Using the CHOgro® High Yield Expression System, exceptional yields of recombinant protein can be obtained by transient transfection of suspension CHO cells. Though transgene expression ceases for the vast majority of cells within one to two weeks post-transfection, a small fraction of transfected CHO cells continues to stably express the recombinant protein through random chromosomal integration of the transfected gene. In this Tip from the Bench, we share a protocol for generating stable cell lines through selection and expansion of single cells or a pool of cells that continually produce high levels of recombinant protein.
To facilitate the selection process, transfection of auxotrophic-marker or antibiotic-resistance genes in cis or trans to the plasmid containing the gene-of-interest is performed. Additional selection pressure, e.g. sublethal doses of MSX on CHO-GS-/- cells, can then be applied to amplify gene copy number and identify high-expressing clones. Generating stable cell lines is a lengthy process (requiring 9-12 weeks), but ultimately has the potential to yield gram per liter quantities of protein with high batch-to-batch consistency for extended periods of time.
The figure below summarizes the protocol for generating stable cell lines in suspension CHO cells using antibiotic selection.
Not all serum-free complete media formulations can support transient transfection. Thus, CHOgro® Expression Medium is recommended for use with this protocol. However, it is not compatible with DHFR-based methods of selection because it contains hypoxanthine and thymidine.
The first critical step for stable cell line generation is determining the optimal antibiotic concentration for selecting stable cell colonies. A kill curve is a dose-response experiment where cells are subjected to increasing amounts of antibiotic to determine the minimum antibiotic concentration that is needed to kill all the cells over the course of one week. Performing a kill curve is recommended with each new cell type or when a new selection antibiotic is used.
Linearize your target plasmids before transfection. When generating a stable cell line, the transfected plasmid undergoes recombination during chromosomal integration. The recombination event can occur within any region of the plasmid, including the gene expression or selectable marker cassettes that might 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 restriction enzyme(s) that cut within these non-essential regions. Prior to transfection, purify the linearized DNA by ethanol precipitation or column purification.
|Table 1. Recommended Transfection Conditions per Well of a 6-well Plate
|CHOgro® Complex Formation Solution
|TransIT-PRO® Transfection Reagent
|Total Plasmid DNA (1 µg/µl)*
|Incubate transfection complexes for no more than 5 min before adding to cultures.
|* If the antibiotic selection marker is in trans of the gene of interest, then maintain a 10:1 ratio of gene-of-interest plasmid over antibiotic-selection plasmid.
The wells containing antibiotic-resistant cells can easily be identified by holding the 96-well plate up to a light. Mark these wells with a permanent marker.
The polyclonal cell culture can be further processed to isolate clones using various techniques such as:
Use of conditioned media or culturing cells in semi-solid media such as soft-agar can increase cell survival. Of the above-mentioned methods, limiting dilution is the most cost-effective and frequently adopted technique; a detailed protocol using limiting dilution to generate monoclonal cell lines is as follows:
To increase the likelihood of obtaining monoclones, limited dilution can be repeated several times.
Questions? Chat, call, or email Mirus Bio Tech Support for advice on generating stable cell lines!