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Systematic excision of vector sequences from the BAC-cloned herpesvirus genome during virus reconstitution
M Wagner1, S Jonjic, U H Koszinowski
1Max von Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie, Ludwig-Maximilians-Universität München, D-81377 Munich, Germany.
Abstract:
Recently the mouse cytomegalovirus (MCMV) genome was cloned as an infectious bacterial artificial chromosome (BAC) (M. Messerle, I. Crnkovic, W. Hammerschmidt, H. Ziegler, and U. H. Koszinowski, Proc. Natl. Acad. Sci. USA 94:14759-14763, 1997). The virus obtained from this construct is attenuated in vivo due to deletion of viral sequences and insertion of the BAC vector. We reconstituted the full-length MCMV genome and flanked the BAC vector with identical viral sequences. This new construct represents a versatile basis for construction of MCMV mutants since virus generated from the construct loses the bacterial sequences and acquires wild-type properties.
Insights
Researchers reconstituted the full-length mouse cytomegalovirus (MCMV) genome using a bacterial artificial chromosome (BAC) system. This improved MCMV BAC construct enables the generation of wild-type viruses, facilitating mutant generation.
Area of Science:
- Virology
- Molecular Biology
- Genetics
Background:
- The mouse cytomegalovirus (MCMV) genome was previously cloned into a bacterial artificial chromosome (BAC).
- The initial MCMV BAC construct resulted in an attenuated virus due to sequence deletions and BAC vector insertion.
Purpose of the Study:
- To reconstitute a full-length MCMV genome within a BAC construct.
- To develop a versatile platform for generating MCMV mutants with wild-type properties.
Main Methods:
- Reconstitution of the full-length MCMV genome.
- Flanking the BAC vector with identical viral sequences within the MCMV genome.
Main Results:
- The reconstituted MCMV BAC construct allows for the generation of infectious virus.
- Virus generated from the new construct loses bacterial sequences and regains wild-type MCMV properties.
Conclusions:
- The improved MCMV BAC system provides a robust basis for creating MCMV mutants.
- This methodology facilitates the study of MCMV genetics and pathogenesis.