Fig. 2

Construction and analysis of pHSVF-CREin. a The infectious clone pHSVF-CREin was made by insertion of the CREin expression cassette into the pHSVF-BFP infectious clone in a process paralleling that described in Fig. 1. The CREin expression cassette was PCR amplified from the pEP-CREin-in template and recombined into pHSVF-BFP BAC vector backbone by lambda RED recombination, resulting in the replacement of the TagBFP coding sequence with that of CREin::kanamycin. In the second recombination step, the kanamycin resistance gene (aphA1) was removed based on partially duplicated sequences in the flanking CREin coding sequence (red boxes), which simultaneously established the contiguous CREin coding sequence and resulted in pHSVF-CREin. Excision of the BAC from pHSVF-CREin was achieved by autonomous expression of Cre recombinase following introduction of the DNA into either Vero or HEK293T cells, resulting in a passage 1 (P1) harvest. b MluI (left) or PvuI (right) restriction analysis of HSV-1 DNA harvested from purified nucleocapsids. Yellow arrow heads indicate restriction fragments that mark the presence or absence of the BAC vector sequences. Size standards are indicated in kb. c Amplification of viral and plasmid DNA using primers designed to detect removal of BAC DNA from the viral genome. The positions of the primer pairs are indicated in panel A