Science & tech · 1987
Can a calcium phosphate protocol half of mouse L(A9) cells?
Yes. Under the reported conditions, 50% of mouse L(A9) cells were stably transformed with the pcDneo marker . Circular DNA was effective, while linear DNA was almost inactive.
Details
The researchers described a simple calcium phosphate protocol and neo marker vectors for highly efficient transformation of mammalian cells. During incubation with the cells, the calcium phosphate–DNA complex gradually forms in the medium and precipitates on the cells.
Crucial factors for efficient transformation were the pH of the buffer used for calcium phosphate precipitation (6.95), the CO2 level during incubation of DNA with the cells (3%), and the amount and form of DNA (20–30 micrograms, circular). In sharp contrast to circular DNA, linear DNA was almost inactive.
Under these conditions, 50% of mouse L(A9) cells could be stably transformed with pcDneo, a simian virus 40-based neo marker vector conferring neomycin resistance. NIH3T3, C127, CV1, BHK, CHO, and HeLa cell lines were transformed at efficiencies of 10–50% using this vector and neo marker-incorporated pcD vectors. The latter were used to construct and transduce expression libraries and to express cloned cDNA in mammalian cells.
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