Fig. 4.

Analysis of genomic integration and expression of pT/Caggs-V12Nras in tumors. Southern analysis was performed on genomic DNA from primary tumors, tumor-derived cell lines, and normal tissue to assess integration of the transposon vector. (A) Genomic DNA was digested with SacI (sites indicated by arrows) to differentiate SB-mediated transposon integrants and randomly integrated plasmids. (B and C) A probe specific to the Caggs promoter was used to identify integration events. To confirm that random integration was occurring within cell lines, a probe specific for the plasmid backbone of pT/Caggs-V12Nras was used on the same blot. (UI WT, uninjected wild-type liver; UI Arf, uninjected Arf liver) (D) Genomic sequences flanking transposon integration sites in tumor-derived cell lines were amplified by linker-mediated PCR. (E and F) Expression of the integrated NRAS transgene was assessed by western blot by using an antibody specific for the EE (Glu-Glu) epitope tag to differentiate between exogenously delivered and endogenous NRAS. Expression was detected in all tumor-derived cell lines and primary tumors from both liver and the tail base but not in normal adjacent tissue (+ and -, 293T cells transiently transfected with pT/Caggs-V12Nras or empty vector; N, normal adjacent tissue; T, primary liver tumor; TT, primary tail tumor).