Figure 2.

Absence of stable episomal DNA from long-term cultures of cells transfected with TR reporter plasmids. (a) Aliquots of FACS-sorted BJAB cells transfected with the indicated reporter constructs were harvested at various time points after FACS sorting (indicated in days above the lanes) and subjected to Gardella gel analysis. Samples were derived from the same cultures shown in Figure 1e. As a control, untransfected BJAB cells were loaded in the lanes labeled BJAB. Plasmid DNA was detected with a probe specific for GFP. The band marked with an asterisk results from unspecific background hybridization, since it is also present in untransfected BJAB cells. (b) Episomal DNA was isolated from the same BJAB cultures described above by Hirt extraction and subjected to PCR amplification of the GFP cassette as described in Methods. (c) Gardella gel analysis of transfected BJAB cells cultures grown under antibiotic selection. BJAB cells were transfected with pGFP, pGTR4, or pGTR4:73 and cultured in the presence of G418. Aliquots of cells propagated for the time periods indicated above the lanes (in days) were examined for the presence of episomal DNA by Gardella analysis. Episomes were detected with a probe specific for the GFP cassette.