Fig. 6

Rescue of recMVA-NoV from recBAC plasmid in DF-1 cells. a Confirmation of gene insertion into the MVA genome by PCR. Insertion of the capsid gene was checked by amplifying the DelVI cassette using DelVI primers for the WT (expected size: 498 bp) and gene-specific primers for the recMVA-BAC-GII.4 (expected size: 1.6 kb). Uninfected DF-1 cells were used as negative control. b Confirmation of RFV clearance by PCR using RFV-specific primers with an expected size of 265 bp. RFV-related band was detected in the first rescue sample; it was not detectable from the 4th passage. Purified RFV was used as positive control. c and d BAC self-excision: After transfection of the shuttle vector into RFV-infected DF-1 cells, the BAC backbone and the GFP cassette were spontaneously lost by passaging: (C) Confirmation of removal of BAC backbone by PCR using specific primers for gfp gene. (D) significant reduction in GFP population indicating the loss of BAC cassette from the recMVA genome. Scale: 100 μm