Transfection of Rodent Myelomas

Dr S.M. Hobbs hobbs at icr.ac.uk
Tue Oct 13 06:37:14 EST 1998


Hello,

Does anyone have a reliable protocol for stable transfection of rodent
myeloma cell lines (NS1, NS0,Y0 etc)?  This seems to be a standard
technique in everyone else's lab except mine.  I've tried every
variation and combination that I can think of: transfection methods,
vectors, promoter/selectable marker combinations, linear vs supercoiled
DNA, feeder cells, media, you name it I've tried it.  Occasionally a
transfection will work well and I get lots of clones but the vast
majority of the time I get absolutely nothing.  A typical
electroporation transfection protocol that I use would be 10^7 cells in
0.8ml serum-free DMEM (also tried PBS) + 10ug supercoiled plasmid DNA
prepared with a QIAGEN endotoxin-free maxiprep kit (although others have
been tried), 10 minutes on ice then a pulse in a 0.4mm cuvette on a
BioRad Genepulser at 250V 960uF, back on ice then plate out on a 24 well
plate.  Allow to grow for 48hr then select with e.g. 1ug/ml puromycin
(although I've tried G418 and XHAT depending on the vector used) feeding
occasionally.  If clones appear they are visible within 1 week. The
cells grow very well in the medium used (DMEM + 10% heat-inactivated
FCS) and when the occasional clone appears they are relatively hardy,
but this is an all too infrequent event.  At one point I had three
transfections, all identical, performed about two days apart with the
same cells/vector etc: one worked giving 50+ clones, the other two
produced nothing.  It does seem to be an all-or-nothing event.

I suspect now that it has something to do with the phase of the moon
when the transfection is carried out, I've run out of other variables. 
If anyone has any suggestions to solve this apparently intractable
problem before I send for the feng shui man to check out the tissue
culture lab I shall be eternally grateful.

Thanks,

Steve Hobbs

hobbs at icr.ac.uk



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