Transfection Of Cells With DNA
28 Jul 04, SP
2. Transient transfection efficiency
3. About stable transfection
4. Transient transfection using calcium phosphate
5. Transient transfection using lipid-based reagents like Fugene and Lipofectamine
6. Generating stable lines
Transfection or the introduction of DNA into eukaryotic cells can be achieved using a variety of methods. E.g., retroviruses can be used to transduce or transfer DNA into the cell's genome (DNA gets integrated). Electroporation and calcium phosphate- and liposome -based deliveries are other methods. The former is easy to perform on cells in suspension while the other two methods are convenient to use with adherent cells. Electroporation however requires more number of cells. Some methods such as the DEAE dextran method, though very good for transient transfection, are inefficient for producing stable cells lines. Electroporation usually leads to stable integration of a single copy of the gene while the other methods usually lead to the integration of multiple copies of the gene (often in tandem and on different chromosomes, which increases the chances of chromosomal rearrangements).
Transient transfection efficiency
In general, any cell that grows can be transfected. However, the transfection efficiency varies from cell type to cell type and from method to method. A high efficiency is needed for experiments such as expression cloning and protein purification. Efficiency may not be optimal and still be okay if, for example, one is merely interested to check if a protein is made at all.
Low transient transfection efficiency may be compensated for by transfecting more cells.
Transfection efficiency depends on, among other things, the passage number (age) of cells, presence of serum, antibiotics, etc., in medium, etc.
Transient transfection efficiency for CHO (W5) cells of calcium phosphate methods is 5-10x less than lipid-based transfection methods.
The DNA that is introduced into the cells can be linear or circular (plasmids). Plasmids cannot propagate in eukaryotic cells unless they are integrated (inserted in chromosmes) in the genome. As a result, and also because of degradation, transfected plasmid DNA dilutes out over 5-6 rounds of cell replication. The transient transfectants can be cultured for longer duration during which stable integration can take place. Integration is a rare phenomemon (1 in 10e4 for mammalian cells), but if there are markers for it, one can select stable cell lines.
Usually the presence of a drug resistance gene (such as one to inactivate neomycin or G418) on the plasmid allows for such selection. If the expression plasmid does not have a selection gene, one can cotransfect a second plasmid that has the selection gene. The cotransfection is done in 5:1 (plasmid with gene of interest: that with the selection gene) molar ratio.
However, drug resistance necessarily does not mean the protein of interest is indeed expressed. One therefore has to pick many clones and confirm protein expression by western analysis, etc.
Some plasmids allow for generation of a fusion, bicistronic message. In such cases, the gene of interest is transcribed but in the absence of a termination signal, the transcription from the plasmid extends beyond the insert and another gene such as GFP that is downstream gets transcribed as well. These transcripts are bicistronic (two coding regions: gene of interest and GFP in the example). An IRES or internal ribosomal entry site upstream of the second gene (GFP) allows the translation of the second coding region (GFP) independently of translation of the first one. A clone that expresses high GFP thus suggests high expression of the first protein as well (indirectly, both proteins coming from same transcript). High expressors can thus be FACS-sorted from the drug resistant, stable population.
Generation of a stable cell line takes many weeks.
Transient transfection using calcium phosphate
Protocol for 293T cells in 10 cm dish
Scale up or down depending on size of dish. Works well for other cell lines such as CHO and 3T3. Performed inside culture hood. Thanks to Kyoungja Hong
All solutions (store at room temperature) should be sterile. Filter before storing aliquots at -20 deg or room temperature. Over time, the pH of solutions might change. For unknown reasons, HBS solutions older than about 6 months should not be used.
1 or 2.5 M calcium chloride
2x HBS (HEPES buffered saline) - pH 7.05-7.10 (crucial; adjusted with NaOH), 280 mM NaCl, 50 mM HEPES (16.4g NaCl and 11.9g HEPES in 1l water)
100x phosphate (Na2HPO4) - 75mM (10.5g in 1l water)
1. At time of transfection, cells should be 40-50% to nearly 100% confluent - they should not be touching each other. A higher surface area is needed for better transfection efficiency. The dish should have 7-9 ml medium. Calcium phosphate based transfection does not work well if using RPMI culture medium.
2. Add 10 ul phosphate solution to 500 ul HBS in a plastic tube (a 10 or 15ml tube is good).
3. In a separate tube, add 120 (48 if using 2.5 M stock) ul calcium chloride to DNA (12ug) in water (volume such that the final volume at the end of this step is 500 ul, with the calcium chloride). The DNA can be in 1 mM or 10 mM Tris buffer but, then, if it is too dilute, its addition may affect the pH and reduce transfection efficiency.
