mutant neo gene for targeting

[Andrei Popov]

d.m. wright wrote:
> 
> I have been trying a gene targeting project in myogenic cells over the
> past two years with no success so far.  We chose to use a mutant (weaker)
> neo gene because at the inception of the project there were a couple of
> reports that using this gene as the selctable marker had allowed the
> targeting of both alleles with one construct.  I have now heard from
> other people that this is very possibly what prevented our success.  Has
> anuone else tried this or heard of other people that have tried it, and
> what the outcome has been?  Also does anyone know if there is a gene
> targeting newsgroup where this would be better placed?
> 
> Thank you,
> 
> D.Wright

Hello,
you did not specify where your construct comes from, or what promoter is being used.
However, at least in  mouse ES cells the NEO-r marker gene construct of Thomas and
Capecci (Stratagene, pMC-NEOpolyA) works well, and as far I know people who made 
modifications on both alleles simply selected for 1 targeted event, then increased the 
G418 conc approximately 2 fold to get gene conversion on the second chromosome.
There is another problem associated with neo from Tn5- there were some reports about
its silencing activity...
If you need this (unmodified) NEO construct, let me know.

Best regards

Andrei