FIG. 1.

Completion of the full-length HCMV BAC and excision of the BAC cassette. (A) In HCMV BAC plasmid pHB-5, the BAC cassette replaces genes US2 through US6 (nt 193360 to 196045). (B) Details of the region of BAC insertion. X, XbaI; N, NsiI; H, HpaI; B, BglII. (C) For construction of HB-5loxP, a loxP site flanked with 2.0-kbp fragments homologous to HCMV and BAC sequences was cloned into pST76K-SR. Site-directed introduction into the HCMV genome by shuttle mutagenesis yielded an additional BglII site and two additional NsiI sites (I). Subsequently, the genes US2 to US6 were introduced by shuttle mutagenesis together with a loxP site and an RP4 origin of transfer (oriT) (II). (D) Cotransfection of AD169-BAC with a plasmid expressing recombinase Cre into permissive cells leads to the removal of the BAC cassette from the HCMV genome. (E) The virus progeny AD169-RV is distinguishable from AD169-wt by XbaI and HpaI restriction sites located next to the loxP site.