DNA transfection experiment using Polybrene
DNA transfection experiment using Polybrene
The Polybrene and DMSO transfection method described below is a modification of the method of Aubin et al. (1997). Using this method, plasmid DNA can be efficiently transfected into Chinese hamster ovary cells and keratinized cells, producing 15 times more transformants than the calcium phosphate-DNA precipitates method. However, there was no significant difference in transfection efficiency between the two methods when high molecular weight DNA was used. This experiment is from the next volume of Molecular Cloning Laboratory Guide (3rd edition) by J. Sambrook and D.W. Russell.
Operation method
DNA transfection experiment using Polybrene
Materials and Instruments
Exponentially growing mammalian cells Move makings For more product details, please visit Aladdin Scientific website.
DMSO(30%) Serum-containing medium Giemsa stain Methanol Polybrene Sodium butyrate DNA to be transfected
Minimum basal 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.
DMSO (30%)/serum-containing medium
Add HPLC-pure or tissue culture-pure DMSO to serum-containing cell growth medium to a final concentration of 30% (V/V) before using DMSO in Step 3.
Giemsa stain (10% m/V)
Prepare with phosphate buffer salt or water prior to use and filter through Whatman No. 1 filter paper.
Methanol
Polybrene (10 mg/ml)
Dissolve Polybrene (Aldrich) in water to a final concentration of 10 mg/ml, sterilize by filtration through a 0.22 mm filter, dispense into small aliquots (0.25 ml), store at -20°C and discard after use.
Sodium butyrate (500 mmd/L) (optional)
In a chemical fume hood, adjust the sodium butyrate storage solution to pH 7.0 with 10mol/L NaOH, remove bacteria by filtration through a 0.22um filter and store at -20°C in 1ml aliquots.
Nucleic acids and oligonucleotides
Dissolve DNA to be transfected, e.g. plasmid DNA, in water to 1ug/ul.
Medium
Minimum Essential Medium (MEM)-α (medium with 10% fetal bovine serum, serum-free medium with selective medium)
Special equipment
Tissue Culture Dish (90 mm)
This method is for culturing cells in 90 mm dishes. If other multiwell plates, cell bottles, or other diameters are used, change the cell concentration proportionally to the amount of reagent used. See Table 16-3.
Additional Reagents
Step 6 of this protocol may require reagents listed in Chapter 17, Protocol 7 or Protocol 1 Auxiliary Protocols.
Cells and Tissues
Exponentially growing mammalian cells
Methods
1. Collect cells (e.g. CHO cells) by trypsin digestion and spread each 5X105 cells on a 90 mm tissue culture dish with 10 ml of MEM-α medium containing 10% fetal bovine serum. The cells were incubated for 18-20 h in a 37°C incubator with 5%~7% CO2 .
2. Replace the original medium with 3 ml of warm medium containing serum, DNA (5ng~40ug, no carrier DNA) and 30ug Polybrene. Mix the DNA with the medium and then add 10 mg/ml Polybrene, and continue to incubate the cells for 6-16 h. Shake the dish every 90 min during the early stage of incubation so that the cells are evenly exposed to the DNA-Polybrene mixture.
3. Aspirate off the medium containing DNA-Polybrene and add 5 ml of medium containing 30% DMSO with serum. Shake the medium gently so that the cells are evenly exposed to the solution and place the dish in an incubator.
4. Incubate for 4 min, remove the dish from the incubator and quickly aspirate off the DMSO medium. Wash the cells once or twice with warm serum-free medium and add complete medium containing 10% fetal bovine serum. If sodium butyrate is required to facilitate transfection, proceed to step 5, if not, incubate the cells in a 37°C incubator with 5% to 7% CO2 for 48 h. Then proceed directly to step 6 (detection of transient transfection) or 7 (stable transfection).
DMSO-treated cells are easy to detach, so be as gentle as possible when discarding solvent-containing medium and adding new medium, e.g., by pipetting the medium along the side wall of the dish during each change.
5.(Optional) To promote transfection of DMSO with Polybrene-treated cells:
a. Add 500 mmol/L sodium butyrate to the culture medium to a final concentration of 2.5~10 mmol/L. b. Add 500 mmol/L sodium butyrate to the culture medium to a final concentration of 2.5~10 mmol/L.
The exact concentration of sodium butyrate depends on the cell type.
b. Incubate the cells in a 37°C incubator with 5%~7% CO2 for 20~24 hours.
c. Discard the sodium butyrate-containing medium and replace it with butyrate-free medium containing 10% fetal bovine serum. The cells were incubated in an incubator.
Sodium butyrate promotes the transient (but not sustained) expression of certain recombinant plasmids in DMSO-treated cells (Aubin et al. 1997), particularly plasmids with the SV40 early promoter/enhancer in ape and human elks (Gorman et al. 1983a).
6. If cells are to be stably transfected, proceed directly to step 7. For transient expression, assay the cells 1-2 days after transfection as described below:
- If E.coli.β-galactosidase-expressing plasmid DNA is used, assay for enzyme activity in the cell lysate according to the procedure outlined in Scheme 7 in Chapter 17. Alternatively, follow the histochemical staining method described in the additional protocol in Scheme 1 of this chapter.
- If a green fluorescent protein expression vector is used, examine the cells with a microscope under 450-490 nm light.
- If other gene products are expressed, the newly synthesized proteins are analyzed by in vivo metabolic markers by radioimmunoassay, immunoblotting, immunoprecipitation, or by measuring 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) digest the cells with trypsin after 24 h of incubation; (3) pool the cells; and (4) redeploy the cells on several dishes.
7. Isolation of stabilized transfectants: After incubating the cells with non-selective medium for 48 h [to allow expression of transferred genes (step 4)], trypsin digest the cells or resurface the cells with appropriate selective medium, or selective reagents can be added directly to the medium. Fluid changes are made every 2 to 4 days for 2 to 3 weeks with the aim of removing dead cellular debris and allowing the growth of resistant cell clones.
8. Thereafter, clones are cloned and independent clones are propagated for use in the assay [for methods, see Jakoby and Pastan 1979 or Spector et al. 1998b (Chapter 86 of the Handbook of Cellular Experiments)].
Cells should be fixed in pre-cooled methanol for 15 min and then stained with 10% Giemsa for 15 min at room temperature, rinsing with running water, so that the number of clones can be recorded.The Giemsa staining solution should be freshly prepared in phosphate buffer or water and filtered through Whatman No. 1 filter paper before use.