High-efficiency transformation of mammalian cells by plasmid DNA.

TLDR

The study presents a simple calcium phosphate transfection protocol and neo marker vectors designed to achieve highly efficient mammalian cell transformation. The protocol forms a calcium phosphate–DNA complex gradually in medium, precipitating on cells, with optimal efficiency at pH 6.95, 3 % CO₂, and 20–30 µg circular DNA. Under these conditions, circular DNA yields 10–50 % transformation in various cell lines, including 50 % in mouse L(A9) cells, whereas linear DNA is almost inactive.

Abstract

We describe a simple calcium phosphate transfection protocol and neo marker vectors that achieve highly efficient transformation of mammalian cells. In this protocol, the calcium phosphate-DNA complex is formed gradually in the medium during incubation with cells and precipitates on the cells. The crucial factors for obtaining efficient transformation are the pH (6.95) of the buffer used for the calcium phosphate precipitation, the CO2 level (3%) during the incubation of the DNA with the cells, and the amount (20 to 30 micrograms) and the form (circular) of DNA. In sharp contrast to the results with circular DNA, linear DNA is almost inactive. Under these conditions, 50% of mouse L(A9) cells can be stably transformed with pcDneo, a simian virus 40-based neo (neomycin resistance) marker vector. The NIH3T3, C127, CV1, BHK, CHO, and HeLa cell lines were transformed at efficiencies of 10 to 50% with this vector and the neo marker-incorporated pcD vectors that were used for the construction and transduction of cDNA expression libraries as well as for the expression of cloned cDNA in mammalian cells.