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[anonymous]

Dear All,

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. Where in a plasmid relative to the transgene is the best position to liniarize?

Transfection: Lipofectamine 2000 or Lipofectamine PLUS

Selection: 500 µg/ml G418 medium added the day after transfection.

Regards,

Morten Præstegaard, Ph.D.

[anonymous]

Hi Morten  !Some remarks that come to my mind :1) I am really surprised that you didn't linearize your vector before transfection. To my mind it's the only way to have an efficient integration of your construct. 2) If you don't linearize, integration could happen but in a very small rate. As far as I know, for vizualizing stable fluorescent cells you need more than a single integration per cell (but gene of resistance against G418 could already function !)3) When linearizing, try different sites (if possible...). In my experiments with EGFPN1 (Clontech), I linearize either with Dra III  or AseI and only the second solution gave results...I hope this will help...ALain