Fancg knock-down in immortalized telomerase-deficient mouse cells with humanlike telomere length (G5 Terc^-/-/Fancg shRNA3 MEFs). (A) GFP shRNA and Fancg shRNA3 MEFs in wild-type and G5 Terc^-/- backgrounds were immortalized following a 3T3 protocol, and specific Fancg interference was assessed by real-time RT-PCR. Extent of interference was similar to primary MEFs. (B) Correlation between RNA interference and protein knock-down was assessed by IP and Western blot. Fancg expression is readily detected in wild-type MEFs and 293 cells transfected with a Fancg vector (lanes 1 and 7, respectively) and absent in Fancg^-/- MEFs (lane 2). Significantly decreased protein expression is observed in Terc^+/+/Fancg shRNA3 MEFs (lane 4) compared with their Terc^+/+/GFP shRNA controls (lane 3) and in G5 Terc^-/-/Fancg shRNA3 MEFs (lane 6) compared with their G5 Terc^-/-/GFP shRNA controls (lane 5). (C) Q-FISH analysis of immortalized Terc^+/+/Fancg shRNA3 and G5 Terc^-/-/Fancg shRNA3 MEFs and their GFP shRNA-expressing controls. Like G5 Terc^-/-/GFP shRNA control telomeres, G5 Terc^-/-/Fancg shRNA3 telomeres showed very heterogeneous telomere length and frequent signal-free ends, indicative of ALT activation in these cells with critically short telomeres and no telomerase activity.