Calcium phosphate-mediated plasmid DNA transfection in eukaryotic cells
Calcium phosphate-mediated plasmid DNA transfection in eukaryotic cells
The calcium phosphate-mediated plasmid DNA transfection of adherent cells described in this method is an improvement on the method established by Jordan et al. (1996) Jordan et al. greatly optimized the calcium phosphate-mediated transfection of Chinese hamster ovary cells with human embryonic kidney 293 cells. This experiment is from the next volume of Molecular Cloning Laboratory Guide (3rd edition) by J. Sambrook D.W. Russell.
Operation method
Calcium phosphate-mediated plasmid DNA transfection in eukaryotic cells
Materials and Instruments
Exponentially Growing Mammalian Cells Move makings For more product details, please visit Aladdin Scientific website.
CaCl2 chloroquine Giemsa stain Glycerol HEPES salt buffer Methanol Phosphate buffer Sodium butyrate TE Plasmid DNA Cell growth medium
Tissue Culture Dish (60 mm) or 12-well plate
Buffers & Solutions
The composition of storage solutions, buffers and reagents is shown in Appendix 1.
Dilute the storage solution to the desired concentration.
CaCl2 (2.5mol/L)
Chloroquine (100 mmol/L) (optional)
Dissolve 52 mg of chloroquine diphosphate in 1 ml of deionized water and filter through a 0.22um filter to remove bacteria. Wrap the tube containing the filtrate in foil and store at -20°C. See step 5.
Giemsa stain (10% m/V)
The Giemsa Solution should be freshly prepared in phosphate buffer or water prior to use and filtered through Whartman No. 1 filter paper.
Glycerol (15% V/V) 1XHEPES salt buffer solubilization (optional)
HEPES Salt Solution Filtered for sterilization and add autoclaved 15% glycerol prior to use. See Step 5.
2XHEPES Salt Buffer
140 mmol/L NaCl
1.5 mmol/LNa2 HPO4-2H20
50 mmol/L.HEPES
Dissolve 0.8 g NaCl,0.027 g Na2HPO4-2H20,1.2 g HEPES in 90 ml of distilled water, adjust pH to 7.05 with 0.5 mol/L NaOH, make up the volume to 100 ml with distilled water, and filter with 0.22um filter to remove bacteria; dispense into 5 ml aliquots and store at -20°C. Use within 1 year. Use within 1 year.
Methanol
Phosphate buffer
Filter and sterilize the solution before use. Store at room temperature.
Sodium butyrate (500 mmol/L) (optional)
Adjust butyric acid storage solution to pH 7.0 with 10 mol/L NaOH in a chemical fume hood. filter through a 0.22um filter to remove bacteria; dispense into 1 ml aliquots and store at -20°C, see step 5.
0.1XTE (pH 7.6)
1 mmol/LTrLi-Cl (pH 7.6)
0.1 mmol/LEDTA(pH7.6)
Filter with 0.22um filter to remove bacteria, dispense and store at 4°C.
Nucleic Acids and Oligosiderophores
Plasmid DNA
Dissolve DNA with 0.1XTE (pH 7.6) to a final concentration of 25ug/ml; 50ul of plasmid solution is required per ml of medium. Plasmid DNA should be purified using a folded column (see Scheme 9 in Chapter 1) or CsCl-ethidium bromide gradient centrifugation (see Scheme 10 in Chapter 1) for maximum transformation efficiency. If plasmid DNA is limited as a starting point, vector DNA can be added to bring the final concentration to 25ug/ml. eukaryotic carrier DNA prepared in the laboratory is usually more efficient for transfection than purchased DNA such as calf thymus or salmon sperm DNA. The carrier DNA is sterilized by ethanol precipitation or chloroform extraction prior to use.
Culture media
Cell growth medium [complete medium and (optimized alternative) selective medium].
Specialty Equipment
Tissue culture dish (60 mm) or 12-well plate
This method is for culturing cells in 60 mm tissue culture dishes or 12-well plates. If other plates, flasks, or other diameters are used, vary the concentration of fines and the amount of reagents proportionally. See Table 16-3.
Auxiliary Reagents
The reagents required for Step 6 are listed in Chapter 6, Option 10, and Chapter 7, Option 8.
Cells and Tissues
Exponentially growing mammalian cells
Methods
1. 24 h before transfection, collect the cells by trypsin digestion and spread the cells on 60 mm tissue culture dishes or 12-well plates with appropriate complete medium at a density of 1x105-4x105cells/cm2. Incubate the cells in a 37°C incubator with 5%~7% CO2 for 20~24 h. Change the solution 1 h before transfection.
For high transfection efficiency, be careful to use exponentially growing cells, and the confluence of cells during transfection should not exceed 80%.
2. Prepare calcium phosphate-DNA precipitation as follows: Mix 100ul of 2. 5mol/LCaCl2 with 25ug of plasmid DNA in a 5 ml sterilized plastic tube, and make up the volume to 1 ml with 0.1xTE (pH 7.6) if necessary. mix the above 2x calcium-DNA solution with an equal volume of 2xHEPES salt solution at room temperature. Mix the solution by rapidly tapping the side walls of the tube and let stand for 1 min.
If a large number of cells are transfected, the volume of the precipitation reaction mixture can be doubled or quadrupled, usually 0.1 ml of calcium-DNA phosphate precipitate per 1 ml of medium in the dish, well or cell vial.
3. Immediately transfer the calcium-DNA phosphate suspension to the cell culture medium of the monolayer of cells described above. Add 0.1 ml of suspension per 1 ml of medium. Gently shake the dish to mix the medium, which will turn a cloudy orange color. Once DNA precipitates are formed, the transfection efficiency will be greatly reduced, so this step should be done as quickly as possible. If cells are treated with chloroquine, glycerol and/or sodium butyrate, proceed directly to step 5.
