Transfection without cytotoxicity
wolfsc at ibms.sinica.edu.tw
Fri Mar 8 06:34:33 EST 2002
Maybe you could reduce the amount of DNA and LF or just the LF. If you don't
want to use transfection reagents (though LFA 2000 has very low cytotoxicity
in my hands, I normally don't change the medium after transfection), you could
use retrovirus and maybe not include polybrene. For your problem, the
generation of a stable producer line could make sense.
Check http://www.stanford.edu/group/nolan/retroviral_systems/retsys.html for
details. (It's the system I am using) together with pLHCX/ pLNCX from Clontech.
3T3 MF are considered to be the "gold standard" for retroviral infection
tests, so your chances are high to get it work. For HeLa you'd need
amprotrophic packaging cells like e.g. PT 67 from Clontech. They have a
comprehensive manual on their website.
> 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.
Dr. Wolfgang Schechinger
Institute of Biomedical Sciences
Academia Sinica, Taipei, Taiwan R.o.C.
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