Biology Reference
In-Depth Information
regulated gene and induction by doxycycline exposes the viral replication program
to the DN mutant. This system allowed us detailed quantitative and qualitative
analysis of the effect of DN mutants of both M50 and M53. In addition, the result of
the random screen on MCMV M50 aided the construction of a DN mutant of the
homolog in HCMV (UL50) (Rupp et al. 2007). We believe that this systematic
approach will facilitate the functional analysis of essential CMV genes.
Concluding Remarks
Cloning large DNA sequences as BACs has become the method of choice for mapping,
sequencing and manipulation of large eukaryotic genomes. Genetic engineering in BACs
is based on homologous recombination and now allows any type of DNA modification.
In addition, for herpesviruses, including CMV, these procedures permit the manipulation
of the infectious genome as a single plasmid. Mutagenesis is safely carried out in
E. coli
and physical controls can be performed prior to virus reconstitution. These are necessary
because CMV genomes contain repetitive sequences that are prone to recombination. In
the past, the investigation of CMV gene functions was limited by the laborious and time-
consuming generation of virus mutants. Using BAC recombineering, this problem is
solved. In the future, more weight has to be placed on careful planning of appropriate
controls. The lack of technical limitations allows the production of a plethora of mutants.
Traceless mutagenesis permits multiple sequential mutagenesis steps and complex engi-
neering procedures. However, only the local targeted mutations can be thoroughly
checked. There is an uncertainty on the numbers of unwanted mutations in other regions
that may occur. These mutations do not need to have a growth phenotype in the cell line
under study but may mar experiments in other cells or in vivo experiments. According to
the law of error propagation, the necessary controls increase with each sequential muta-
genesis step. Since at the level of plasmids each step can be controlled by sequencing, as
many steps as possible should be done on subcloned target regions. With regard to BAC
engineering, it may be advisable to decide on a smaller number of BAC engineering steps
rather than on perfect sequence correction.
Acknowledgements
The work was supported by the Deutsche Forschungsgemeinschaft through
SFB 455, SPP New vaccination strategies, SPP1175 and the VCI.
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