Rapalog-induced chemical dimerization experiments

To perform chemical-induced dimerization (CID) experiments we will first establish cell lines expressing the FRB-Fis1 and FKBP-GFP-GOI (FKBP-GFP fused to our gene of interest).

HeLa cells are consecutively transduced with lentivirus for FRB-Fis1, which also expresses mitochondrially targeted monoKeima (mt-mKeima), and then with FKBP-GFP-GOI. See the lentivirus transduction protocol for further details.

Once cell lines are established, cells are seeded in 6-well plates the day before the experiment (density: 800k per well).

Induce FKBP-FRB dimerisation by treating the cells with 50nanomolar (nM) Rapalog A/C hetero-dimerizer rapalog (635057, Takara) for 24h 0m 0s.

After 24h, collect cells by trypsinization and take the cells to the FACS facility for mt-mKeima analysis.

Analyze the cells by flow cytometry, using the mt-mKeima ratio (561/405) as a readout with an LSR Fortessa Cell Analyzer (BD Biosciences).

Gate the cells for GFP/mt-mKeima double-positive cells, and collect 10,000 events for this population.

Analyze data using FlowJo.