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Viral bacterial artificial chromosomes: generation, mutagenesis, and removal of mini-F sequences.

Tischer BK, Kaufer BB - J. Biomed. Biotechnol. (2012)

Bottom Line: Maintenance and manipulation of large DNA and RNA virus genomes had presented an obstacle for virological research.BAC vectors provided a solution to both problems as they can harbor large DNA sequences and can efficiently be modified using well-established mutagenesis techniques in Escherichia coli.Furthermore, we address common mutagenesis techniques that allow modification of BACs from single-nucleotide substitutions to deletion of viral genes or insertion of foreign sequences.

View Article: PubMed Central - PubMed

Affiliation: Institut für Virologie, Freie Universität Berlin, Berlin, Germany. k.tischer@fu-berlin.de

ABSTRACT
Maintenance and manipulation of large DNA and RNA virus genomes had presented an obstacle for virological research. BAC vectors provided a solution to both problems as they can harbor large DNA sequences and can efficiently be modified using well-established mutagenesis techniques in Escherichia coli. Numerous DNA virus genomes of herpesvirus and pox virus were cloned into mini-F vectors. In addition, several reverse genetic systems for RNA viruses such as members of Coronaviridae and Flaviviridae could be established based on BAC constructs. Transfection into susceptible eukaryotic cells of virus DNA cloned as a BAC allows reconstitution of recombinant viruses. In this paper, we provide an overview on the strategies that can be used for the generation of virus BAC vectors and also on systems that are currently available for various virus species. Furthermore, we address common mutagenesis techniques that allow modification of BACs from single-nucleotide substitutions to deletion of viral genes or insertion of foreign sequences. Finally, we review the reconstitution of viruses from BAC vectors and the removal of the bacterial sequences from the virus genome during this process.

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Overview of techniques that facilitate the insertion of a sequence of interest (soi) into a target site. Boxes of same color represent identical sequences.
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Related In: Results  -  Collection


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fig3: Overview of techniques that facilitate the insertion of a sequence of interest (soi) into a target site. Boxes of same color represent identical sequences.

Mentions: Several strategies that allow the insertion of a sequence of interest (soi) including reporter genes, fluorescent tags, or foreign, viral antigens into BAC constructs have been developed (Figure 3). One approach utilizes a transfer construct that contains the soi and a positive selection marker flanked again by two loxP or FRT recognition sites. The construct is inserted into the target site by Red recombination. In a second step, the selection marker can be removed by the induction of the Flp or Cre recombinase, while one recognition site remains in the BAC construct downstream of the soi (Figure 3(a)) [7].


Viral bacterial artificial chromosomes: generation, mutagenesis, and removal of mini-F sequences.

Tischer BK, Kaufer BB - J. Biomed. Biotechnol. (2012)

Overview of techniques that facilitate the insertion of a sequence of interest (soi) into a target site. Boxes of same color represent identical sequences.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3303620&req=5

fig3: Overview of techniques that facilitate the insertion of a sequence of interest (soi) into a target site. Boxes of same color represent identical sequences.
Mentions: Several strategies that allow the insertion of a sequence of interest (soi) including reporter genes, fluorescent tags, or foreign, viral antigens into BAC constructs have been developed (Figure 3). One approach utilizes a transfer construct that contains the soi and a positive selection marker flanked again by two loxP or FRT recognition sites. The construct is inserted into the target site by Red recombination. In a second step, the selection marker can be removed by the induction of the Flp or Cre recombinase, while one recognition site remains in the BAC construct downstream of the soi (Figure 3(a)) [7].

Bottom Line: Maintenance and manipulation of large DNA and RNA virus genomes had presented an obstacle for virological research.BAC vectors provided a solution to both problems as they can harbor large DNA sequences and can efficiently be modified using well-established mutagenesis techniques in Escherichia coli.Furthermore, we address common mutagenesis techniques that allow modification of BACs from single-nucleotide substitutions to deletion of viral genes or insertion of foreign sequences.

View Article: PubMed Central - PubMed

Affiliation: Institut für Virologie, Freie Universität Berlin, Berlin, Germany. k.tischer@fu-berlin.de

ABSTRACT
Maintenance and manipulation of large DNA and RNA virus genomes had presented an obstacle for virological research. BAC vectors provided a solution to both problems as they can harbor large DNA sequences and can efficiently be modified using well-established mutagenesis techniques in Escherichia coli. Numerous DNA virus genomes of herpesvirus and pox virus were cloned into mini-F vectors. In addition, several reverse genetic systems for RNA viruses such as members of Coronaviridae and Flaviviridae could be established based on BAC constructs. Transfection into susceptible eukaryotic cells of virus DNA cloned as a BAC allows reconstitution of recombinant viruses. In this paper, we provide an overview on the strategies that can be used for the generation of virus BAC vectors and also on systems that are currently available for various virus species. Furthermore, we address common mutagenesis techniques that allow modification of BACs from single-nucleotide substitutions to deletion of viral genes or insertion of foreign sequences. Finally, we review the reconstitution of viruses from BAC vectors and the removal of the bacterial sequences from the virus genome during this process.

Show MeSH
Related in: MedlinePlus