Applications: Stable Cell Line Generation

Select and Expand Clones Post-Transfection

1. At 48-72 hours post transfection, add the selection antibiotic at the high, optimal and low dose to each of the transfected T75 flasks
2. As a control to assess the antibiotic response side by side in non-transfected 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 (see table below)
3. Change media containing selection antibiotic 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. Since the efficiency of stable integration into the genome is quite low, surviving cells should be allowed to expand in the T75 flask to ensure that the selected clones are not unstable
4. Keep replacing media containing selection antibiotic twice a week until the cells in the T75 flask reach high confluence. At this point, they can be frozen down as a polyclonal line

Polyclonal to Monoclonal Selection

The polyclonal cell culture can be further processed to isolate monoclones using different techniques such as:

• Limiting dilution: The goal using this method is to isolate each individual cell that carries selection by plating them at very low cell densities (< 1 well per well in 96 well plates) and expand colonies from those single cells in separate wells. This is a cost-effective yet tedious process; additionally, certain cell types do not survive the limited dilution step due to a need for secreted factors from neighboring cells. An alternative in such cases is to use conditioned media and/ or 2X serum to increase cell attachment and survival. Occassionally, culturing cells in semi-solid media such as soft-agar and methylcellulose might help, particularly in case of suspension cells
• Cloning rings and trypsin discs: If limited dilution does not work, the cloning process might need to be carried out at higher cell concentrations and repeated a few times to ensure monoclonality. Using cloning rings and/or trypsin discs is a viable option for adherent cells in this scenario. In this method, selected cells are seeded sparsely but not at limiting dilution in 10 cm dishes and allowed to expand and form discernible colonies for 2-3 weeks. The individual colonies can then be trypsinized and transferred to another smaller culture vessel using either cloning rings or trypsin discs for monoclonal expansion
• Fluorescence activated cell sorting (FACS): This method can be employed if a detectable marker is expressed on the cells post-transfection. Single cells can be isolated using FACs and replated to generate a monoclonal lineage
• Automated clone picking: More sophisticated instrumentation based methods, e.g. ClonePix™ technology (Molecular Devices) allows a completely automated process of high producer clone identification and expansion

Of all the above-mentioned methods, limiting dilution is the most cost-effective and frequently adopted technique; a detailed protocol using limiting dilution to generate monoclonal cell lines is as follows:

Identify single clones by limiting dilution and expansion

1. 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)
2. Assess the number of cells per well after 18-24 hours and note the wells with only 1 cell
3. 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
4. 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. Repeating the limited dilution step 3-4 times ensures that there are no false-positive monoclones

Transfer clones and assess expression

1. 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)
2. 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
3. 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

1. 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
2. Once expanded, freeze down cell stocks using appropriate freezing medium lacking the selection antibiotic. A common freezing medium is 10% Dimethyl Sulfoxide (DMSO) plus normal growth medium
3. 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

STEP 1: Antibiotic Kill Curve

STEP 2: Stable Transfection

For stable cell line generation, use Mirus' high quality cell-culture grade selection antibiotics that are easy-to-use:

All Mirus' broad spectrum plasmid DNA transfection reagents can be used for stable cell line generation. Visit the product pages for more information on each product:

• TransIT-X2® Dynamic Delivery System
• TransIT®-2020 Transfection Reagent
• TransIT®-LT1 Transfection Reagent

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