Stable transfection reagents and techniques
Stable transfection, sometimes called permanent transfection, is the integration of plasmid DNA into the chromosome of cancer cell’s DNA. The creation of a stable cell line enables the researcher to analyze long term effects of the introduced gene. Downstream studies incorporating the stable cell line includes overexpression of the gene insert for protein production, cell based assays to determine apoptosis or incorporation of a fluorescent/luminescent marker in order to track cell growth when surgically implanted in a mouse xenograft model.
Although understood to be a rare event, there are many ways to get your plasmid of interest into your cells. One method is to use a lipid based transfection reagent, such as one of the many cell line transfection kits offered by Altogen Biosystems. For hard to transfect cells, electroporation is a viable alternative to mechanically transfer the plasmid DNA construct into the cancer cells.
The most important aspect of your experiment is to make sure your plasmid backbone contains an antibiotic resistance marker in order select for positively transfected cells. The cells will need to be maintained under constant antibiotic selection in order to force the cell population into a majority of positively transfected cells. Here are additional considerations of a stable transfection:
- Takes a couple of weeks to generate a stable cell line
- Difficult to create primary stable cells due to inability to continue passaging primary cells
- Unless single clones are selected and expanded, there could be clonal variations due to variations in site of integration in each cell
- Antibiotic selection pressure needs to be maintained