Conditional OPN Knockdown in MDA-MB-231 Cells

Parent breast cancer MDA-MB-231 cells and subclones were cultured in RPMI 1640 medium (Invitrogen, Karlsruhe, Germany) supplemented with 10% fetal calf serum (FCS), 100 ng/mL doxycycline (added to deactivate the tetracycline-controlled transactivator, tTA), 2 mM l-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin (Invitrogen, Karlsruhe, Germany) in standard cell culture flasks (TPP, Trasadingen, Switzerland) in a humidified incubator at 37 °C and 5% CO₂. The human breast adenocarcinoma cell line MDA-MB-231 was obtained from ATCC (Manassas, VA, USA).
Two breast cancer cell clones with conditional OPN knockdown (O1 and O2) were generated by recombinase-mediated cassette exchange (RMCE) from parent MDA-MB-231 cell clone by using plasmid constructs and by following a previously-described procedure [15 (link)].