Recombination-Mediated Genetic Engineering of a Bacterial Artificial Chromosome Clone of Modified Vaccinia Virus Ankara (MVA) - Genetic Engineering: Recent Developments in Applications - page 45

containing pBELO-BAC11, a LoxP recombinase site and a GFP reporter gene
driven by a late poxviral promoter (see Figure 1) was inserted into the deletion III
locus of MVA by conventional recombination in infected CEF cells. Cells infected
with this recombinant virus, referred to at MVA-BAC-parent, were treated with
isatin- β -thiosemicarbazone (IβT) β T) to inhibit viral hairpin resolution and promote
genome concatemerisation [59]. After transformation of E. coli with DNA from
these cells, dozens of chloramphenicol-resistant colonies were observed regardless
of treatment of infected cells with IβT. β T. Unexpectedly, transfection of cells with
pCI-Cre prior to infection in an attempt to induce recombination between LoxP
sites to produce circular molecules from head-to-tail concatemers [53] dramati-
cally decreased the number of colonies obtained, probably due to cytotoxicity of
the transfection reagent. Alternatively, it is possible that cryptic LoxP sites such as
those present in mammalian genomes [60] resulted in illegitimate recombination
events.
Colonies were screened for potential full-length clones by PCR at five loci
spaced along the genome, and candidates were further characterised by restriction
mapping with HindIII and XhoI and pulsed-field gel electrophoresis after diges-
tion with FseI to excise the BAC cassette (data not shown). No full-length clones
were isolated from pCI-Cre transfected samples (of 5 colonies screened), but from
untransfected cells, four apparently full-length clones were obtained, three de-
rived from I β T-treated cells (of 24 colonies screened) and one from untreated
cells (of 18 colonies screened). This frequency is very similar to that observed for
VAC-BAC [53].
bAc rescue
The next step was to “rescue” the BAC clones to infectious MVA using a Fowlpox
virus helper to provide transcriptional machinery. The BHK cell line, which is
the only mammalian cell line known to support MVA replication, is ideal for
this purpose since it is non-permissive for the host-range restricted avipoxviruses.
Following transfection into BHK cells infected with Fowlpox virus, all four of
the MVA-BAC clones were converted to infectious MVA able to replicate and
form plaques on BHK and CEF cells. We have not conducted formal growth rate
analysis of the resulting viruses, but viral titres of 8.0×10 8 to 2.4×10 9 pfu/mL (fi-
nal volume ~0.5 mL) were achieved following sucrose cushion purification from
BHK cultures totalling 1500 cm 2 . The titrations were performed on CEF cells,
in which Fowlpox virus can replicate, and absence of significant residual Fowlpox
virus was demonstrated by X-Gal staining for the LacZ marker gene present in
the helper virus.