Biology Reference
In-Depth Information
mutants need to be analyzed individually. The random transposon mutagenesis of
the BACs targets the entire genome and null mutants of the respective genes
discriminate essential from nonessential genes. Yet, these insertion libraries cannot
be used for the functional characterization of a coding sequence. Comprehensive
mutant pools of subcloned genes can be obtained through different random muta-
genesis procedures. However, a large set of these mutants has to be introduced one
by one into the CMV genome lacking the gene of interest to analyze their effect in
the context of virus replication. Therefore, we developed a strategy combining a
comprehensive Tn7-based linker-scanning mutagenesis of isolated genes (Biery
et al. 2000) with fast reinsertion of mutants at an ectopic position into the viral
genome by FLP/FRT-mediated site-specific recombination (Fig. 6) as described
Fig. 6
Scheme of the strategy for random muta-
genesis of an essential viral gene in the viral
genome context. Part I: In the first step, the viral
gene of interest (
gray box
) is subcloned into a
rescue plasmid (
rescue
) containing one FRT site
(
open box with gray triangle
) This plasmid is
subjected to an in vitro Tn7-based random muta-
genesis procedure, leading to a mutant library
with 15-bp insertions (
black box
) through the
target plasmid. This mutant library is trans-
formed into special
E. coli
strain (PIR) that is
permissive for the rescue plasmid and single
clones are screened by PCR or followed by
sequencing to identify insertions within the
ORF under study. Part II: To reinsert the gene
mutants into the viral genome lacking the gene
of interest, the respective deletion mutant-BAC
and a FLP recombinase-expressing plasmid
(FLP) are maintained in normal
E. coli
strain
(DH10B) and transformed with the rescue plas-
mids. FLP recombinase mediates site-specific
recombination between the FRT sites and uni-
fies the BAC and the rescue plasmid. Combined
selection identifies the recombinant BACs with
the inserted rescue plasmid because the rescue
plasmid itself cannot be maintained in normal
E.
coli
. The FLP-expressing helper plasmid is
removed by elevated temperature. Part III:
Subsequently, BAC DNAs are isolated and
transfected one by one into eukaryotic cells for
virus reconstitution and cells are screened for
viral plaques
