Expression of functional, recombinant mammalian proteins often requires expression in mammalian cells (see Single Cell Cloning of a Stable Mammalian Cell Line). If the expressed protein needs to be made frequently, it can be best to generate a stable cell line instead of performing repeated transient transfections into mammalian cells. Here, we describe a method to generate stable cell lines via electroporation followed by selection steps.
This protocol will be limited to the CHO dhfr– Urlaub et al. (1983) and LEC1 cell lines, which in our experience perform the best with this method.
Electroporation is a popular technique to introduce foreign DNA into host cells. The DNA enters the cells following a quick electric pulse that generates temporary openings in the cell membrane. After electroporation, selection steps must be followed to allow for cells containing only the target DNA to grow.
While outside the scope of this protocol, we have listed some other cell lines and appropriate selection markers in Table 17.1 . Stable cell lines also can be generated by other transfection methods (e.g., using lipid-like transfection reagents or calcium phosphate transfection. See also Rapid creation of stable mammalian cell lines for regulated expression of proteins using the Gateway ® Recombination Cloning Technology and Flp-In T-REx ® lines) followed by appropriate selection steps.
Summary of selection method
| Cell type | Transfection method | Selection markers | Round 1 selection | Days until die-off begins | Round 2 selection |
|---|---|---|---|---|---|
| CHO dhfr– | Electroporation | pSV2-dhfr | Alpha MEM, 5% D-FBS | 12–14 | Alpha MEM, 5% D-FBS, MTX (0.1–0.4 mM) |
| LEC1 | Electroporation | pcDNA3.1, pSV2-dhfr | Alpha MEM, 5% FBS, 0.5 mg ml −1 geneticin | 15–17 | Alpha MEM, 5% D-FBS, MTX (0.025–0.1 mM) |
| HEK 293 GnTi- | PEI | pcDNA3.1 | DMEM:F12, 5% FBS, 2 mg ml −1 geneticin | 7–10 | N/A |
| CHO-S | PEI | pcDNA3.1 | DMEM:F12, 5% FBS, 0.5 mg ml −1 geneticin | 7–10 | N/A |
Two different selection methods are described here for the first round of selection immediately following electroporation. The first selection method relies on co-transfection with the pcDNA3.1 vector, which carries the neo gene that confers resistance to geneticin, an aminoglycoside antibiotic (Southern and Berg, 1982). It is generally not necessary for the gene of interest and the selectable marker to be on the same plasmid. Positively transfected cells are selected by growth in the presence of geneticin. The second selection method relies on dihydrofolate reductase (DHFR) activity (Wigler et al., 1980) and its inhibitor methotrexate (MTX). DHFR selection works best with the cells lacking DHFR activity, such as CHO dhfr–. But cells having wild-type DHFR activity can also be selected with high MTX concentrations ( Table 17.1 ). The DHFR/MTX selection step can be repeated with higher concentrations of MTX until a desirable level of protein expression is achieved.
Stable cell lines often lose their protein expression with time as a result of a heterogeneity in the transfected population of cells. A more homogeneous population of cells can be obtained by limiting dilution cloning or picking individual colonies of drug-resistant cells.
MEM α (containing Earl’s Salts and L-glutamine, but no ribonucleosides, deoxyribonucleosides, NaCO3; Invitrogen 12000)
DMEM/F12 (with L-glutamine, but no HEPES, NaHCO3; Invitrogen 12500) Freestyle™ 293 medium (Invitrogen 12338-026) Hybridoma SFM (Invitrogen 12045) pcDNA 3.1 (Invitrogen) pSV2-dhfr (ATCC)Note Some of the stock solutions come with the pH indicator phenol red. This supplement does not affect the application and might be useful if the researcher wishes to visualize any pH changes that can occur in the solutions over time. In the case of non-CO2 incubators (e.g., when scaling-up the production of adherent cells in roller bottles), HEPES-buffered media can be used to keep the pH stable.
