silence a gene by insertion of an antibiotic casette

Marina Verkhovskaya verkhovs at penger.helsinki.fi
Mon Jan 5 05:06:44 EST 1998


 I am trying to silence a gene by inserting a kanamycin cassette  by
allelic replacement into the chromosomal gene.  What I have done is the
following
The gene to be silenced was cloned in a 3.2 kb. A kanamycin cassette was
introduced into the gene in such a way that 1.2 and 0.6 kb of the target
DNA remained flanking the cassette. The fragment with insert was cloned
into a suicide plasmid which is conjugative but cannot replicate in the
strain to be mutated. The plasmid contained an ampicillin resistance
gene and the cells were also kanamycin resistant (maybe a possible
problem, see below). The plasmid was introduced into the recipient by
conjugation and transconjugants were selected on the basis of the
antibiotic markers.
I got several exconjugants that were kanamycin resistant but very
suspiciously all were amp sensitive. I analyzed several of them by PCR
and the gene of interested was intact, kanamycin was not inserted. I
checked the recipient and donor cells for resistance and the pass the
test (recipient was kan and amp sensitive). One possibility is that
cells are getting spontaneous kan resistance or worse that kan is
inserted inspecifically somewhere else, by double crossover that could
be at least in theory, unlikely.
I wonder if someone run into the same kind of problem. One possible
solution is to change the marker.
Please if someone has had similar experience or any suggestion, all help
will be very much appreciated.

Blanca Barquera
Dept.Medical Chemistry
University of Helsinki
email: barquera at rock.helsinki.fi




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