Figure 2

Establishment of an autonomous replicon. Transfection of CHO cells with pEPI or linearized vectors was performed using the lipid-based transfection reagent FuGENE 6 (Roche). Subsequently the establishment of the episome in the cell nucleus was monitored 6 h (A), 24 h (B) and after a selection period of 10 days (C, D) post transfection. The episome (green) was visualized by pEPI FISH. To-Pro-3 was used for DNA counterstaining (red) in (A-D). Maximum intensity projections were rendered from a set of 5 mid serial sections. The cellular contour was outlined with a white line in (A, B). (A) Vesicles containing numerous vector molecules occurred within the cytoplasm 6 h post transfection, whereas no signals were observed within the nucleus. (B) A strong intranuclear pEPI signal indicating a large number of vector molecules was observed 12 h post transfection, while cytoplasmic localization of pEPI was no longer observed. (C) In the majority of cases, cells transfected with the linearized vectors displayed FISH signals consistent with integration of the vector at a single chromosomal locus (D) Solely a limited number of distinct pEPI signals was observed after a selection period of 10 days suggesting that only a minor portion of the episome is stably established.