gene replacement

Mikhail Alexeyev malexeyev at biost1.thi.tmc.edu
Fri Dec 8 15:05:25 EST 1995


In article <4a9i4u$fuu at mserv1.dl.ac.uk>, Jose Nieto (Path)
<jn10001 at mole.bio.cam.ac.uk> wrote:

> dear netters,
> 
> I have mutated a cloned gene from a large conjugative plasmid
> and now I intend to replace the mutated gene in its original
> position. There's no way I can do that by cut and paste cloning
> so I must do it by in vivo gene replacement. Any one has
> direct experience on that kind of experiment. Any suggestions
> will be appreciated,
> 
 
Although it is difficult to suggest anything without knowing the details,
you may try putting your mutated gene in one of the of the sacB-containing
vectors like one described by Kaniga (Gene 109:137-141; 1991) or other
conditionally lethal gene (Skrzypek Plasmid, 1993 29:160-163), mobilize
(electroporate) your vector into recipient, select for cointegration and
then induce the lethal gene to select for second recombination event. If
you choose introduction of your mutated gene into the recipient by
conjugation, you may want to select against donor E. coli using minimal
media and prototrophic recipient or recipient with different from the
donor prototrophic requirements if recipient plasmid can replicate in
E.coli. All the standard stuff.

Alternatively, if mutation phenotype is known and is selectable or atleast
easily screenable, you may inactivate the wt gene with insertion of
mini-transposon bearing your mutated gene. It worked for me in the case of
inactivation of the chromosomal lacZ with transposon bearing lacZdeltaM15.
Of course, you will need recA- background reduce homologous recombination.


Regards,
M. Alexeyev



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