Calcium Phosphate-DNA Co-precipitation Method:
The calcium phosphate-DNA co-precipitation technique is a widely used method for transfecting adherent mammalian cells. In this approach, DNA forms a precipitate with calcium phosphate, which then attaches to the cell surface and is internalized by the cell. Only a small fraction (1–5%) of the internalized DNA reaches the nucleus, and less than 1% is integrated into the host genome, leading to stable expression. The gene transduction efficiency is typically around 10-4. This method is versatile and can be applied to any DNA fragment, making it suitable for both transient and long-term gene expression in mammalian systems.
1. Reagents Preparation:
(1) 2x HBS: Dissolve 1.63 g NaCl, 1.19 g Hepes, and 0.023 g Na2PO4·2H2O in water to make 100 mL. Adjust pH to 7.1 and store at 4°C.
(2) 2 mmol/L CaCl2: Prepare using sterile filtration.
(3) TE Buffer: 0.1 mmol/L EDTA and 1 mmol/L Tris-HCl, pH 8.0.
(4) G418 Solution: Dissolve 1 g of G418 in 1 mmol/L Hepes solution, dilute to 10 mL, filter sterilize, and store at 4°C.
(5) G418 Selection Medium: Add G418 to DMEM with 10% FBS at a final concentration of 200–800 mg/L. It is important to pre-test the optimal G418 concentration that kills more than 50% of the recipient cells within 10–14 days.
2. Experimental Procedure [Method 1]:
(1) Donor DNA Preparation: Extract DNA using standard methods and resuspend it in TE buffer at a concentration of 40 μg/mL.
(2) Culturing Recipient Cells: Choose cell lines without human Alu sequences, such as NIH3T3 mouse fibroblasts. Seed cells at 2 × 104 cells/cm² in DMEM with 10% FBS. Incubate at 37°C, 5% CO2 until they reach 50–70% confluence, usually one day before transfection.
(3) Preparing DNA-Calcium Phosphate Precipitates:
1. Mix 200 μL of donor DNA (40 μg/mL) with 220 μL of PSV2-neo plasmid DNA (20 μg/mL) and 250 μL of 2x HBS.
2. Transfer 500 μL of the mixture to a silanized tube and slowly add 3.1 mL of 2 mmol/L CaCl2, mix gently for 30 seconds.
3. Incubate at room temperature for 30 minutes. The solution will become slightly cloudy, indicating successful precipitation. Use immediately for transfection.
(4) Transfecting Recipient Cells:
1. Replace the medium 4 hours before transfection with fresh DMEM. Add 0.5 mL of the DNA-calcium phosphate precipitate to a 5 mL culture in a 25 mL flask.
2. Incubate for 24 hours or longer to allow DNA uptake.
3. Replace the medium and continue culturing for another 24 hours to induce gene expression.
4. Switch to G418 selection medium (800 mg/L) and include non-transfected control cells.
5. After 3–5 days, most control cells should die. Change the selection medium to 200 mg/L G418 every 3–4 days.
After two weeks, drug-resistant clones should appear in the transfected cells. These clones are expanded, cloned, and further characterized to establish stable cell lines.
In this experiment, both PSV2-neo and the target DNA were co-transfected. This allows the cells to acquire neomycin resistance, enabling selection even if the oncogene does not show immediate transformation. The presence of the neo marker also facilitates the detection of successfully transfected cells. By introducing PSV2-neo, researchers can efficiently select and expand transformed cell lines using G418, providing a powerful tool for functional studies.
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