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

Martin_Messerle Martin.Messerle at rhein-neckar.de
Sun Jan 14 15:52:39 EST 1996


Dear colleagues,
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.
My first aim is to delete certain genes from the genome, in the long
term, however, it would be advantageous to perform even such subtle
manipulations like amino acid substitutions.
I THINK THIS IS NOT ONLY A PROBLEM FOR ME OR FOR VIROLOGISTS BUT FOR
EVERYBODY WHO IS DEALING WITH LARGE DNA FRAGMENTS AND GENOMES.
Some people (for example Nat Sternberg from DuPont NEN) have proposed
transposon mutagenesis for P1 clones. But this is not specific enough
for my purposes.
In the moment I can think of two possibilities. The first idea is to
protect certain EcoRI sites with oligonucleotides against EcoRI
methylation and then to delete or substitute the corresponding EcoRI
fragment. I am not sure whether this is generally applicable. To my
knowledge triple helix formation with oligonucleotides is only
possible for specific sequences (CT-rich). ANY COMMENTS OR SUGGESTIONS
TO THIS POINT?
The second idea is that the mutagenesis has to be performed by
homologous recombination in E.coli. In former times when cloning
strategies were not as sophisticated as nowadays scientist performed
astonishing manipulations with this technique (for example of the
E.coli genome). My feeling is that the knowledge of this technique has
been lost (at least in this generation of Ph.D. students).
SO I AM LOOKING FOR ANYBODY WHO CAN GIVE ME ADVICE FOR MY PROBLEM.
ACTUALLY I AM WILLING TO COOPERATE WITH EVERYBODY WHO WILL CONVINCE ME
THAT HE GOT A SOLUTION FOR THIS PROBLEM.

If you prefer to send my an EMAIL message I would appreciate if you
could send it to the following address:

uk691mm at genius.embnet.dkfz-heidelberg.de

Thanx in advance

Dr. Martin Messerle
Department of Medical Virology
University of Heidelberg
69120 Heidelberg
Germany

  






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