Transfection without cytotoxicity

Frank O. Fackelmayer Frank at Fackelmayer.de
Thu Mar 21 10:14:09 EST 2002


Two things might be helpful to consider.
First, try electroporation. Although it does cause death of some cells,
those cells will not attach to the plate well, and can be washed away
the day after transfection. Electroporation DOES NOT, however, cause apoptosis.
Second, when the control construct causes as much cell death as your
test protein, I presume the DNA is not clean enough for transfection.
Impurities in the DNA preparation often cause cell death, especially if
the cells are susceptible. ALWAYS use the best DNA purification
possible. Experience has shown me that CsCl maxiprep DNA is superior to
DNA purified by any other means. Try it and you´ll understand what I mean.

Frank



Martin Thompson wrote:
> 
> I was wondering if anyone knows of a high efficiency way of transfecting
> cell lines (such as NIH 3T3's and HeLa's) without significant cytotoxicity.
> I am currently investigating a protein that seems to cause apoptosis.  I am
> using transient transfections, but the control transfection (empty vector)
> appears to be causing almost as much death as the test transfection (which
> seems to be considerable).  I think that is most probably caused by the
> transfection reagent I am using (Lipofectamine 2000).
> Any suggestions would be appreciated.
> 
> Thanks,
> 
> Marty.
> 
> <http://www.biowww.net/forum/read.php?f=1&i=5344&t=5344>




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