4. Above volumes can be scaled up in the same tube if transfecting more than one dish.
5. Add HBS-phosphate solution dropwise to the DNA-calcium tube while vortexing at low speed (1 or 2 of dial). You can also keep flicking the tube while adding HBS - the important thing is contiuous mixing. Make sure all solutions are at 22-25 deg C and not colder.
6. Let the solution stand for 10-20 minutes at room temperature.
7. Vortex at high speed for 2-3 seconds.
8. Pipet dropwise, all over, the cell culture dish. Swirl the dish. Incubate. If possible, incubate in a low CO2 (2-4%) incubator till step 9. If you have made solution for more than one dish, vortex again for 2-3 seconds before picking the mix to put on the second plate and so on.
9. Aspirate medium after 16 (12-24) hours and add fresh medium.
Depending on cell type and your need, the time can vary from 4-24 hours. Some cells cannot survive long exposure to the calcium-DNA precipitate. You may rinse the dish in step 9 with PBS before adding fresh medium. Shocking some cells (such as some CHO lines) at step 9 may dramatically increase transfection efficiency. To do so, 4-6 hours after transfection, 10% glycerol containing medium (2ml) is layered on the cells after aspiration. After 3 minutes, 5ml PBS is layered and the dish swirled. Everything is aspirated and the dish is rinsed twice with PBS before fresh medium is added.
Calcium phosphate transfection, tough easy, can become finicky and fail. Most common reason for that is change in pH of the HBS. Over time, CaCl2 can also go bad. To quickly test if these solutions are okay, mix 500ul of the 2X HBS and 10ul phosphate solution with 500ul 250mM CaCl2. A fine precipitate that is visible under the microscope should form.
The BES transfection method uses an alternate buffer system and is very effective (10-50%) at generating stable lines (but only equally effective for transient transfection).
2.5M CaCl2 - 183.7g CaCl2.2H20 in 500 ml water - filter
2x BES - 50mM N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, 280mM NaCl, 1.5mM Na2HPO4 (pH 6.95) - adjust final pH to 6.95 with 1N NaOH [for 200 ml, add 2.14g BES to 150 ml deionised, distilled water. After adding NaCl and Na2HPO4, pH will be around 5.75. Add 1.3 ml 2N NaOH to bring pH to 6.95. Add water to final 200 ml.]
Aliquots that can be rethawed and refrozen can be stored at -20 deg.
1. Add 50 ul CaCl2 to DNA (20-30ug) in water so final volume is 500 ul.
2. Add 500 ul of 2x BES. Mix and let stand at RT for 10-20 min.
3. Add dropwise to 10cm plates and swirl.
4. Incubate in 3% CO2 at 34-35 deg for 12-24h.
5. Rinse with PBS, add medium and incubate as normally for 2-3 days.
Transient transfection using lipid based reagents
A number of reagents (Fugene, Lipofectamine, etc.) are available, most of them work well with most cell lines (CHO, 293T, etc.). It is best to follow the manufacturer's suggested protocol (see 'Product manuals and inserts' section of this wiki for PDF documents).
In general, these transfections are inefficient in the presence of serum and antibiotics. Some reagents, like Lipofectamine 2000, are more toxic to some cells like CHO (thus transfection should be performed at 90-100% confluence).
Typical method for CHO cells in 35mm dish, using Fugene 6 or Lipofectamine 2000
Scale up or down depending on dish size.
1. Cells should be 40-60% (Fugene) or 80-100% (Lipofectamine) confluent.
2. In a 1.5ml eppendorf tube, pipet 100ul (Fugene) or 250 ul (Lipofectamine) serum free medium (alpha MEM, OptiMEM, etc.).
3. Add the 6ul (Fugene) or 10ul (Lipofectamine) reagent to the tube and mix by gently flicking the tube.
4. Incubate at RT for 5 but no more than 30 minutes.
5. In separate tube with 100ul (Fugene) or 250ul (Lipofectamine) serum free medium, pipet 1ug (Fugene) or 4ug DNA (Lipofectamine) [if cotransfecting in 1:1 ratio, use 2ug each of two plasmids, of approximately same size, and not 4ug each] and mix by flicking.
