Most cell biology experiments utilize transient transfection protocols that afford peak gene expression between 24-96 hours post transfection. However, if sustained gene expression is required for longer periods of time, generation of stable cell lines is a viable option. Furthermore, stable cell lines selected through limited dilution provide a genetically homogenous and clonal population.
Stable cell line generation is made possible by the use of positive selection markers such as hygromycin, G418/Geneticin, zeocin, and blasticidin antibiotic resistance. Selection markers can be delivered using the same plasmid that contains the gene of interest (in cis), or on a separate plasmid (in trans) that needs to be co-transfected with the plasmid containing the gene of interest. The cis approach is generally easier and has a higher likelihood of producing drug-resistant stable transfectants that express the gene of interest. The trans method of co-transfection is a good alternative in instances where the target construct does not have the antibiotic-resistance gene in the vector backbone. In such cases, a plasmid mixture containing 5 to 10 parts gene expression plasmid and 1 part antibiotic selection marker plasmid can be introduced into cells. This plasmid ratio helps ensure that the selected cells will express both the gene of interest and the selection marker.
Stable cell line generation protocol
The protocol for generating stable cell lines requires several steps as shown below:
- Generate a kill curve to determine the optimal selection antibiotic concentration
- Transfect cells with desired plasmid construct(s)
- Select and expand stable polyclonal colonies
- Identify single clones by limited dilution and expansion
- Transfer clones and assess expression
- Expand and freeze down high expressing clones
Generate a kill curve to determine the optimal selection antibiotic concentration
The first critical step for stable cell line generation is determining the optimal antibiotic concentration for selecting stable cell colonies. A kill curve is a dose-response experiment where the cells are subjected to increasing amounts of antibiotic to determine the minimum antibiotic concentration that is needed to kill all the cells over the course of one week. Performing a kill curve is recommended with each new cell type or when a new selection antibiotic or different lot of selection antibiotic is used.
- Plate cells in 0.5 ml complete growth medium per well in a 24-well tissue culture plate one day prior to introducing antibiotic selection. Ideally cells should have reached high confluence (~60-80%) prior to adding the selection antibiotic. Typical cell density ranges are as follows:
- Adherent cells: 0.4–1.2 × 105 cells/well.
- Suspension cells: 1.6–2 × 105 cells/well.
- Add increasing amounts of the appropriate antibiotic such as G418 to duplicate wells of cells plated in complete media. Include a no-antibiotic control. For example, add 0, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, and 1000 µg/ml selection antibiotic to duplicate wells of cells plated in complete growth media.
- Replace media with selection antibiotic every 2-3 days for up to a week. Examine the culture every day for signs of visual toxicity. Determine the following antibiotic doses:
- Low dose - the antibiotic concentration at which minimal visual toxicity is apparent even after 7 days of antibiotic selection
- Optimal dose - the lowest antibiotic concentration at which all cells are dead after one week of antibiotic selection
- High dose - the antibiotic concentration at which visual toxicity is evident within the first 2-3 days of antibiotic selection (all cells dead by 7 days)
Transfect cells with desired 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. 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 here. 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 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.
- 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.
Select and expand stable polyclonal colonies
- At 48-72 hours post transfection, add the selection antibiotic at the high, optimal and low dose to each of the transfected T75 flasks.
- As a control to assess the antibiotic response side by side in untransfected cells in the 6-well tissue culture plate, add the antibiotic at the same concentrations that are used for the T75 flasks (as indicated in the table below). Include a no antibiotic control.
| Well No. |
Antibiotic dose |
| 1 |
No antibiotic |
| 2 |
Low dose of antibiotic |
| 3,4 |
Optimal dose of antibiotic in each well |
| 5,6 |
High dose of antibiotic in each well |
- Change media every 2-3 days. Examine the cells for visual toxicity daily. Typically, most of the cells that have not integrated the transfected plasmid will die while the cells that have undergone plasmid integration will survive by 9 days post-transfection. Surviving cells should be allowed to expand in the T75 flask.
- Replace media with antibiotic twice a week. When the cells in the T75 flask reach high confluence, they can be frozen down as a polyclonal line. Cells can also be plated for selection of single cell clones using the protocol below.
Identify single clones by limited dilution and expansion
- Plate the polyclonal cells from the selection step at a density of 10 cells/ml in a 96-well tissue culture plate adding 100 µl per well (i.e., 1 cell per well).
- Assess the number of cells per well after 18-24 hours and note the wells with only 1 cell.
- After the 4th day, assess the number of colonies per well in the wells that only had one cell at the initial assessment. Assume each colony is clonal. Only wells with 1 colony per well should be considered monoclonal.
- After these wells are identified, continue to verify colony number every week until the well has reached high confluence. Note: If a monoclonal population is highly critical for your experimental set-up, the dilution step can be repeated a second time.
Transfer clones and assess expression
- Expand selected single-colony wells in the 96-well tissue culture plate to high confluence and transfer to a 12-well tissue culture plate. GFP positive clones could be assessed in the 96-well tissue culture plate, but others should not be assessed for expression yet (depending on the reporter assay).
- Once the 12-well tissue culture plate clones have expanded to high confluence, they can be passaged to a 6-well tissue culture plate. A small portion of the cells should be assessed for expression of the target protein at this time point.
- Propagate a small portion of selected cells for 50-90 doublings to confirm stability of expression by verifying expression of the target gene at multiple time points.
Expand and freeze down high expressing clones
- Once expression is verified, clones of interest can be scaled up to larger volumes (e.g. a T75 flask). Depending on the cell type, most single cell clones should reach high cell densities by 2 weeks (e.g. in HEK 293 cells); some slow growing clones can take up to 4 weeks for complete expansion.
- Once expanded, freeze down cell stocks using appropriate freezing medium lacking the selection antibiotic.
- Upon establishing your target monoclonal stable cell line, a lower amount of antibiotic can be used for maintenance. It is critical to follow the passage number of stable cell lines since the stability of clonal cell lines might vary. Some clones may lose expression after several passages. Freeze down samples from early passage to prolong their use after thawing.