Note Catalog numbers are from the US website of Invitrogen and may differ on other local websites.
Step 2 Lec1 Growth Medium: Alpha MEM + 5% FBS
| Add 50 ml FBS to 1 l of Alpha MEM |
CHO dhfr– Growth Medium: Alpha MEM + 5% FBS + HT solution
| Add 50 ml FBS and 10 ml of 100× HT solution to 1 l of Alpha MEM |
Steps 4–6 50 mg ml −1 Geneticin ® (active)
| Add enough active Geneticin to 50 ml of water to make a 50 mg ml −1 stock solution. Each lot of Geneticin will have a different active concentration. For example, if a 5 g bottle of Geneticin has an active concentration of 750 μg mg −1 of powder, it contains an active weight of 3.75 g Geneticin. Therefore dissolve the 5 g of powder in 75 ml of water to obtain an active concentration of 50 mg ml −1 . Mix to dissolve, pass through a 0.2 mm filter to sterilize, and dispense aliquots into sterile tubes. Store short term at 4 °C or long term at −20 °C |
Lec1 Selection Medium
| Add 50 ml FBS and 10 ml of 50 mg ml −1 Geneticin to 1 l of Alpha MEM |
CHO dhfr– Selection Medium
| Add 50 ml D-FBS to 1 l of Alpha MEM |
Step 6 30 mM MTX
| Component | Final Concentration | Stock | Amount |
|---|---|---|---|
| Methotrexate | 30 mM | 0.136 g | |
| Alpha MEM | 9.5 ml | ||
| NaOH | 50 mM | 1 M | 0.5 ml |
Mix well until MTX is dissolved and pass through a 0.2 μm filter to sterilize. Store in sterile aliquots at −20 − C
| Add 33.3 μl of 30 mM MTX to 1 ml Alpha MEM |
MTX selection concentrations
| Component | Final concentration | Stock | Amount |
|---|---|---|---|
| MTX | 25 nM | 1 mM | 12.5 μl |
| MTX | 50 nM | 1 mM | 25 μl |
| MTX | 75 nM | 1 mM | 37.5 μl |
| MTX | 100 nM | 1 mM | 50 μl |
| MTX | 200 nM | 1 mM | 100 μl |
| MTX | 300 nM | 1 mM | 150 μl |
| MTX | 400 nM | 1 mM | 200 μl |
| MTX | 500 nM | 1 mM | 250 μl |
| MTX | 1 μM | 1 mM | 500 μl |
Add the indicated amount of MTX to 500 ml Alpha MEM containing 5% FBS
Before transfection, sterile high-quality DNA must be prepared. The vector containing the appropriate expression promoter (see Molecular Cloning) and the gene of interest should be transformed into a recA- strain of E. coli (see Transformation of Chemically Competent E. coli or Transformation of E. coli via electroporation) and then the plasmid DNA isolated (see Isolation of plasmid DNA from bacteria). Commercially available, endotoxin-free kits for large-scale plasmid DNA isolation produce sufficiently high-quality DNA. High-quality DNA is characterized as having an OD260/280 ratio between 1.88 and 1.92, an OD260/230 ratio of 2.1–2.2, and a concentration above 0.5 mg ml −1 (see Explanatory Chapter: Nucleic Acid Concentration Determination).
All steps are carried out using sterile technique in a laminar flow hood. Solutions should be sterile filtered through 0.22-μm filters. All plastic and glassware, if not purchased as sterile, should be autoclaved twice. Cell growth media are warmed to 37 °C prior to contact with cells. Before electroporating, cells should have undergone at least five passages from thawing. Grow LEC1 or CHO dhfr– cells to 80% confluency in a T75 flask. One T75 flask at 80% confluence yields enough cells for 1–2 electroporations. No residual trypsin can be present during electroporation.
| Preparation | 1 week |
|---|---|
| Protocol | ~1–3 months |