Method for mammalian cell transfection using 25-hydroxycholesterol
AssigneeThe Regents of the University of California
InventorsDavid H. Spear, Peter A. Edwards
Life of this patent
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Abstract
The present invention relates to a process, a cell culture medium, and a kit for the rapid, high-efficiency transfection of mammalian cells with exogenous DNA. The process comprises incubating a cell culture in the presence of a transfection medium comprising a serum that is different from the serum in the normal growth medium used to grow the cells, and DNA to produce transfected cells. In a preferred embodiment the normal growth medium comprises fetal bovine serum and the transfection medium comprises a serum such as human, calf, horse, lamb, or pig serum. The transfection medium may further comprise an hydroxylated sterol such as 25-hydroxycholesterol.
Claims10
A process for transfecting a mammalian cell with exogenous DNA comprising:
- growing a mammalian cell in a growth medium comprising fetal bovine serum;
- replacing the growth medium comprising fetal bovine serum with a transfection medium comprising a second serum supplemented with 25-hydroxycholestrol, where the second serum is not fetal bovine serum;
- adding a calcium phosphate-DNA precipitate solution to the transfection medium over the cell to form a calcium phosphate-DNA precipitate/medium mixture;
- washing the cell;
- and
- incubating the cell in the presence of a growth medium.
A process for transfecting a mammalian cell with exogenous DNA comprising:
- growing a mammalian cell culture in a medium supplemented with fetal bovine serum;
- removing the medium and supplement from the cell;
- adding a transfection medium supplemented with 10%, by volume, of a serum and 2.5 .mu.g/ml 25-hydroxycholesterol to a mammalian cell culture, wherein the serum is selected from the group consisting of human, newborn calf, calf, horse, lamb, and pig sera;
- adding a calcium phosphate-DNA precipitate solution to the transfection medium over the cell to form a calcium phosphate-DNA precipitate/medium mixture;
- incubating the cell in the presence of the calcium phosphate-DNA precipitate/medium mixture at 35.degree. C. to 37.degree. C. in an atmosphere of 3% to 5%, by volume, carbon dioxide for less than 6 hours;
- washing the cell;
- and
- incubating the cell in the presence of medium supplemented with fetal bovine serum at 37.degree. C. in an atmosphere of 5%, by volume, carbon dioxide.
Dependencies are inferred from the claim text.