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Applications | Stable Cell Line Generation

Stable Cell Line Generation

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, generation of stable cell lines is a viable option. Furthermore, stable cell lines selected through limiting dilution/colony-picking provide a genetically homogenous and clonal population.

A.Stable Cell Line Generation GFPB.Stable Cell Line Generation Data

Stable Cell Line Generation and Characterization. HEK 293 cells stably expressing EGFP were generated through transient transfection of EGFP and neomycin plasmid DNA vectors. Monoclonal populations were selected by limiting serial dilution and gene stability was verified for at least ten passages. Cells were assessed by fluorescence and phase microscopy (A) and flow cytometry (B).

Stable cell line generation is made possible by the use of positive selection markers such as G418, hygromycin B, puromycin resistance, etc.. 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 increase the likelihood 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:

  1. Antibiotic Kill Curve: Titration of selection antibiotic to determine the concentration required to kill cells that are expressing the transgene (1 week)
  2. Transfection: Introduce the plasmid construct(s) into cells (2 days)
  3. Selection and expansion: Determine polyclonal and monoclonal populations of cells (8-10 weeks)
Timeline for Stable Cell Line Generation

Timeline for Stable Cell Line Generation. Stable cell line generation can take a total of 9-12 weeks to establish the cell line. It is ideal to first determine the optimal antibiotic concentration for selection for each cell type; this can take up to 1 week. Following this, the target plasmids can be transfected for two days and antibiotic selection can be applied. Once the cells are under adequate selection, clone picking and expansion is the final and most time consuming part of stable cell line generation. After the stable cell line is generated, it is preferable to verify stable expression from a few cell passages before freezing stocks.

Determining 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; the optimal concentration is cell type dependent. A kill curve is a dose-response experiment where the cells are subjected to increasing amounts of antibiotic to determine the minimum antibiotic concentration 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.

  1. 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: 0.8–3.0 × 105 cells/ml
    • Suspension cells: 2.5–5.0 × 105 cells/ml
  2. 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. Please refer to the antibiotic specific range of concentrations below.
    Selection AntibioticWorking concentration range
    G4180.1 -2.0 mg/ml
    Hygromycin B100 – 500 µg/ml
    Puromycin0.25 – 10 µg ml
  3. Replace media containing 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

 

Antibiotic Kill Curve Illustration

 

G418 Titration for Determining Working Selection Concentration. Increasing amounts of the antibiotic such as G418 (0-1000 µg/ml) is added to duplicate wells of cells plated in complete media in a 24 well plate. Visual toxicity is assessed for upto a week to determine low, optimal and high doses for antibiotic selection.

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 here.

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.

  1. 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
  2. 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).
  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.
  4. 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.

General TransIT Transfection Protocol Workflow Illustration

 

TransIT® Transfection Reagents are Ideal for Virus Production. All TransIT® Transfection Reagents are low toxicity and do not require a media change. Save time and money by adding formed complexes directly to cells in media containing serum and avoiding unnecessary media changes.

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

 

Well No.Antibiotic dose
1No antibiotic
2Low dose of antibiotic
3,4Optimal dose of antibiotic in each well
5,6High dose of antibiotic in each well

 

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

Products for Stable Cell Line Generation

 

TransIT-X2 Transfection Reagent Sample

TransIT-X2® Dynamic Delivery System

A high efficiency, low toxicity transfection reagent for large RNA delivery

  • Efficiency –Exceptional broad spectrum transfection
  • Versatility –Cutting edge delivery of plasmid DNA and small RNAs
  • Technology – Novel, non-liposomal, polymeric delivery

TransIT-LT1 Transfection Reagent Sample

TransIT®-LT1 Transfection Reagent

A low toxicity, broad spectrum DNA transfection reagent

  • Broad Spectrum DNA Delivery – Achieve high expression in many cell types, including hard to transfect cell lines
  • Low Cellular Toxicity – Maintain cell density and reduce experimental biases
  • High Efficiency Delivery – Achieve expression in a large population of cells

TransIT-2020 Transfection Reagent Sample Size

TransIT®-2020 Transfection Reagent

A high performance, animal-free, broad spectrum DNA transfection reagent

  • Broad Spectrum DNA Delivery – Achieve high expression in many cell types, including hard to transfect cell lines
  • Gentle to Your Cells – Balances high efficiency nucleic acid deliver and low cellular toxicity
  • Animal Origin Free – High performance with maximum compatibility
 

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