Use of Recombination-Mediated Genetic Engineering for Construction of Rescue Human Cytomegalovirus Bacterial Artificial Chromosome Clones

Figure 1

Toledo ROI Deletion Mutant BAC Clone Generation. A kanamycin resistant gene (a) is amplified by primers containing 40-bp homology to the sequences flanking the region of interest (ROI) in Toledo BAC (b) to create the kanamycin resistant cassette (c). In order to generate an ROI deletion mutant BAC clone (ROI  Δ), DY380 E. coli carrying WT Toledo BAC (d) must undergo electrocompetent cell preparation and induction for recombination gene expression (e). The kanamycin resistant cassette is then electroporated into the electrocompetent DY380 harboring WT Toledo BAC (f). Upon homologous recombination, the ROI will be replaced by the kanamycin resistant gene, generating the Toledo ROI Δ mutant BAC clone (g).