Figure 3

PCR analysis of plasmid DNA transfected into the packaging cell line GPenv-AM12. A. Analysis of tandemly integrated plasmid DNA. Amplification was performed with a 5' primer specific to neo sequences (T1, unique for pDHEneo) and a 3' primer specific to SV40 early promoter region (T2, unique for pDΔpbsSVpuro). Membrane was hybridized with 3' neo specific probe generated from a 150 bp SalI-ClaI fragment of pDHEneo. ST is GPenv-AM12 virus-producing cell clone ST2-1 generated by sequential transfection of pDHEneo and pDΔpbsSVpuro, and CT is cell clone CT2 generated by cotransfection of the same vectors. Molecular size markers are indicated on the right of the Southern blot. Similar results were obtained when four cell clones were analyzed. B. Control of amplification. Primers specific to the 5'- and 3'-end of neo gene (CND and CNR, respectively) were used to generate PCR products (1.63 kb) from ST and CT DNA samples. Membrane was hybridized with the same probe as in A. PCR products obtained from 200 and 40 ng of ST DNA sample (line 1 and 2); PCR products obtained from 200 and 40 ng of CT DNA sample (line 3 and 4). The result shows that specific PCR products could be amplified both from ST and CT DNA samples with this set of primers.