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      High-efficiency transformation of mammalian cells by plasmid DNA.

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      Molecular and Cellular Biology

      American Society for Microbiology

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          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.

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          Author and article information

          Journal
          Molecular and Cellular Biology
          Mol. Cell. Biol.
          American Society for Microbiology
          0270-7306
          1098-5549
          August 01 1987
          August 1987
          August 1987
          August 01 1987
          : 7
          : 8
          : 2745-2752
          10.1128/MCB.7.8.2745
          367891
          3670292
          © 1987
          Product

          Molecular medicine, Neurosciences

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