Calcium Phosphate-DNA Co-Precipitation Method:
The calcium phosphate-DNA co-precipitation technique is a widely used method for introducing DNA into mammalian cells. In this process, the DNA forms a precipitate with calcium phosphate, which can then adhere to the cell membrane and be internalized by the cell. However, only about 1% to 5% of the DNA enters the cytoplasm, and less than 1% of that makes it to the nucleus. Of that small fraction, only a tiny portion integrates into the host genome, leading to stable expression. The transfection efficiency is generally around 10-4, making this method suitable for both transient and long-term gene expression studies. It is particularly effective for adherent cells and is one of the most commonly used methods in molecular biology.
Materials Required:
1. Reagents Preparation:
(1) 2x HBS: Dissolve 1.63g NaCl, 1.19g Hepes, and 0.023g Na2PO4.2H2O in water to make 100ml. Adjust pH to 7.1 and store at 4°C.
(2) 2mmol/L CaCl2: Prepare and filter sterilize.
(3) TE Buffer: 0.1mmol/L EDTA and 1mmol/L Tris-HCl (pH 8.0).
(4) G418 Solution: Dissolve 1g G418 in 1mmol/L Hepes solution, dilute to 10ml with water, filter sterilize, and store at 4°C.
(5) G418 Selection Medium: Prepare DMEM with 10% FBS and add G418 to a final concentration of 200–800 mg/L. Before use, test the appropriate concentration that kills over 50% of the cells within 10–14 days.
2. Experimental Procedure [Method 1]:
(1) Donor DNA Preparation: Isolate DNA using standard extraction methods and dissolve it in TE buffer at a concentration of 40 µg/mL.
(2) Culturing Recipient Cells: For oncogene transfer, choose cell lines without human Alu sequences, such as NIH3T3 mouse fibroblasts. Seed cells at 2 × 104/cm2 in DMEM with 10% FBS, incubate at 37°C, 5% CO2, until they reach 50–70% confluence.
(3) Preparing DNA-Calcium Phosphate Precipitate:
• Mix 200 µL donor DNA (40 µg/mL) with 220 µL of 2×HBS and 250 µL of PSV2-neo plasmid DNA (20 µg/mL). PSV2-neo is typically used at 1–2 mg/L.
• Add 3.1 mL of 2 mmol/L CaCl2 to 500 µL of the above mixture, mix gently for 30 seconds, and let stand at room temperature for 30 minutes.
(4) Transfecting the Cells:
• Replace the culture 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 and mix gently.
• Incubate at 37°C, 5% CO2 for 24 hours or longer to allow DNA uptake.
• Replace the medium and continue culturing for 24 hours to induce gene expression.
• Switch to G418 selection medium (800 mg/L) for screening. Include non-transfected control cells.
• After 3–5 days, most control cells will die. Replace with 200 mg/L G418 and change the medium every 3–4 days.
• After 2 weeks, drug-resistant clones appear. Expand and clone these cells to establish a stable cell line.
In this experiment, both the PSV2-neo plasmid and the foreign DNA are co-transfected. This allows the recipient cells to acquire neomycin resistance, enabling the selection of successfully transfected cells. Even if the introduced oncogene does not show immediate transformation, the presence of the neo marker can still be detected. This method is useful for establishing cell lines through G418 selection and is especially valuable for studying gene function and expression in mammalian systems.
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