Generation of A1-L4 cell lines. (A) The Ad5 major late transcription unit L4 region. During expression from the major late promoter (MLP), leaders 1, 2 and 3 (the tripartite leader; TPL) are spliced to alternative acceptor sites to generate L4 mRNAs encoding 100K, 22K, 33K and the structural protein pVIII; L4-22K and -33K proteins share the same N-terminus but have distinct C-termini due to the presence of an intron in the L4-33K ORF. L4-22K and -33K are also expressed from the L4 promoter early in infection. (B) The L4 cassette within pShuttle100/22/33KFLAG, used for RMCE to generate the A1-L4 cell population. Numbers indicate Ad5 genome positions. LoxP: DNA target sequence for Cre recombinase; FLAG: C-terminal epitope tag on the 33K open reading frame; HygR; hygromycin-resistance gene. (C) 293TETOFF, Clone A1 and the uncloned A1-L4 population, 21 days post-recombination, were grown without Dox for 3 days with daily media changes and the level of GFP analysed by FACS. M1 shows the percentage of cells expressing GFP above the threshold set by 293TETOFF cell background fluorescence. (D) Clone A1 cells and the uncloned A1-L4 cell population were analysed for L4-100K expression by western blot analysis. The positions to which proteins of known molecular mass migrated are indicated (kDa). (E) To confirm correct insertion of the L4 cassette in clone A1-L4 obtained from the A1-L4 population, genomic DNA from these and control (A1) cells was used as template for PCR amplification using one primer to the tetracycline-regulated PBi promoter and another to the leader 3 sequence within the TPL of the L4 cassette; -ve, PCR negative control. All lanes shown derive from the same exposure of a single gel with irrelevant lanes excised for clarity of presentation. The positions to which DNA size markers migrated are indicated (bp).