In vitro knockout?
ladasky at my-deja.com
Fri Jan 4 17:57:58 EST 2002
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
> > 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
> > 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
Thanks again for your help!
John J. Ladasky Jr., Ph.D.
Department of Biology
Johns Hopkins University
Baltimore MD 21218
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