Biology Reference
In-Depth Information
compared to
wt
. To generate the phenotypic reversion, first a rescue plasmid is gener-
ated by cloning the
wt
gene into an FRT transfer plasmid that carries an FRT site and
can only be maintained in special
E. coli
strain by a conditional origin of replication.
This rescue plasmid is then reinserted into the mutant genome using the FRT/Flp
system. In contrast to the commonly used approaches, in which the FRT sites are
located on the same DNA molecule and mediate deletions or inversions, here the FRT
sites induce intermolecular recombination, resulting in the unification of the mutant
BAC and the rescue plasmid. The recombinants are selected by the antibiotic resistance
of the rescue plasmid and the recombinants are reconstituted for analysis of the pheno-
type. Ectopic reinsertion, like
trans-
complementation, will not restore polar effects at
the site of mutation. However, this approach has advantages compared to protein
trans-
complementation by cells: (a) cell toxicity of the viral gene product is not an issue;
(b) the gene expression is controlled by the virus life cycle; (c) Complementation
works in any cell-type since the complementing gene is expressed from the virus
and permits the analysis of in vivo phenotypes; and (d) it does not require cumber-
some establishment of
trans-
complementing cell lines. Ectopic
cis
-complementation
system has been established in the context of both MCMV and HCMV and was
proven for essential and nonessential genes in vitro and in vivo (Borst et al. 2001;
Bubeck et al. 2004; Bubic et al. 2004).
Genetic Analysis of Essential Genes
Comprehensive Mutational Analysis of Essential CMV Genes
Detailed genetic analysis of genes involved in DNA replication and packaging,
morphogenesis and egress of infectious particles, requires expression of mutant
genes in the viral context, where the relevant functions are expressed in operational
conditions. High-resolution genetic analysis has been demonstrated by elegant
pool screens for genetic foot-printing of viral genes, subgenomic fragments, and
even of complete viral genomes up to 10 kbp in size (Laurent et al. 2000;
Rothenberg et al. 2001). These screens discriminate between virus mutants that
replicate and mutants in which essential functions are affected and therefore
cannot be retrieved. The transfection of viral DNA corresponding to the size of a
CMV genome is not yet efficient enough for pool reconstitution. Therefore, CMV
Fig. 5
(continued)
The wt gene can be introduced into the mutant viral BAC at a neutral second
site. By FLP-mediated site-specific recombination of an FRT transfer plasmid carrying the wt
gene including regulatory sequences and the mutant BAC genome with an FRT site (
black triangle
in a square
), the wt gene product can be expressed from the mutant genome itself. After transfec-
tion of permissive cells with this revertant BAC genome, a homogenous population of revertant
virus is reconstituted. Only protein
trans-
complementation (
c
) and ectopic
cis
- complementation
(
d
) allow the formal confirmation that the mutated gene product (and not possible other
cis
-effects
of the mutated sequence) is responsible for the observed phenotype
