Figure 1

Schematic outline of the Flip-Flop HSV-BAC system for the generation of exogenous promoter driven HSV vectors. Prior to recombinant virus construction, a promoter of interest (Pr) is inserted into the MCS of the promoterless shuttle vector pFLS-ICP4 to generate the ICP4 expression cassette driven by that promoter. The prototype HSV-BAC vector plasmid, pM24-BAC contains the genome of replication-deficient ICP4-deletion mutant d120. In the first step of the Flip-Flop HSV-BAC system, the shuttle vector pFLS-Pr is integrated into the loxP site on pM24-BAC, using Cre recombinase, and then electroporated into E. coli to obtain the integrated pM24-BAC-shuttle. The second step is performed in mammalian E5 cells, which carry the ICP4 gene and are permissive for ICP4- mutant replication. When FLPe recombinase expression plasmid pCAGGS-FLPe is co-transfected with pM24-BAC-shuttle to E5 cells, the FRT-flanked segment on the oversized vector (marked with orange arrow), containing the BAC vector and stuffer sequences, is excised by the recombinase. The reduction in genome size permits efficient packaging and production of the recombinant virus (bM24-Pr). As pM24-BAC-shuttle is oversized (>166 kb), the vector containing BAC sequences cannot be packaged efficiently into the HSV virion. BAC: BAC backbone and replication origin (mini F plasmid sequences), Cm: Chloramphenicol resistance gene, EGFP: Enhanced Green Fluorescence Protein gene, Km: Kanamycin resistance gene, LacZ: β-galactosidase gene, Stuffer: Stuffer sequence from bacteriophage lambda, ICP4: HSV ICP4 coding sequence, Pr: Exogenous promoter of interest, US: Unique short sequence of HSV, UL: Unique long sequence of HSV, Open circle: R6Kγ plasmid replication origin, ICP6 5': 5' portion of the HSV ICP6 coding sequence, ICP6 3': 3' portion of the HSV ICP6 coding sequence, Closed triangle: loxP recombination site, Open triangle: FRT recombination site.