Fig 3.

Effect of transcription on ssODN-mediated gene targeting in wild-type ES cells. (A) A single copy of a tetracycline (Tc)-controllable mutant neo reporter gene was stably integrated into the Rosa26 locus of wild-type ES cells. In the resulting ROSA-TET cell line, binding of tTA to the hCMV*-1 promoter induces expression of a neo-IRES-Venus reporter gene in culture medium without Tc. (B) Tc-induced inhibition of Venus expression in ROSA-TET ES cells carrying a wild-type (WT) or mutant (mut) neo reporter gene. Cells were cultured for 24 hrs in medium without tetracycline (−Tc; panels a, b, c and d) or medium plus tetracycline (+Tc; panels e, f, g and h) before visualization of Venus fluorescence through a YFP filter (panels b, d, f and h). Corresponding bright field images are shown in panels a, c, e and g, respectively. (C) Quantitative RT-PCR showing Tc-induced inhibition of neo expression in ROSA-TET ES cells carrying a wild-type (WT) or mutant (mut) neo reporter gene. Mouse β-actin mRNA levels were used for normalization. (D) Correction of the mutant neo gene in ROSA-TET ES cells by ssODN 4n (sense orientation) or ssODN 4nas (antisense orientation) in −Tc medium (transcription ‘on’) or +Tc medium (transcription ‘off’). Targeting efficiency is the number of G418-resistant colonies per 10⁶ cells that were plated after ssODN exposure. Error bars represent the S.D. of nine independent experiments.