In vitro knockout?

John Ladasky ladasky at my-deja.com
Tue Jan 1 01:32:10 EST 2002


Greetings, fellow Bionetters,

It has been a while since I've visited here.  Hope you all are doing
well.

I am interesting in knocking out a gene, but I don't want to have to
go through the trouble of generating a transgenic animal to perform
the knockout.  It hardly looks like it's worth the great expense in
time and money for the small amount of data I expect to obtain.

What I would like to do is to transfect a cell line with a plasmid
construct similar to that used to make transgenic mice.  This plasmid
would consist of a region homologous to my gene of interest, and would
include a marker for negative selection which would have to be spliced
off by recombination, like the traditional thymidine kinase gene which
renders cells sensitive to HAT medium.

Before you say, "wait, you're wasting your time, you'll only get a
heterozygous KO in vitro," let me provide you with one critical extra
piece of information: the gene I'm trying to knock out is located on
the X chromosome.  If I work with a cell line which is genetically
male (XY), I believe that ONE recombination event would be all that I
would need to effect the knockout.

Someone must have thought of this before.  I've been combing Medline
to try to find a reference to this technique.  Maybe I haven't come up
with the right combination of key words.  Has anyone seen a reference
to an X-chromosome somatic cell knockout?  Or can you comment on the
validity of this strategy?

Oh, and if by some remote chance I AM the first person to recognize
this strategy, remember: you saw it here first.  :^)

Thanks for your input!

--
John J. Ladasky Jr., Ph.D.
Department of Biology
Johns Hopkins University
Baltimore, MD 21218




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