help in transiently transfect cultured B- and T- cell lines

Thomas Chiles chilesT.hermes.bc.edu
Fri Feb 10 13:10:04 EST 1995


In article <D3FFMp.6K at acsu.buffalo.edu>, jtyl at ubvms.cc.buffalo.edu wrote:

> I would like to hear experiences on how to transiently transfect
cultured B- and T- cell lines.  Thanks very much.
> 
> Joseph Lau
> lau at sc3101.med.buffalo.edu

Dear Joe, 
I can only comment on B cells.  Primary cultures of murine B cells are
very resistant to many conventional transfection methods.  My lab (and
others) has found lipofection, DEAE-dextran, electroporation etc. does not
work.  However, John Monroe has recently published a paper in Mole. Cell
Biol. 15(2)1086 in which he and Steve McMahon have successfully employed a
DEAE-dextran method for the transfection of plasmid-reporter genes into
primary B cells.  However, they first had to produce B cell blasts (LPS
stimulation for 72 hr.). These cells can then be transfected and rested
for 24 hr (which affords arrest in G1 phase of the cell cycle), and then
stimulated with anti-IgM or PMA.  We have used this method and it works.
Second, transformed B cell lines are usually easy to transfect.  Tom
Rothstein and I have published a method in J. Immunol. 149:825. that
describes AP-1-dependent reporter gene studies in the murine BAL-17 B
lymphoma cells.  This DEAE-dextran method also works in WEHI-231 cells. 
We have also found that B cells contain de-acetylases and thus once you
obtain cell extracts after transfection, you should heat the extract to
75oC for 20 min (if you are monitoring CAT activity). David Scott has
published several papers that employ antisense oligonucleotides as a means
to block c-myc expression in,I believe, murine CH 2 cells.

Hope this helps.

Thomas Chiles
Boston College
ChilesT at hermes.bc.edu



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