DEAE-dextran-mediated high-efficiency transfection assay
DEAE-dextran-mediated high-efficiency transfection assay
Two forms of the classical DEAE-dextran transfection method are introduced here. The first one (the main option), in which the cells are exposed to high concentrations of DEAE-dextran for a short time, has a higher transfection efficiency, but is more toxic to the cells. The second (alternative: DEAE-dextran-mediated transfection: enhancement of cell viability), in which the cells are exposed to a lower concentration of DEAE-dextran for a longer period of time, results in a lower transfection efficiency but a higher cell viability. This experiment is from the next volume of the Molecular Cloning Laboratory Guide (3rd edition) by [American] J. Sambrook D.W. Russell.
Operation method
DEAE-dextran-mediated high-efficiency transfection assay
Materials and Instruments
Exponentially growing mammalian cells Move makings For more product details, please visit Aladdin Scientific website.
Chloroquine diphosphate DEAE-dextran phosphate buffer Tris-buffered salt solution with glucose Plasmid DNA Cell growth medium
Tissue Culture Dish
Buffers & Solutions
The composition of storage solutions, buffers and reagents is shown in Appendix 1.
Dilute the storage solution to the desired concentration.
Chloroquine diphosphate (100 mmol/L)
Dissolve 60 mg of chloroquine diphosphate in lml of deionized water. Filter the solution through 0.22um filter paper. Store the filtrate in foil-wrapped test tubes at -20°C.
See Chloroquine Diphosphate in the information panel
DEAE-dextran (50 mg/ml)
Dissolve 100 mg of DEAE-dextran (relative molecular mass = 500,000; Pharmacia) in 2 ml of deionized water. The solution is autoclaved on a 15 pai (1.05 kg/cm2 ) liquid cycle for 20 min. High pressure also aids in the solubilization of the polymer.
The original DEAE-dextran used for transfection had a molecular mass greater than 2x106 (McCutchan and Pagano 1968). This DEAE-dextran is no longer available, but some laboratories occasionally stock it. The transfection efficiency of this old batch of high molecular mass DEAE-dextran is higher than that of the low molecular mass polymers used today.
Phosphate buffer solution (PBS)
Filter and sterilize before use and store at room temperature.
Tris-Buffered Salt Solution with Dextrose (TBS-D)
Add 20% (m/V) glucose to TBS solution (prepared with water, autoclaved or filtered for sterilization) prior to use. The final concentration of radose should be 0.1% (V/V).
Nucleic acids and oligonucleotides
Plasmid DNA
For maximum transfection efficiency, plasmid DNA should be purified by chromatography columns (see Scheme 9 in Chapter 1) or by density gradient centrifugation with CsCl?ethidium bromide (see Scheme 10 in Chapter 1).
Medium
Cell growth medium (complete medium and serum-free medium)
Specialty Equipment
Tissue culture dishes (60 mm or 35 mm)
This method is suitable for culturing cells in 60 mm or 35 mm fertile dishes. If other multiwell plates, cell bottles, or other suitable sizes of Petri dishes are used, change the cell concentration proportionally to the amount of reagents. See Table 16-3.
Additional Reagents
The reagents required for step 8 of this protocol are listed in Chapter 6, Protocol 1 and Chapter 7, Protocol 8.
Cells and Tissues
Exponentially growing mammalian cells 
Methods
1. 24 h before transfection, harvest exponentially growing cells by trypsin digestion and transfer to 60 mm dishes at a density of 105 cells/dish (or 5x104 cells/35 mm dish). Add 5 ml (3 ml for 35 mm dish) of complete medium and incubate the cells at 37°C for 20-24 h with 5%-7% CO2.
Cells should be 75% full grown at the time of transfection. If the cell growth is less than 12 h before transfection, the cells will not be easy to wall, and they can be easily detached after adding DEAE dextran.
2. Prepare DNA/DEAE-dextran/TBS-D solution by mixing 0.1-4ug of superhelical or circular DNA with 1 mg/ml TBS-D dissolved DEAE-dextran.
0.25 ml solution per 60 mm dish; 0.15 ml solution per 35 mm dish.
The amount of DNA required to achieve a high level of transient expression depends on the nature of the construct, and the appropriate amount should be worked out in pre-tests. If the construct carries a replicon that can function in the transfected cells (e.g., SV40 early region promoter/replication start), 100-200 ng of DNA per 105 cells is sufficient; if there is no replicon, more DNA is needed (1ug of DNA per 105 cells).
3. Aspirate off the medium and wash the cells twice with pre-warmed (37°C) PBS and once with pre-warmed TBS-D.
4. Add DNA/DEAE-dextran/TBS-D solution (250 per 60 mm dish 150ul per 35 mm dish). Gently shake the dish so that the solution evenly covers the cell layer. Return the dishes to the incubator and incubate for 30~90 min (the incubation time depends on the sensitivity of each batch of cells to the DNA/DEAE-dextran/TBS-D solution). Every 15-20 min, remove the dish from the incubator, shake it gently, and examine the cell morphology under a microscope. If the cells are still adherent to the wall, continue to incubate. Stop the incubation when the cells start to shrink and become round.
5. Aspirate off the DNA/DEAE-dextran/TBS-D solution. Wash the cells once with pre-warmed TBS-D and once with pre-warmed (37°C) PBS, taking care not to aspirate the transfected cells.
6. Add 5 ml (per 60 mm dish) or 3 ml (per 35 mm dish) of pre-warmed medium containing haematocrit and chloroquine (final concentration 100umol/L) and incubate for 3-5 h at 37°C with a CO2 concentration of 5%-7%.
Treatment of cells with chloroquine increased the transfection efficiency by a fold, probably because chloroquine inhibits lysosomal hydrolases from degrading DNA (Lutbman and Magnusson 1983), but it should be noted that the mixture of DEAE-dextran and chloroquine produced severe cytotoxic effects. Therefore the maximum duration of exposure of DEAE-dextran-treated cells to chloroquine should be determined in a preexperiment (see Chloroquine diphosphate in the information column for details).
7. Aspirate the medium and wash the cells 3 times with serum-free medium. Add 5 ml (per 60 mm dish) or 3 ml (per 35 mm dish) of pre-warmed serum-containing medium and incubate for 36-60 h at 37°C in a solution chamber with a CO2 concentration of 5% to 7%, then assay for transient expression of transfected DNA.
The incubation time of specific cell lines and constructs should be optimized.
8. To detect transient expression of imported DNA in transfected cells, harvest the cells 36-60 h after transfection. RNA or DNA is analyzed by hybridization, and newly synthesized proteins are analyzed by in vivo metabolic markers by radioimmunoassay, immunoblotting, immunoprecipitation, or determination of the enzymatic activity of cell extracts.
To minimize differences in transfection efficiency between dishes, it is preferable to (1) transfect several dishes with each construct; (2) trypsin-digest the cells after 24 h of incubation; (3) pool the cells; and (4) respread the cells on several dishes. 
