TRANSFECTION METHODS - SUMMARY
There are 4 different methods of transfection currently used in the unit: Calcium Phosphate, DEAE-Dextran, Electroporation and Lipofectin. The uses of these different methods is listed below.
|Calcium Phosphate*||CHO cells,
other adherent lines
|Stable (transient)||Low level expression can be
seen in transient assays.
Glutamine synthetase system.
|DEAE-Dextran||C0s-7 cells||Transient (stable)|
|Electroporation||Y3, NIH3T3, RBL
|Stable||Works with many cell lines.|
|Lipofectin||L-cells, Cos-7||Stable||Reported to rival electroporation for transfection levels in many cell lines.|
*Calcium phosphate precipitation does not work in RPMI, due to the high concentration of phosphate within the media. Therefore plate cells out in DMEM immediately prior to transfection.
Separate protocols for the first three methods are listed. There has been very little use of Lipofectin in the unit and only the method sheet is included. The general principle behind Lipofectin (and other liposome based methods) is the use of a liposome formulation that interacts spontaneously with DNA to form lipid-DNA complexes with complete DNA entrapment. These liposomes fuse with cell membranes and mediate efficient uptake and subsequent expression of the DNA. According to the manufacturer's literature this method is between 5- and 100- fold more efficient than other methods.
Which method do I choose?
The method of choice if one is not using either the glutamine synthetase-CHO cell system or the transient Cos cell system is probably electroporation.