Editing 293E Protein Production (section)

=== <br>'''Reagents'''  ===

Cell lines, 293E or 293-6E (NRC-BRI), maintenance cells 

See Culture of HEK293-EBNA1 Cells for Production of Recombinant Proteins (this issue) for preparation. The presence of G418 does not interfere with transfection. 

Use appropriate culture medium listed below, depending on cells used. 

Culture medium for 293E cells (e.g., LC-SFM medium [Invitrogen]) 

Supplement with 1% (v/v) heat-inactivated serum and 0.1% (w/v) Pluronic F-68 prior to use. 

Culture medium for 293-6E cells (e.g., F17 culture medium [Invitrogen] or HyQ SFM4Transfx-293 [HyClone]) 

Add 10 mL of Pluronic F-68 stock solution per liter of culture medium to a final concentration of 0.1% (w/v) prior to use. 

cautionPhosphate-buffered saline (PBS) 

Plasmid DNA of interest, purified 

Prepare using CSH Protocols articles Preparation of Plasmid DNA by Alkaline Lysis with SDS: Maxipreparation or Preparation of Plasmid DNA by Large-Scale Boiling Lysis. 

Plasmid, fluorescent protein-expressing (optional; see Step 6) 

Adding a plasmid encoding a fluorescent protein (e.g., green fluorescent protein [GFP]) to 5% in the transfection mixture does not significantly alter expression of the gene of interest and allows visual (or quantitative, if using flow cytometry) confirmation of transfection efficiency. A few GFP-positive cells can be detected as early as 3-4 h post-transfection using a fluorescence microscope. 

Pluronic F-68, 10% stock solution (w/v) (Invitrogen) 

cautionPolyethylenimine (PEI), linear (1 mg/mL) 

TN1 medium, prewarmed (e.g., FreeStyle medium or LC-SFM containing 20% [w/v] Tryptone N1 [Organotechnie]) (optional; see Step 10) 

Supplement with 0.1% (w/v) Pluronic F-68 and filter-sterilize prior to use.<br>Equipment 

Containers, plastic (e.g., Tupperware) 

Flasks, Erlenmeyer, shake, plastic, disposable (Corning) 

Hemocytometer 

Incubator preset to 37°C, humidified, 5% CO2 

Microscope, fluorescence (optional; see Step 6) 

Paper towels 

Plates, six-well, disposable (Nunc) 

Shaker, orbital 

Tubes, 1.5- or 15-mL 

Vortex mixer<br>