Hello everyone,
I m testing a transcription factor Y for its ability to repress a well known target promoter X. In so doing, When I co-transfect the plasmid containing the promoter X (expressing for a destabilized fluorescent protein: the reporter gene) and the trascription factor Y, the experiment works well!
When I generate a stable cell line that stably carry the promoter X and I transiently transfect in this cells the transcription factor Y in order to observe a repression (decrease of the fluorescence) the experiment does not work! Someone can help me?
Thx
Marco
Two initial ideas to consider:
1. In transient transfection, only a small percentage of the cells are transfected, but most take up both plasmids. Repression works. When you transfect the stable line, all the cells have the reporter, but only a small percentage take up the repressor plasmid. You can't detect repression above the signal from the majority of the cells.
2. In selecting for the stable, the promoter for the reporter gene changed and/or is affected by the position of integration.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Cell Biology
- Culture Cells
- Cell Line