In vitro knockout?

[John Ladasky]

Thanks, Ashok and Pamela, for your replies.

Pamela Norton <pamela.norton at mail.tju.edu> wrote in message news:<020120021538346231%pamela.norton at mail.tju.edu>...
> In article <slrna33in8.86o.aiyar at ebv.mimnet.nwu.edu>, Ashok Aiyar
> <aiyar at ebv.mimnet.northwestern.edu> wrote:
> 
> > On 31 Dec 2001 22:32:10 -0800,
> >     John Ladasky (ladasky at my-deja.com) wrote:
> > 
>  Original query snipped
> > 
> > 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.  

Right.  I have several lines available to me which are apparently
diploid, however.

> > Second, the efficiency of homologous recombination is very low.

Yes, this can be a problem.  Thanks to Pamela's leads I have John
Sedivy's references in hand now.

> > 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.

Neat trick, but I need to perform microscopy and identify organelles,
thus I need to work with an adherent cell line.  I guess I'll just
have to put up with the low frequency of homologous recombination.

> This is certainly a good suggestion. Let me point out that knockout
> mice start out as cultured ES cells - I'm guessing that you wish to use
> a specific cell line? 

My criteria for the cell line is that it be human, male, and adherent.
 My preliminary studies have been done in HeLa.  I can't easily do the
X-chromosome knockout in HeLa, because it's female.  I've been looking
through our liquid nitrogen stock, and the ATCC and Coriell databases.
 I have a few candidates on hand.

> At least in the ES cells, although the targeted
> disruptions are initially hemizygous (excepting X/Y in male cells), it
> is possible to obtain a second event for many (but not all) genes. 

With considerable effort!

> From memory, I believe that John Sedivy has published a good deal on
> obtaining homologous recombinants in cultured cells.
> 
> > 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.  

One of Sedivy's papers reports that increasing the concentration of
G418 used in selection, to ~5 mg/ml (!), cuts down on the frequency of
incorrect insertions.

> > We are looking at other methods of disrupting
> > gene expression.  Right now, RNAi looks promising.
> > 
> > Regards,
> > Ashok
> 
> Also suggest that you consider RNAi.

Flossie Wong-Staal visited Hopkins a few months ago.  From her I
learned about hammerhead ribozymes.  It looks like a pretty good
"knock-down" method, but I'm also interested in replacing the
wild-type gene with a mutant.  As such, I still favor replacement by
homologous recombination.

Thanks again for your help!

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