Calcium phosphate coprecipitation

Various techniques for neuronal transfection have been reported including viral transfer, DNA/calcium-phosphate coprecipitation, electroporation, microinjection, biolistics, and liposomes. Although the ideal transfection method has not been developed, we describe a neuronal transfection method with LF2000, a commercially available cationic lipid reagent. This method yields consistent transfection efficiency without any time consumption, experienced culture technique, or cell toxicity. 

Calcium phosphate coprecipitation was one of the first chemical methods widely used to transfer DNA into ceUs in culture. To accompHsh this, calcium chloride and a phosphate buffer such as HEPES-buffered saHne or BES-buffered saHne is mixed with DNA. The result is a precipitate that distributes throughout the ceU culture. Uptake of the precipitate, and consequently the DNA, is beHeved to be achieved through phagocytosis [12]. An important issue affecting transfection efficiency for this method is the pH of the phosphate buffer solution. The pH of the buffer should be maintained between 6.95 as in BES-buffered saHne and 7.1 as in HEPES-buffered saHne. In addition, the quaHty of the precipitate must be uniform and removed after incubation. Calcium phosphate coprecipitation works for both transient and stable transfections in numerous ceU Hnes, both adherent and suspension. 

Calcium phosphate coprecipitation was one of the first chemical methods widely used to transfer DNA into cells in culture. To accomplish this, calcium chloride and a phosphate buffer such as... 

Walters RW, Duan D, Engelhardt JF, Welsh MJ. Incorporation of adeno-associated virus in a calcium phosphate coprecipitate improves gene transfer to airway epithelia in vitro and in vivo. J Virol 2000 74 535-540. 

Walters R, Welsh M. Mechanism by which calcium phosphate coprecipitation enhances adenovirus-mediated gene transfer. Gene Ther 1999 6 1845-1850. 

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