Stable Transfection of NIH-3T3 and 3T3-L1
Morten Præstgaard
We are trying to make stable transfectants in NIH 3T3 and 3T3-L1 cells. We
are successful in getting our transgene (GFP-fusions) into the cells.
However, after some time under selection (G418 mostly) we loose the
expression of our GFP-fusion, but retain resistance to G418. Does anyone
have suggestions how to avoid this problem?
Conditions:
Plasmid: CMV-promoter, G418 resistance - we do not liniarize, but this is
one of the things we want to try next.
Transfection: Lipofectamine 2000 or Lipofectamine PLUS
Selection: 500 5g/ml G418 medium added the day after transfection.
Regards,
Morten Prfstegaard, Ph.D.
Research Scientist
Direct Tel: +45 44437524
BioImage A/S
Moerkhoej Bygade 28
2860 Soeborg
Denmark
Tel: +45 44433444
Fax: +45 44437505
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Frank O. Fackelmayer
The CMV promoter is known to sometimes be inactivated by DNA
methylation. I´m not sure if the nature of the expressed protein can
influence inactivation, but I guess cells are clever enough to shut down
expression of a protein they don´t like... Inactivation by DNA
methylation takes some time, so you might get good expression in the
beginning, but upon subcloning of stable transfectants expression may go
down. If that is the reason for your problem, there is little you can do
about it but exchange the promoter with a different one.
Of course it is also possible that your gene is destroyed upon
integration into the plasmid. As you do not linearize, the plasmid may
open up at any point, thereby affecting your gene in several cases. Of
course this problems gets worse with the size of your insert. There will
always be clones, however, where the plasmid opens at "neutral" sites,
giving you at least some high expressers.
Hope this helps,
Frank
