In vitro knockout?

[Pamela Norton]

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.  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.
> 

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? 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. 

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

Also suggest that you consider RNAi.

Good luck,

Pam Norton