6. Mix the two solutions by pipeting DNA into the reagent solution.
7. Incubate for 15-25 minutes at room temperature.
8. Meanwhile, aspirate medium from dish and rinse atleast once with serum free medium. Add 0.5ml (Lipofectamine) or 0.8ml (Fugene) serum free medium to the dish.
9. Add the DNA-reagent solution from step 7 to the dish.
10. Incubate for 4-24 hours (usually 12 hours).
11. Aspirate old medium and add 2ml fresh medium with serum and antibiotics as desired.
Generating stable lines
Some cells cannot grow as single a single cell; i.e., they need to be in a colony or be surrounded by other cells. It is generally impossible to get such single cells to grow into a stable cell line.
After selection for stable transfectants, the entire surviving population can be used as a whole or individual clones may be isolated. Not all surviving cells express the gene of interest. Thus, a stable population is guaranteed to have expression of your gene, though at a lower level, while an isolated clone may not express the gene at all or may express it at very high levels. Isolated clones thus need to be analyzed for expression levels.
A stable population may be sorted (by FACS for example) to get rid of non or low expressors.
1. Transient transfect as above.
2. After final step, allow cells to grow for 24 hours.
3. Aspirate old medium and add fresh medium containing the selection agent (G418, hygromycin, etc.). For CHO cells, we use 0.5-1.5mg/ml G418 or 0.2-0.5 mg/ml hygromycin B. For other lines and/or antibiotics, check with others.
4. It will take 2-3 generations before cells start dying (appear as floating under microscope). Cells that have the selection marker (and thus hopefully the gene of interest) integrated in them will survive. Keep growing the cells for 10-14 days (for CHO), changing medium (with selection agent) every 3-4 days. Observe them under microscope to rule out any contamination with microbes.
5. After 10-12 days, colonies should be visible. When they are a mm or so in diameter (500-1000 colonies), they can be picked up. Alternately, all colonies can be scraped or trypsinized off the plate and replated in a new dish or moved to rollers (for suspension cultures) if a stable population is desired..
6. Picking a colony is required if aiming for individual clone isolation. You need to pick up atleast 10 clones.
— After last step of transient transfection, allow cells to grow for 24 hours or so. Then remove the cells by scraping or trypsinization. Count the density of cells.
— Plate multiple 10 cm dishes with varying number of cells - 1000, 10,000, 100,000 and so on. If transfection efficiency was good, enough colonies, that are physically well separated from each other, will be obtained in the smaller-number dishes. Grow for 10-12 days. Cells that loosely adhere to each other and to the plate may get dislodged from their colony (and form too many satellite colonies or contaminate other colonies) if the plates are disturbed too much while handling during this period.
— To pick up, carefully aspirate medium. Then rinse two times with medium (taking care not to dislodge colonies).
— Layer 10 ml medium (for 10 cm dish) and use the tip of a glass Pasteur pipette (attached to a rubber suction bulb) to scrape over a colony 4-5 times while gently aspirating (cells being dislodged will be aspirated).
— Decant the cells into 4-6ml polypropulene tubes making final volume to 2ml with medium (with selection agent).
— After 5-7 days, a layer of cells should have grown at the bottom of the tubes.
— Resuspend the cells after trypsinization (see protocol elsewhere in this wiki) and transfer to 10ml rollers.
7. As soon as possible, freeze atleast 2 vials of cells (isolated clones or stable populations).
PEI transfection CHO (by Hung-Hsiang)
Plate at 3.4x106 cells in your choice of medium, 37 deg., o/n so that it will cover more than 80 % of the 10 cm plate. The reason of seeding cells at high density is for getting large amount of your target protein by transient transfection. If you just want to have stable cell line by PEI transfection, I will go down to 1.7x106 per 10 cm plate.
(Tube A) Prepare DNA and mix with 500 ul (alpha-MEM+10 % FBS or serum free medium or saline buffer)
(Tube B) Prepare another 1.5 microtube and add 500 uL of same medium as in no. 1, then add suitable amount of PEI directly into medium.
Keep DNA:PEI as ratio of 1ug:3ul. 6-well: 1 ug/transfection
Add everything in Tube B to Tube A. Vortex.
Incubate for 15 min at RT.
Replace with fresh medium w/o antibiotic prior to transfection.
Add the DNA-PEI complex dropwise into the plate and mix well but gently
Incubate 37 deg, 24 h or longer.
Next day check transfection efficiency by western blot or other methods. After o/n transfection, you will observe some cells floating which is normal due to the cell high seeding density.