Stable cell line generation is made possible by the use of positive selection markers such as G418, hygromycin B, puromycin resistance, etc.. Selection markers can be delivered using the same plasmid that contains the gene of interest (in cis), or on a separate plasmid (in trans) that needs to be co-transfected with the plasmid containing the gene of interest. The cis approach is generally easier and has a higher likelihood of producing drug-resistant stable transfectants that express the gene of interest. The trans method of co-transfection is a good alternative in instances where the target construct does not have the antibiotic-resistance gene in the vector backbone. In such cases, a plasmid mixture containing 5 to 10 parts gene expression plasmid and 1 part antibiotic selection marker plasmid can be introduced into cells. This plasmid ratio helps increase the likelihood that the selected cells will express both the gene of interest and the selection marker.
Stable cell line generation protocol
The protocol for generating stable cell lines requires several steps as shown below:
- Antibiotic Kill Curve: Titration of selection antibiotic to determine the concentration required to kill cells that are expressing the transgene (1 week)
- Transfection: Introduce the plasmid construct(s) into cells (2 days)
- Selection and expansion: Determine polyclonal and monoclonal populations of cells (8-10 weeks)