In vitro knockout?

Ashok Aiyar aiyar at ebv.mimnet.northwestern.edu
Tue Jan 1 09:42:48 EST 2002


On 31 Dec 2001 22:32:10 -0800,
    John Ladasky (ladasky at my-deja.com) wrote:

> 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.

If you use HSV-TK, you can select against it with ganciclovir -- easier 
than HAT.
 
> 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.

John,

Lots of groups have made heterologous and homologous knock-outs in
cell-lines.  There are two issues that you should consider.  First
most cell-lines are not diploid.  Second, the efficiency of homologous
recombination is very low.

One method used to get around this is to make knockouts in the
chicken bursal B cell-line DT40.  Antibody diversity in chickens
is largely generated by somatic recombination, and this cell-line
performs homologous recombination with high frequency.

A PubMed search of "DT40 homologous recombination", or "DT40
targeted disruption" will give you a large number of references to
peruse.

I have attempted homologous recombination in an EBV-immortalized
human B lymphoblastoid cell-line, using double-selection -- i.e. 
against HSV-TK, and for G418 resistance.  I screened about 100 clones 
by PCR, and none of them were correct insertions .... but your luck
could be better.  We are looking at other methods of disrupting
gene expression.  Right now, RNAi looks promising.

Regards,
Ashok
-- 
Ashok Aiyar, Ph.D.
Assistant Professor                    email: a-aiyar at northwestern.edu
Department of Microbiology-Immunology           office: (312) 503-2524
303 E. Chicago Avenue, WARD 4-123                  lab: (312) 503-2542
Northwestern University, Chicago, IL 60611         fax: (312) 503-1339




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