Transfect Cells with Target Plasmid Construct(s)
While performing the kill curve (1 week), optimize transfection conditions in a T75 flask by transfecting a reporter plasmid (such as a GFP encoding plasmid) into cells at high confluence, e.g. 80%. Determine the appropriate dose of plasmid (5-15 µg) and transfection reagent (15-45 µl) in a T75 flask. Observe reporter gene expression (GFP) and toxicity at regular time points over at least a 48 hour period, optimally for 72 hours. Specific tips on optimizing DNA transfection can be found at Tips from the Bench - Optimize DNA Transfection. Use the optimal DNA and transfection reagent dosage for generating stable transfectants.
Optional: Linearize your target plasmids before transfection. When generating a stable cell line, the transfected plasmid undergoes recombination during chromosomal integration. The recombination event can occur within any region of the plasmid, including the gene expression or selectable marker cassettes that might disrupt their function. To increase the likelihood that recombination will occur in non-essential plasmid regions, such as the bacterial replicon or bacterial marker gene, linearize the plasmid with restriction enzyme(s) that cut within these non-essential regions. Prior to transfection, purify the linearized DNA by ethanol precipitation, size exclusion or column purification.
- For each individual stable cell line to be created, plate cells in three T75 flasks and one 6-well tissue culture plate approximately 18–24 hours before transfection such that they reach high confluence (~60-80%) at the time of transfection. Typical cell density ranges are as follows:
- Adherent cells: 0.8–2.4 × 105 cells/ml of complete media
- Suspension cells: 3.2–4 × 105 cells/ml of complete media
- Leave the 6-well tissue culture plate untransfected. This will serve as an untransfected control and is important as a reference during the selection process (Step 3).
- Transfect the plated cells with 5-15 µg of total plasmid DNA per T75 flask. If the antibiotic selection marker is on a separate plasmid than the gene of interest, then maintain a 10:1 ratio of “gene of interest” plasmid over “antibiotic selection” plasmid.
- Do not expose cells to the selection antibiotic until 48-72 hours post transfection to avoid low cell viability. A media change can be performed at 24 hours post transfection, if needed.
The above procedure works well for routinely transfected cell types. For hard-to-transfect cells, another method to generate stable cell transfectants is via lentivirus or retrovirus transduction. In this case, antibiotic resistance harboring virus particles generated after transfection of producer cell types such as HEK293T are used to transduce cells that can then be selected for virus integration. Details on virus production can be found here.