Figure 5.

Recombinant virus generation after FLPe recombinase excision of BAC sequence. (a) EGFP and LacZ marker expression by recombinant viruses harvested after transfection of pM24-BAC (prototype HSV-BAC) or pM24-BAC-null DNA with or without the FLPe recombinase plasmid. E5 cells were transfected with indicated DNA and 6 days after transfection, viruses were harvested and titered on E5 cells. Detection of EGFP was performed without fixation before LacZ staining. When pM24-BAC alone was transfected, all harvested viruses formed EGFP-positive plaques none of which were LacZ positive. When pM24-BAC-null alone was transfected, the viruses formed both EGFP- and LacZ-positive plaques. Sizes of the plaques were smaller than those of viruses harvested from pM24-BAC transfected cells. When pM24-BAC-null and pCAGGS-FLPe were co-transfected, all harvested viruses formed LacZ-positive plaques and most of them were EGFP-negative. A very few number of EGFP-positive plaques, which were smaller in size than EGFP-negative plaques, were observed in lower dilution wells. All photos are at same magnification. (b) Titer of EGFP-positive and LacZ-positive viruses harvested in each transfection group (error bars show standard deviation. N = 3). Ratios between titers of EGFP-positive and LacZ-positive virus in each group are shown below. (c) Single-step growth assay of viruses harvested in Fig 5b. 8 × 10³ of E5 cells plated in 48-well plates were infected at an MOI of 2.5. Progeny viruses were harvested 24 or 38 hours after infection and titered on E5 cells. The virus yield was divided by the number of infected cells (Virus burst).