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

nanooe from gmail.com via methods%40net.bio.net (by nanooe from gmail.com)
Thu Aug 9 07:04:41 EST 2007


Did you try Microporation? www.microporator.com. Slightly different
take on electroporation so may have advantages for explants?



On Aug 8, 4:00 pm, Tom Anderson <ucga... from ucl.ac.uk> wrote:
> While we're on the subject ofelectroporation,
>
> 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.sest... from uniurb.it. Hi and thanks again
>
> > Piero
>
> > -----Messaggio originale-----
> > Da: methods-boun... from oat.bio.indiana.edu
> > [mailto:methods-boun... from oat.bio.indiana.edu] Per conto di DK
> > Inviato: martedì 7 agosto 2007 23.51
> > A: meth... from magpie.bio.indiana.edu
> > Oggetto: Re: How to efficiently transfect U937 cells with a plasmid
> > vectorexpressing shRNA
>
> > In article <mailman.546.1186502638.11350.meth... from net.bio.net>, "Prof.
> > Piero Sestili" <piero.sest... 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 eitherelectroporation(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 withelectroporation. 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.ander... from ucl.ac.uk




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