One way of physically introducing DNA into cells is as DNA adsorbed to microprecipitates.
- DNA of a gene to be put into cells and DNA of a selectable marker gene are dissolved in a phosphate buffer. The two genes are on separate fragments.
- A solution of calcium chloride is added slowly to the DNA. A very fine precipitate (calcium phosphate) forms with the DNA adsorbed to it.
- The solution is incubated briefly with cells in tissue culture plates. After rinsing, cells are cultured further.
- Selection for the marker gene is then applied.
- Most of the surviving cell foci (posessing the marker gene) also contain the target gene. The genes usually appear to have integrated as tandem arrays at the same location.
- Though technically this procedure is transformation (as practiced with bacterial cells) it is called transfection to prevent confusion with use of transformation to indicate conversion to uncontrolled growth.
- Transfection with microprecipitates is a standard method for integrating an isolated gene into nuclear DNA of cultured mamalian cells.
- The method can't be used with cultured plant cells, presumably because these are surrounded by cell walls.
- It can be used in making transgenic organisms if the target cells are embryonic stem cells.
- The transfected genes wind up in environments different from their normal environment and the positions of integration of the genes in different transfectant cell lines differ. Expression levels vary widely. Variation can be reduced by the addition of MAR's to the target gene.
- Transfection is but one way of putting DNA into living cells.
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This is page 432 of Molecular Genetics by Ulrich Melcher, ©1997-2000
E-mail inquiries to U. Melcher------------Last Updated: 3 October, 2000