Recombination-Mediated Genetic Engineering of a Bacterial Artificial Chromosome Clone of Modified Vaccinia Virus Ankara (MVA) - Genetic Engineering: Recent Developments in Applications - page 48

insertion arose during production of the MVA-BAC parent, or represents isola-
tion of a rare genotype present at low levels in the source MVA. Evidence for
genetic heterogeneity within poxvirus strains [64], [65](personal communication,
R. Regnery) favours the latter possibility.
Deletion of MVA Genes by BAC Recombineering
Recombination mediated genetic engineering (recombineering) permits modifi-
cation of BAC DNA by homologous recombination in E. coli. We used the sys-
tem described by Warming et al. [55], which is based on stringent temperature-
sensitive expression of the λ Red genes exo, bet and gam coupled with GalK-based
positive and negative selection. A similar approach, not using GalK, has been
described for VAC-BAC by Domi and Moss [54], who also verified the stability
of their Vaccinia virus BAC clone after induction of the λ Red system. Five genes
reported to affect MVA immunogenicity or Vaccinia virus immunogenicity or vir-
ulence were selected from the literature for deletion by insertion of GalK in place
of the ORF (see Table 2). Long oligonucleotide primers were used to introduce
50 bp homology arms at the 5 ′ and 3 ′ ends of GalK by PCR and these products
were electroporated into heat-induced SW102 cells carrying MVA-BAC prior
to selection on galactose minimal medium, as described [55]. Before rescue by
transfection into Fowlpox virus infected BHK cells, as above, the modified BACs
were checked by restriction digest with HindIII and XhoI and by sequencing two
PCR products spanning the junctions at the GalK insertion. In only one case (the
A46R deletion), was any mutation detected: a primer-derived single nucleotide
deletion in the intergenic region downstream of the insertion. This mutation was
ignored as it did not fall within any predicted regulatory sequences (promot-
ers or terminators). GalK deletion mutant BACs were also checked for absence
of undeleted contaminants by PCR across the deleted locus. All five modified
GalK-carrying MVA-BACs converted into infectious virus, and again, though we
did not formally compare growth rates, all were amplified to titres of 2.8×10 8 to
2.6×10 9 pfu/mL (final volume ~0.5 mL) following sucrose cushion purification
from 1500 cm 2 of BHKs.
Table 2. MVA-BAC genes deleted by GalK recombineering.