Antibiotic selection in mammalian cell culture is a fundamental laboratory technique used to isolate specific cells that have successfully taken up and incorporated foreign genetic material. This method leverages selective agents to differentiate between modified and unmodified cells. It provides a reliable way to obtain pure populations of genetically altered cells, and is foundational for various downstream applications, enabling deeper investigations into cellular functions and disease mechanisms.
Purpose of Antibiotic Selection
Antibiotic selection primarily creates stable cell lines, where foreign DNA has become a permanent part of the host cell’s genome. This foreign DNA includes a gene of interest along with an antibiotic resistance gene. The resistance gene acts as a marker, allowing only cells that have successfully integrated the desired DNA to survive in the presence of a specific antibiotic. This process ensures the resulting culture consistently carries and expresses the introduced genetic information.
Stable cell lines are used for long-term gene expression studies, enabling researchers to observe the sustained effects of a gene over time. They are also employed in the production of therapeutic proteins, where consistent protein output from a genetically engineered cell line is important. Furthermore, stable cell lines aid in drug discovery, allowing for high-throughput screening of potential drug candidates, and in functional genomic screens, where stable knockdown or overexpression of genes helps elucidate their biological roles. The consistency and reproducibility offered by stable cell lines are thus foundational for reliable experimental outcomes.
Common Selective Agents
Several common selective agents are used in mammalian cell culture, each targeting specific cellular processes and requiring a corresponding resistance gene for survival.
G418 (Neomycin)
G418, also known as Neomycin, is an aminoglycoside antibiotic that inhibits protein synthesis by interfering with ribosomal function in eukaryotic cells. Resistance to G418 is conferred by the neomycin phosphotransferase (neo) gene, which inactivates the antibiotic through phosphorylation.
Puromycin
Puromycin is an antibiotic that rapidly kills eukaryotic cells by disrupting protein synthesis, specifically by causing premature chain termination during translation. Cells acquire resistance through expression of the puromycin N-acetyltransferase (pac) gene, which chemically modifies the antibiotic, rendering it inactive.
Hygromycin B
Hygromycin B is an aminocyclitol antibiotic that interferes with protein synthesis by inhibiting translocation and promoting mistranslation on the ribosome. The hygromycin phosphotransferase (hph) gene provides resistance by phosphorylating hygromycin B, thereby preventing its inhibitory action on the ribosome.
Blasticidin S
Blasticidin S is a nucleoside antibiotic that inhibits protein synthesis in both prokaryotic and eukaryotic cells by interfering with peptide bond formation. Resistance is conferred by the blasticidin S deaminase (bsd) gene, which detoxifies the antibiotic by deaminating it. Each of these agents, coupled with its specific resistance gene, offers a valuable tool for selecting genetically modified mammalian cells.
General Protocol for Selection
The general protocol for antibiotic selection begins with the introduction of foreign DNA into mammalian cells, a process known as transfection. This involves delivering an expression vector, which contains both the gene of interest and the chosen antibiotic resistance gene, into the host cells. Common transfection methods include lipid-based reagents, electroporation, or viral transduction, each chosen based on the cell type and experimental requirements. Following transfection, cells are typically allowed a recovery period, 24 to 72 hours, in a standard growth medium without any selective agent. This allows the cells to recover from the transfection procedure and begin expressing the newly introduced genes, including the antibiotic resistance gene.
After the recovery phase, antibiotic selection is initiated by adding the appropriate selective agent to the culture medium at a predetermined concentration. Non-transfected cells, which lack the resistance gene, will begin to die off due to the toxic effects of the antibiotic. The culture is then regularly monitored, typically every 2-3 days, to observe the progressive death of susceptible cells and the emergence of resistant clones. During this phase, the medium containing the selective antibiotic is refreshed periodically to maintain its concentration and remove dead cell debris.
As non-resistant cells clear, small colonies of surviving, resistant cells will become visible, often appearing within one to three weeks depending on the cell line and antibiotic. These resistant clones are then carefully isolated, often by picking individual colonies, or the entire surviving population is passaged into larger culture vessels. This expansion phase allows the stable cell population to grow sufficiently for downstream applications. Continuous culture in the presence of the selective agent ensures that only cells stably harboring the foreign DNA continue to proliferate, establishing a homogeneous and genetically stable cell line.
Ensuring Successful Selection
Ensuring successful antibiotic selection requires careful attention to practical considerations and troubleshooting strategies. A primary step involves determining the optimal antibiotic concentration for a specific cell line, often achieved through a “kill curve” experiment. This experiment exposes cells to varying concentrations of the antibiotic to identify the lowest concentration that effectively kills all untransfected cells within 7-14 days, while allowing resistant cells to thrive. Using an insufficient concentration can lead to incomplete selection, where non-resistant cells persist.
Several factors influence the efficiency of selection. The specific cell type being used plays a significant role, as different cell lines exhibit varying sensitivities to antibiotics and different rates of foreign DNA integration. Transfection efficiency, which is the percentage of cells successfully taking up the foreign DNA, directly impacts the number of initial resistant cells available for selection. A higher vector copy number, meaning more copies of the resistance gene are integrated per cell, can sometimes lead to more robust resistance.
Maintaining strict aseptic technique throughout the entire process is also important to prevent bacterial or fungal contamination, which can compromise cell viability and interfere with selection. Common issues include incomplete selection, where too many non-resistant cells survive, often due to an insufficient antibiotic concentration or too short a selection period. Conversely, excessive cell death, even of resistant cells, might indicate an antibiotic concentration that is too high, or a cell line that is particularly sensitive. Adjusting the antibiotic concentration, extending the selection period, or optimizing transfection parameters can help resolve these challenges.