Tips from the Bench: Transfection Tip
Generate Stable Cell Lines
Optimize Selection of Stable Transfectants
Positive selection markers such as hygromycin, neomycin, zeocin, and blasticidin resistance are required for the generation of stable cell lines expressing your gene of interest. There are two possible locations for the selection marker:
- Within the gene expression plasmid, or
- On a separate plasmid that is co-transfected with the gene expression plasmid
The first approach is generally easier and has a higher likelihood of producing drug resistant stable transfectants that express the gene of interest. Co-transfection is also an effective method when a plasmid mixture containing 5 to 10 parts gene expression plasmid and 1 part selection marker plasmid is introduced. These ratios help ensure that the selected cells will express both the gene of interest and the marker.
Linearize Your Plasmids Before Transfection
When generating a stable cell line, the transfected plasmid undergoes non-homologous recombination upon integration into a chromosome. The recombination event can occur within any region of the plasmid, including the gene expression or selectable marker cassettes. Recombination within these critical regions will disrupt their function. To increase the likelihood that recombination will occur in non-essential plasmid regions, such as the bacterial replicon or bacterial marker gene, linearize the plasmid with appropriate restriction enzyme(s). Prior to transfection, purify the linearized DNA by ethanol precipitation or column purification.