Figure 1.

Schematic overview of the EBV shuttle system. A linearized DNA fragment consisting of an F factor plasmid and two flanking regions. A and B of EBV are transfected into the B95.8 cell line, which is latently infected with EBV. Homologous recombinations occur via the regions A and B to generate a B95.8 cointegrate that encompasses the gene for hygromycin resistance (hyg) and green fluorescent protein (GFP) together with the F factor replicon. Cells that contain such a cointegrate B95.8/F factor survive under hygromycin selection. Preparation of circular DNA from these cells and its transfection into an appropriate E. coli strain establishes the B95.8/F factor molecule in E. coli for further genetic modifications. The B95.8/F factor DNA can be amplified and isolated from E. coli in microgram quantities to be used for transfection into EBV-negative cells, i.e., 293. Upon hygromycin selection, cell lines that carry the B95.8/F factor molecule as extrachromosomal copies can be established. Induction of the lytic phase of EBV’s life cycle yields viruses that carry the B95.8/F factor molecule as genetic information. Infection of primary B cells leads to their immortalization and B95.8/F factor-positive B cell lines.