Dynamin conditional knockout fibroblasts: Tamoxifen inducible Knockout method
Pietro De Camilli, Daehun Park
Abstract
This cell line was described and characterized in the following paper: Ferguson, S.M., Raimondi, A., Paradise, S., Shen, H., Mesaki, K., Ferguson, A., Destaing, O., Ko, G., Takasaki, J., Cremona, O., O’Toole, E., De Camilli P. Coordinated actions of actin and BAR proteins upstream of dynamin at endocytic clathrin-coated pits. Developmental Cell 17, 811-822, 2009. PMID: 20059951]. This procedure describes tamoxifeninducible KO method using this cell line.
Attachments
Steps
Protocol
Culture the cells at 37°C and 5% CO2 in DMEM containing 10% FBS, 100U/ml penicillin, 100mg/mL streptomycin.
When cells reach 80-90% confluency, detach cells from the dishes using trypsin-EDTA and split the cells 1:4 and add 2micromolar (µM) 4-hydroxy-tamoxifen along with fresh culture medium. Incubate the cells for 48h 0m 0s (48 hours). A dish with no 4- hydroxy-tamoxifen is used as a control.
After 48h 0m 0s, split the cells once again to prevent cell overcrowding. Add 300nanomolar (nM) 4-hydroxy-tamoxifen for 72h 0m 0s.
Check the depletion of dynamin using immunofluorescence or Western blot.
