I'm having troubles maintaining my fluorescence, either RFPtag or ZsGreen, expression after a few days under selection with G418. I'm working with MCF7, T47D and NIH3T3. They transfect ok...not as nice as HEL293. However, the fluorescence in MCF7, T47D and NIH3T3 fades after a couple of days under selection whereas in HEK293 is quite strong. As two side notes: 1) the fluorescence in MCF7, T47D and NIH3T3 starts lower than in HEK293 (~30-40% lower), and 2) the promoter is CMV from pCMV6-Entry vector from Origene. The tags are C-terminal and correctly in frame. The fluorescence fades even without a cloned gene (empty vector). Any help or suggestion is very much welcomed. Thanks!
You may be experiencing some unwanted cytotoxicity from your fluorescent proteins. Over time this will cause selection for the cells with lower expression levels. I definitely documented this in an earlier study called "A noncytotoxic DsRed variant for whole-cell labeling" (http://www.nature.com/nmeth/journal/v5/n11/full/nmeth.1264.html), with both stably and transiently transfected mammalian cells. You may have success with different fluorescent proteins, such as superfolder GFP or DsRed-Express2.
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