- Calcium phosphate transfection is a widely used and cost-effective method for introducing plasmid DNA into eukaryotic cells. It is particularly favored for its simplicity and affordability, especially in academic research settings. This method works by creating a fine precipitate of calcium phosphate that traps the DNA molecules. When this precipitate is added to cultured cells, the DNA is taken up via endocytosis, allowing for either transient or stable gene expression depending on the experimental design and selection conditions.
- The success of calcium phosphate transfection depends heavily on precise control of certain parameters, most notably the pH of the buffer used. The transfection mixture is typically prepared by combining the plasmid DNA with a calcium chloride solution, and then carefully adding this mixture to a HEPES-buffered saline solution (2× HBS). This step must be done slowly and with proper mixing to allow the formation of a fine, homogeneous precipitate. The ideal pH of the 2× HBS is around 7.05 to 7.12; even slight deviations can significantly reduce transfection efficiency or increase toxicity.
- Once the precipitate has formed, it is added dropwise to the cells, which should be at approximately 70–80% confluency to ensure optimal uptake and minimal toxicity. The cells are then incubated, usually at 37°C with 5% CO₂, for several hours. Depending on the sensitivity of the cells, the medium may be replaced after 6 to 8 hours to minimize cytotoxic effects. Gene expression from the transfected DNA can typically be observed within 24 to 72 hours, using reporter assays or protein detection methods.
- After endocytosis, the DNA-calcium phosphate precipitates are trapped within endocytic vesicles. For successful transfection, the DNA must escape these vesicles to reach the cytoplasm and eventually the nucleus. The mechanism of escape is not fully understood but is believed to involve the buffering capacity of calcium phosphate, which can raise the pH inside the vesicles. This pH shift may destabilize the vesicle membrane or interfere with lysosomal acidification, leading to vesicle rupture. Additionally, mechanical stress from the growing precipitate and osmotic swelling may contribute to membrane disruption. These events enable the release of DNA into the cytosol, from where it can enter the nucleus, particularly during mitosis when the nuclear envelope temporarily disassembles.
- Although calcium phosphate transfection is highly efficient in certain cell types like HEK293 and COS cells, it can be variable or ineffective in others, particularly primary or suspension cells. Moreover, the method can sometimes be toxic, especially if the precipitate is too coarse or if the buffer conditions are not optimal. Nevertheless, with careful optimization and proper controls, calcium phosphate remains a valuable technique for DNA delivery in molecular and cell biology research.
