Electroporating tissue in suspension was Re: R to DK: How to efficiently transfect U937 cells with a plasmid vectorexpressing shRNA

[Tom Anderson]

While we're on the subject of electroporation,

Has anyone ever tried, and moreover had any success at, electroporating
chunks of tissue in suspension? I'm working with an explant culture
system, and it would be incredibly useful to be able to put genes into the
explants before culturing them. At present, i can do this with viruses,
but this is a bit cumbersome, and i haven't got any chemical transfection
method, or nucleofection, to work.

Dissociating the explants and electroporating the cells is a non-starter -
the cells don't come out right unless the explants are intact.

tom

On Wed, 8 Aug 2007, Prof. Piero Sestili wrote:

> we have something like this guide but there's nothing "amazing"....so
> yes, please send your one to the public address or, if you prefer to
> piero.sestili from uniurb.it. Hi and thanks again
>
> Piero
>
> -----Messaggio originale-----
> Da: methods-bounces from oat.bio.indiana.edu
> [mailto:methods-bounces from oat.bio.indiana.edu] Per conto di DK
> Inviato: martedì 7 agosto 2007 23.51
> A: methods from magpie.bio.indiana.edu
> Oggetto: Re: How to efficiently transfect U937 cells with a plasmid
> vectorexpressing shRNA
>
> In article <mailman.546.1186502638.11350.methods from net.bio.net>, "Prof.
> Piero Sestili" <piero.sestili from uniurb.it> wrote:
> >We are trying to transfect U937 cells with a plasmid vector expressing
> >shRNA. However we are obtaining a very low transfection efficiency
> >using either electroporation (which is not the best solution since we
> >need that transfected cells retain their viability for some weeks) or
> >lipofectin.  Is there anybody who knows some nice alternative method or
>
> >trick to increase the efficiency?  thanks a lot,
>
> The highest efficiency is practically always with electroporation. The
> trick is that it needs to be properly optimized. If you would
> you be willing to go this route, I can post an "electroporation
> optimization guide" that I've used to transfect all kinds of cells and
> help many people over many years.
>
> DK

-- 
Tom Anderson, MRC Laboratory for Molecular Cell Biology, UCL, London WC1E 6BT
(t) +44 (20) 76797264   (f) +44 (20) 76797805   (e) thomas.anderson from ucl.ac.uk