Fig. 5 (continued) The wt gene can be introduced into the mutant viral BAC at a neutral second site. By FLP-mediated site-specific recombination of an FRT transfer plasmid carrying the wt gene including regulatory sequences and the mutant BAC genome with an FRT site (black triangle in a square), the wt gene product can be expressed from the mutant genome itself. After transfec-tion of permissive cells with this revertant BAC genome, a homogenous population of revertant virus is reconstituted. Only protein trans-complementation (c) and ectopic cis-complementation (d) allow the formal confirmation that the mutated gene product (and not possible other cis-effects of the mutated sequence) is responsible for the observed phenotype mutants need to be analyzed individually. The random transposon mutagenesis of the BACs targets the entire genome and null mutants of the respective genes discriminate essential from nonessential genes. Yet, these insertion libraries cannot be used for the functional characterization of a coding sequence. Comprehensive mutant pools of subcloned genes can be obtained through different random muta-genesis procedures. However, a large set of these mutants has to be introduced one by one into the CMV genome lacking the gene of interest to analyze their effect in the context of virus replication. Therefore, we developed a strategy combining a comprehensive Tn7-based linker-scanning mutagenesis of isolated genes (Biery et al. 2000) with fast reinsertion of mutants at an ectopic position into the viral genome by FLP/FRT-mediated site-specific recombination (Fig. 6) as described

Fig. 6 Scheme of the strategy for random mutagenesis of an essential viral gene in the viral genome context. Part I: In the first step, the viral gene of interest (gray box) is subcloned into a rescue plasmid (rescue) containing one FRT site (open box with gray triangle) This plasmid is subjected to an in vitro Tn7-based random muta-genesis procedure, leading to a mutant library with 15-bp insertions (black box) through the target plasmid. This mutant library is transformed into special E. coli strain (PIR) that is permissive for the rescue plasmid and single clones are screened by PCR or followed by sequencing to identify insertions within the ORF under study. Part II: To reinsert the gene mutants into the viral genome lacking the gene of interest, the respective deletion mutant-BAC and a FLP recombinase-expressing plasmid (FLP) are maintained in normal E. coli strain (DH10B) and transformed with the rescue plas-mids. FLP recombinase mediates site-specific recombination between the FRT sites and unifies the BAC and the rescue plasmid. Combined selection identifies the recombinant BACs with the inserted rescue plasmid because the rescue plasmid itself cannot be maintained in normal E. coli. The FLP-expressing helper plasmid is removed by elevated temperature. Part III: Subsequently, BAC DNAs are isolated and transfected one by one into eukaryotic cells for virus reconstitution and cells are screened for viral plaques

above for ectopic cis-complementation (see Sect. 4.6). The wt gene is subcloned in an FRT containing rescue plasmid. This construct is subjected to random Tn mutagenesis, as described for small plasmids. Mutants are sequenced and a comprehensive set of mutants is selected. Recombination between the FRT transfer plasmids carrying the mutants and the BAC lacking the gene of interest, mediated by the FLP recombinase, provides genomes that are cis-complemented by the mutant set. The complemented genomes are tested one by one for virus rescue. In combination with standard biochemical or cell biological assays, this procedure allowed genetic analysis of essential gene functions of MCMV at high resolution (Bubeck et al. 2004; Lotzerich et al. 2006). The method easily maps functionally important sites in essential viral proteins.

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