Calcium Phosphate Transfection
Calcium phosphate co-precipitation has been a popular transfection method since its introduction in the early 1970s (Graham and van der Eb, 1973) because the components it requires are easily available and inexpensive. Furthermore, the technique is easy to master, it is effective with many types of cultured cells, and it can be used for both transient and stable transfection of a variety of cultured cell types. However, calcium phosphate co-precipitation is prone to variability due to its sensitivity to slight changes in pH, temperature, and buffer salt concentrations, and can be cytotoxic to many types of cell cultures, especially of primary cells. In addition, it is unsuitable for in vivo transfer of nucleic acids to whole animals, and it shows relatively poor transfection efficiency compared to other chemical transfection methods such as lipid-mediated transfection.
The principle of calcium phosphate co-precipitation involves mixing DNA with calcium chloride in a buffered saline/phosphate solution to generate a calcium-phosphate–DNA co-precipitate, which is then dispersed onto cultured cells. Calcium phosphate facilitates the binding of the condensed DNA in the co-precipitate to the cell surface, and the DNA enters the cell by endocytosis. Aeration of the phosphate buffer while adding the DNA-calcium chloride solution helps to ensure that the precipitate that forms is as fine as possible, which is important because clumped DNA will not adhere to or enter the cell as efficiently.
Mix DNA with calcium chloride and add in a controlled manner to a buffered saline/phosphate solution.
Incubate at room temperature to generate a precipitate of extremely small, insoluble particles containing condensed DNA.
Add the DNA-calcium phosphate co-precipitate to cells, which adhere to the cell membrane. The co-precipitate enters into the cytoplasm via endocytosis.
Assay cells for transient gene expression or select for stable transfection.
Calcium-phosphate co-precipitation workflow.