Sustained LMP2A expression prevents apoptotic death of EBV-transformed B cells. (A) Schematic representation of the LMP2A gene locus in 2190 EBV, which carries 2 loxP sites located upstream of the LMP2A promoter (pLMP2A) and downstream of the first exon of LMP2A. This exon is unique to LMP2A and encodes its amino-terminal signaling domain,⁸  whereas the 12 putative transmembrane domains and a short cytoplasmic terminus are encoded by exons 2 through 8, which are shared by the carboxy coterminal LMP2B protein whose transcript is expressed from the LMP2B promoter (pLMP2B). To monitor the status of the LMP2A gene, the PCR primers (P1, P2) were used, which gave rise to PCR products of 1220 bp and 418 bp in length with the parental LMP2A⁺ 2190 EBV and the Cre-deleted LMP2A⁻ variant, respectively. (B) The p3233 expression plasmid is schematically shown, which encodes the truncated NGF receptor (NGF-R) as a phenotypic marker³⁵  and the site-specific recombinase Cre from a bicistronic transcript. (C) Cre-mediated deletion of LMP2A could be monitored by PCR as early as 2 hours after transfection of p3233. Lanes indicated as floxed LMP2A, deleted LMP2A, and H₂O are PCR controls. The lane indicated as “survivors” refers to PCR analysis of 2190-infected B cells (labeled Cre⁺ in panel F) at 6 weeks after transfection. (D) Transient transfection of the expression plasmid p3233 and FACS analysis of LMP2A⁺ 2190 EBV LCLs expressing the truncated NGF-R at their cell surface 18 hours after transfection. LCLs established with the prototypic 2089 WT EBV gave comparable results (data not shown). (E) FACS sorting of successfully transfected LMP2A⁺ 2190 EBV LCLs with the topmost 5% expression levels of NGF-R (Cre⁺/NGF-R⁺) versus NGF-R⁺–depleted (Cre⁻/NGF-R⁻) cells. (F, top panel) The absolute numbers of cells in the different fractions were determined by FACS analysis at indicated days after transfection. BD Biosciences CaliBRITE beads were used as an internal volume standard as described previously.²¹  Cells in the lymphocyte gate according to forward and sideward scatter criteria were included in the analysis and set to 100% 24 hours after transfection. EBV-infected LCLs infected with LMP2A⁺ 2190 EBV carrying the floxed LMP2A allele are indicated (2190, black lines) as well as LCLs infected with the prototypic 2089 WT EBV strain (2089, gray lines). Transfected and sorted Cre⁺/NGF-R⁺ cell fractions are indicated (Cre⁺) and compared with cells depleted from NGF-R⁺ cells by sorting (Cre⁻). (Bottom panel) Cre-transfected (Cre⁺/NGF-R⁺) LCLs infected with either 2089 WT EBV or LMP2A⁺ 2190 EBV were analyzed for annexinV staining by FACS at the time points indicated.