In-Vitro Mutagenesis of BACs, PACs or Cosmids - any Suggestions ?

[Peter Wang]

(Martin_Messerle) wrote:
>I managed to clone the large genome of a herpesvirus into a BAC vector
>and actually I am able to reconstitute the virus after transfection of
>the BAC plasmids into eukaryotic cells.
>My intention is to mutagenize the genome of the virus in vitro or in
>E.coli, to reconstitute recombinant viruses and to analyze the
>function of the mutagenized genes.
>...
>The second idea is that the mutagenesis has to be performed by
>homologous recombination in E.coli. ...

Your posting reminded me of the following paper:

M. Tripodi, S. Perfumo, R. Ali, L. Amicone, C. Abbott, R. Cortese. 
(1990) "Generation of small mutation in large genomic fragments by 
homologous recombination:  description of the technique and examples of 
its use".  Nucleic Acids Research 18:6247-6251.

They used recombination to engineer genes contained in cosmids.
I don't have any personal experience with the techniques, however.

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Peter Wang, M.D., Ph.D.
MRC Centre for Protein Engineering,
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