Chen C, Okayama H
Laboratory of Cell Biology, National Institute of Mental Health, Bethesda, Maryland 20892.
Mol Cell Biol. 1987 Aug;7(8):2745-52. doi: 10.1128/mcb.7.8.2745-2752.1987.
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.
我们描述了一种简单的磷酸钙转染方案和新霉素标记载体，可实现哺乳动物细胞的高效转化。在该方案中，磷酸钙-DNA复合物在与细胞孵育期间于培养基中逐渐形成，并沉淀在细胞上。获得高效转化的关键因素是用于磷酸钙沉淀的缓冲液的pH值（6.95）、DNA与细胞孵育期间的二氧化碳水平（3%），以及DNA的量（20至30微克）和形式（环状）。与环状DNA的结果形成鲜明对比的是，线性DNA几乎没有活性。在这些条件下，50%的小鼠L(A9)细胞可用基于猿猴病毒40的新霉素（新霉素抗性）标记载体pcDneo进行稳定转化。使用该载体以及用于构建和转导cDNA表达文库以及在哺乳动物细胞中表达克隆cDNA的掺入新霉素标记的pcD载体，NIH3T3、C127、CV1、BHK、CHO和HeLa细胞系的转化效率为10%至50%。
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