Selectable markers are of course useful when screening cells that have been successfully transfected, but achieving stable transfections is difficult. You need to get your plasmids into the nucleus of the cells you are using, and that requires having additional cell marker signals to be present in the complexes (if you are doing chemical transfections). If you want to get higher efficiency, find a transfection reagent compatible with the cell line you are using, or use a modern generic (like https://altogen.com/product/altofect-transfection-reagent/).

You can transfect the plasmids together. After the stipulated time you can replace the media with bot antibiotics for the selection of stable cells. If there is no selection marker, you may require vital staining followed by cell sorting. Generally almost all eukaryotic expression vectors contain selectabe markers.

Selectable markers are of course useful when screening cells that have been successfully transfected, but achieving stable transfections is difficult. You need to get your plasmids into the nucleus of the cells you are using, and that requires having additional cell marker signals to be present in the complexes (if you are doing chemical transfections). If you want to get higher efficiency, find a transfection reagent compatible with the cell line you are using, or use a modern generic (like https://altogen.com/product/altofect-transfection-reagent/).

Hi,

To produce stable cell lines mostly in hard-to-transfect cells, the lentiviral systems (e.g. 3rd generation) is a very good approach to reach this goal.

If u have access to BSL2 laboratory, you can use lentiviral system (e.g. 3rd generation) for transduction in order to establish a stable cell line.

All of the required plasmids are available from Addgene site (https://www.addgene.org/) also. No need expensive equipment for virus islation neither if you use the PEG precipitation protocol despite of ultracentrifugation.

I have some experience in lentivirus propagation and transduction. If you need any help just feel free to write! I have got detailed protocols for the entire process (from transduction to clone screening)

Bests,

Tamas