Transgene expression control through ribozyme switch (96-well plate version)¶
Materials¶
Cells
Plates
DMEM supplemented with 10% FBS
PEI
DNA containing the transgene-ribozyme switch cassette
Small-molecule inducer such as theophylline or guanine (check the concentration that you will need)
Procedure¶
Day 1: Seed cells¶
Seed the cells at a density of 2.5-4k cells/well in a 96-well plate
Incubate the cells at 37C overnight
Day 2: Transfection¶
Transfect 100μg of a plasmid expressing transgene-ribozyme switch cassette per well with PEI by following the general transfection protocol. If your small molecule or the solvent has cytotoxicity, using a lower PEI:DNA ratio of 3:1 is recommended.
Incubate the cells at 37C for 4-6 hours
Remove the media from the wells carefully to not lose the cells, and add 100μl of fresh media that contains the small-molecule inducer
Incubate the cells overnight
Note
If you are using ON-switch, where the ribozyme activity is inactivated by the small molecule (stabilizing the transcript), you don’t need to treat the cells with the small molecules at 4-6 hpt, you can do this next day.
If you are using OFF-switch, where the ribozyme activity is activated by small the molecule (destabilizing the transcript), you MUST do the small-molecule treatment at 4-6 hpt so that transcribed mRNA binds with the molecule before being translated to protein.
Note
Be careful when changing the media for 293T cells—they easily detach, especially in a 96-well plate.
Day 3 or 4¶
After 24 hours post small-molecule treatment, you should be ready for analysis via microscopy or flow cytometry.
A 24-hour incubation with the small molecule should be generally enough to measure the output transgene expression, but longer incubations of 48 or 72 hours may improve detection.