Inducible OGT Knockout Cells

We have generated a cell line in which OGT can be deleted upon addition of 4-hydroxytamoxifen (Kazemi et al., 2010). We are happy to share this cell line; however, we are required to issue an MTA. Your request should be sent to Dr Zachara ([email protected]). Requests for the MEFs carrying the LoxP flanked OGT gene should be made to Jamey Marth (UCSD).

Breaking out the cells: If you break these cells out into a 150mM dish, they are typically 80-90% confluent in three days. At this point we make freezedowns, as these cells lose their transgene slowly during passaging. We do not use cells past passage 20.
  • Thaw cells rapidly at 37oC
  • Plate into complete media (DMEM 1g/L glucose, 10% FBS, Pen-Strep).
  • Maintain in a water jacketed incubator in 5% CO2 at 37oC
  • Replace media at 6-8 hours (once the cells have attached to the plate)
Freezing cells down: Cells are resuspended at 1x10^6 cells per ml in 5% DMSO in DMEM 1g/L glucose, 10% FBS, Pen-Strep. Cells are aliquoted into cryotubes and placed in a Freezing Container (Mr. Frosty, PC, clear, for 18 x 1,2/2,0 ml Tubes, with blue Lid). The container is placed at -85oC for 24hours, before the cells are transferred to liquid nitrogen storage.

  • Day 1 (am): Split cells at 5x10^5 cells per 100mm plate (experimental) and 1x10^6 cells per 150mm plate (stock).
  • Day 1 (pm, no less than 6h post-split): Add 4-hydroxytamoxifen (4OHT; to 500nM) or ethanol (vehicle).
  • Day 2 (pm, 24h post step 2): Aspirate media and replace with 10mls (experimental) or 20mls (stock) of DMEM 10% FBS, Pen/Strep.
  • Day 3: Initiate experiment. Your experiment should finish no later than the time you added the 4HT on day 1.
Other considerations:
  • These cells have been maintained continuously in DMEM 1g/L glucose, 10% FBS, Pen-Strep.
  • Higher doses of 4HT, or prolonged doses of 4HT, have off target effects leading to decreased OGT and O-GlcNAc levels. Thus: 1) it is important to wash out the 4OHT; 2) Use no use more than 500 nM 4OHT; and 3) it is important to maintain the GFP control cell line and determine that your desired phenotype is NOT observed in these cells when treated wit 4OHT.
  • Cells exhibit a growth defect and significant cell death after 48h. Experiments should be initiated and finished before this time-point.
  • Both cell lines should fluoresce green, allowing you to determine the % transfection. Over time we notice that fewer cells are carry the transgene, the only solution is to break out a new “low passage number” freeze down.
Relevant product numbers:
  • DMEM, low glucose, pyruvate (Gibco, 10-014-CV)
  • FBS (Sigma, F4135)
  • Penicillin Streptomycin (Corning, 30-001-CI )
  • 4-hydroxytamoxifen (Sigma, H6278)
Key citations:
This is the paper describing stable transduction of the OGT F/y MEFs with Cre-ERT2-GFP or GFP:
Kazemi et al., 2010, PMID: 20926391

This is the paper describing the generation of the OGTf/y MEFs
O’Donnell et al., 2004, PMID: 14749383