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. 1997 Jun 10;94(12):6450–6455. doi: 10.1073/pnas.94.12.6450

Figure 5.

Figure 5

107/402-T-based episomes replicate extrachromosomally in human cells after direct in vivo gene transfer. (A) Map of pRC/CMV.107/402-T and the location of primers 1 and 2 used to amplify a 1.6-kbp region of this plasmid extending from the bovine growth hormone polyadenylylation site to the neomycin-resistance gene. Asterisks denote DpnI restriction sites. (B) PCR-based episomal replication assay. Southern blot analysis of PCR amplification products derived from HT-1376 cells transfected either in vivo (lanes 3 and 4) or in vitro (lane 5) with pRC/CMV or pRC/CMV.107/402-T. Before PCR amplification, 1 μg of total cellular DNA was digested with DpnI. Samples were then subjected to 32 cycles of PCR amplification. Amplification of 3 pg of pRC/CMV.107/402-T (positive control) is shown in lane 1, and the negative control, lacking template DNA, is shown in lane 2. The hybridization probe was generated from a pair of internally nested primers.