Transfection of mammalian cell lines is usually accomplished with calcium phosphate or liposomes. Transfection by liposomes is rapid and has high efficiency, but is more expensive. The procedure is very simple in essence, but there are a large number of parameters which can be tweaked, including ratio of DNA to liposomes, amount of DNA per well, time of incubation etc. Note that some liposomes with artificial lipids are toxic to cells and cannot be used in large concentrations or for too long; this is an area where more may not be better.
Commercially supplied liposomes usually come with protocols for optimization of the procedure. These are not reliable, despite the manufacturer's claims. In real life, cell lines are highly variable and the optimal conditions may well be quite different from recommended ones. Below is a protocol which worked quite well for transfecting pEGFP into CHO cells; it may be used as a generic example. It is important to always include a non-transfected control to see if the control is killed under selection.
|Plate cells on a 6 well plate one day beforehand so that the cells are 40% to 70% confluent the next day.||The cells must not be completely confluent or the transfected cells will have no room to grow even when under selection. If there are not enough cells, on the other hand, the low toxicity of the liposomes may kill some more cells, resulting in too small a cell number for efficient growth.|
|Plate||Growth area (cm3)||Relative area||Seeding number one day prior to transfection||Volume of media (ml)|
|6 well||9.4||5 x||1-4 x 105||2.0|
|12 well||3.83||2 x||0.4-2 x 105||1.0|
|24 well||1.88||1 x||2-8 x 104||0.5|
|96 well||0.32||0.2 x||0.5-2 x 104||0.1|
|Put 2-6 ml of liposomes in a tube and 1-2 mg of DNA in another tube. Add 100 ml of serum-free media to each tube, then add one tube to the another. Leave the 200 mmixture to incubate at room temp for 15min for the liposomes to package the DNA. Repeat for each well.||Efficient packaging takes place when the liposomes and DNA are both dissolved, do not add one to the other directly. Serum proteins destabilize many types of liposomes, hence serum-free media should be used. Certain serum-stable liposomes can be used if the cell line is very sensitive to serum-deprivation.|
|Wash the cells once with with PBS. Add 0.8ml of serum-free media to the liposome mixture and overlay the mixture onto the well. Incubate at 37 °C and 5% CO2 for 1hr.||Usually, I find that incubation time is not important, hence I use the shortest reasonable time. Start optimizing the ratio of DNA to liposomes and the amount of DNA per well first before varying incubation time.|
|After 1hr, discard transfection mixture, wash the cells with PBS and add growth media. Incubate overnight.||The growth media gives the cells some time to recover and produce the enzyme responsible for resistance. However, sometimes the selection media can be used immediately with no detrimental effects.|
|The next day, replace the growth media with selection media.|