In some cases, higher transfection efficiencies can be achieved by removing the medium and then adding the calcium phosphate-DNA directly to the exposed cells. Then, incubate the cells at room temperature for 15 min, and add the medium again.
4. If the transfected cells are not treated with transfection promoter, incubate the cells in a 37°C incubator with 5%~7% CO2 for 2~6 h, then aspirate the medium and DNA precipitates. Add 5 ml of pre-warmed complete medium at 37°C and return the cells to the incubator for 1~6 days. Continue with step 6 to detect transient expression of transfected DNA or proceed directly to step 7 if the transformation is stable.
5. Treatment of cells with chloroquine in the presence of calcium phosphate-DNA precipitates or exposure of cells to glycerol and sodium butyrate after aspiration of the co-precipitation solution promotes DNA uptake.
Treatment of cells with chloroquine
Chloroquine is a weak base that presumably acts by inhibiting the degradation of DNA by intracellular lysosomal hydrolases (Luthman and Magnusson 1983). The sensitivity of cells to the toxicity of chloroquine limits the concentration and duration of chloroquine added to the medium, and the optimal concentration of chloroquine required for different cells is determined empirically (see information column Chloroquine diphosphate).
a. Add 100 mmol/L chloroquine at 1:1000 directly to the culture medium before or after adding calcium phosphate-DNA precipitates to the cells.
b. Incubate cells in a 37°C incubator with 5% to 7% CO2 for 3 to 5 hours.
Most cells can survive chloroquine for 3-5 h. Chloroquine-treated cells tend to be vesicular.
c. After incubating the cells with DNA and chloroquine, remove the medium, wash the cells with phosphate buffer and add 5 ml of pre-warmed complete medium. Incubate the cells in an incubator for 1~6 days. Continue with step 6 to detect transient expression of transfected DNA or proceed directly to step 7 to obtain stable transformants.
Treat cells with glycerol
This can be done after chloroquine treatment. Due to the different sensitivity of cells to glycerol toxicity, the optimal treatment time (30 s to 3 min) should be tested before use.
a. 2-6 h after addition of calcium phosphate-DNA precipitates to the cells (± chloroquine), aspirate the medium and wash the cell layer with phosphate buffer. b. The cells should be treated with glycerol. c. The cells should be treated with glycerol. d. The cells should be treated with glycerol.
b. Add 1.5 ml of 15% glycerol 1xHEPES salt buffer to each cell dish and incubate the cells at 37°C according to the optimal time tested beforehand.
c. Aspirate off the glycerol and wash the cells once with phosphate buffer.
d. Add 5 ml of pre-warmed complete medium. Incubate the cells in an incubator for 1-6 days. Continue with step 6 to detect transient expression of transfected DNA or proceed directly to step 7 to obtain stable transformants.
Sodium butyrate treatment of cells
The mechanism of action of sodium butyrate is uncertain; however, it is an inhibitor of histone deacetylation (Lea and Randolph 1998), and it is hypothesized that sodium butyrate leads to the peracetylation of histones to form chromatin structures that tend to transcribe foreign plasmid DNA (Workman and Kingston 1998).
The following steps were performed: a. After glycerol shock treatment, 500 mmol/L sodium butyrate was added directly to the growth medium (step d of glycerol treatment). b. After glycerol shock treatment, 500 mmol/L sodium butyrate was added directly to the growth medium. The concentration of sodium butyrate used varies by cell type. Example:
CV-1 10 mmol/L
NIH-3T 37 mmol/L
HeLa 5 mmol/L
CHO 2 mmol/L
Concentrations required for other cell lines depend on experience.
b. Incubate cells in an incubator for 1-6 days. Continue with step 6 to detect transient expression of transfected DNA, or proceed directly to step 7 to obtain stable transformants.
6. To detect transient expression of imported DNA after cell transfection, harvest cells 1-6 days after transfection. Hybridization for analysis of DNA or RNA Newly synthesized proteins are analyzed by in vivo metabolic labeling by radioimmunoassay, immunoblotting, immunoprecipitation, or determination of the enzymatic activity of cell extracts.
To minimize differences in transfection efficiency between dishes, it is best to (1) transfect several dishes with each construct; (2) trypsin-digest the cells after 24 h of incubation; (3) pool the cells; and (4) redeploy the cells on several dishes.
7. Isolation of stable transfectants
a. Incubate cells in non-selective medium for 24~48 h to allow sufficient time for transfected DNA to be expressed. b. Incubate cells in non-selective medium for 24~48 h to allow sufficient time for transfected DNA to be expressed.
b. Trypsin digest the cells and respread the cells in selective medium, or add selective medium directly.
c. Change the medium every 2~4 days for 2~3 weeks to remove dead cell debris and promote the growth of resistant cells.
d. Clone independent colonies, propagate them, and use them in assays (for methods, see Jakoby and Pastan 1979 or Spector et al. 1998b [Chapter 86 of the Handbook of Cellular Experiments]).
e. Cells are fixed with pre-cooled methanol for 15 min, then stained with 10% Giemsa for 15 min at room temperature and rinsed under running water so that the number of cell clones can be recorded.
The dilution of transfected cells is determined according to the stable transfection efficiency, and repopulating cells produces independent clones, which can vary by several orders of magnitude from cell line to cell line (see, e.g., Spandidos and Wilkie 1984). Transfection efficiency is determined by the cell type [even different clones of the same cell line or different numbers of passages may differ significantly (Corsaro and Pearson 1981, Van Pel et al. 1985)], the nature of the introduced DNA and the efficacy of the associated transcriptional control signals, and the amount of DNA used for transfection. 
