treatment, respectively, Student’s <i>t</i>-test). Data is based on images representatively shown in (B); undifferentiated cells were manually counted using three different fields of view that were counted twice. (<b>F</b>) The untreated ES cell populations shown in (B) were differentiated as EBs with (+) or without (-) 20 µM GCV. After 14 days of differentiation dissociated EBs were immunostained with Oct-3/4 (red) and Hoechst (blue) indicating Oct-3/4-positive cells (ES cells) and nuclei, respectively. Representative images are shown (n=3).