Creating a stable transfected cell line involves introducing foreign genetic material into cells and ensuring its permanent presence and expression across generations. This process is instrumental in various biological investigations, enabling consistent and long-term studies of gene function. Stable cell lines also play a significant role in biotechnology, particularly for producing recombinant proteins and developing therapeutic agents.
Fundamentals of Stable Transfection
Stable transfection involves integrating foreign DNA into a host cell’s genome, ensuring its permanent presence and expression. Unlike transient transfection, where introduced DNA is temporary, stable transfection results in the foreign DNA replicating with host chromosomes and passing to daughter cells. This permanent integration makes stable cell lines valuable for long-term experiments, ensuring consistent protein production and reproducibility.
Stable cell lines are pursued when sustained gene expression is required, enabling prolonged studies of gene function, large-scale production of therapeutic proteins, and the development of cell-based assays for drug discovery. Their consistent genetic makeup provides a defined and homogenous system for research.
Designing the Expression Vector
The initial step in generating a stable cell line involves constructing an appropriate expression vector, which serves as the carrier for the genetic material to be introduced into the host cell. This vector typically consists of several essential components. Foremost is the gene of interest, which encodes the specific protein or RNA molecule intended for expression within the cell.
Driving the expression of the gene of interest is a promoter, a DNA sequence that initiates transcription. Common promoters utilized in mammalian expression vectors include the Cytomegalovirus (CMV) promoter, Simian Virus 40 (SV40) promoter, and the Phosphoglycerate Kinase (PGK) promoter. These promoters ensure that the gene of interest is actively transcribed into messenger RNA (mRNA) within the host cell.
Another important component is the selectable marker gene, which allows for the identification and isolation of cells that have successfully integrated the foreign DNA. These markers typically confer resistance to a specific antibiotic, such as the neomycin resistance gene (neoR) for G418, or the puromycin N-acetyltransferase (pac) gene for puromycin. When cells are exposed to the corresponding antibiotic, only those expressing the selectable marker survive, while non-transfected or transiently transfected cells are eliminated. For example, neoR inactivates G418, and pac neutralizes puromycin, both preventing their toxic effects. The selectable marker can be on the same plasmid as the gene of interest or on a separate co-transfected vector, with the former generally offering a higher likelihood of co-integration and selection.
Cell Transfection and Selection
Once the expression vector is designed, it must be introduced into the host cells through a process called transfection. Several methods are available for this, each with its own advantages depending on the cell type.
Lipofection, a widely used chemical method, involves mixing DNA with cationic lipids to form lipoplexes, which cells internalize through endocytosis. This method offers high efficiency, broad applicability, and reproducibility.
Electroporation is a physical method using brief, high-voltage electrical pulses to create temporary pores in the cell membrane, allowing DNA entry. It applies to many cell types, including those difficult to transfect chemically, and can yield stable transfectants. A potential drawback is associated cell death from the electrical shock.
Viral transduction uses modified viruses, such as lentiviruses or retroviruses, to deliver and integrate the gene of interest into the host cell’s genome. These vectors are effective for achieving stable, long-term gene expression.
Following transfection, the important step of selection begins to isolate the rare cells that have stably integrated the foreign DNA. This process involves culturing the cells in a medium containing the antibiotic corresponding to the selectable marker gene in the expression vector. Non-transfected cells or those with only transient expression will succumb to the antibiotic’s effects, while the stably transfected cells, expressing the resistance gene, will survive and proliferate.
Before full-scale selection, a “kill curve” experiment determines the optimal antibiotic concentration to eliminate all untransfected cells, usually within one week. Antibiotic selection is typically applied 48 to 72 hours after transfection, allowing cells to recover and express the resistance gene. Regular media changes are necessary during this period to replenish nutrients and remove dead cells.
Characterizing Stable Clones
After the selection process yields a population of antibiotic-resistant cells, it is important to characterize these stable clones to confirm successful gene integration and expression. Verification of gene integration into the host cell’s genome can be achieved through techniques such as genomic Polymerase Chain Reaction (PCR), junction PCR, Southern blotting, or Fluorescence In Situ Hybridization (FISH). These methods confirm the physical presence of the foreign DNA within the cell’s chromosomal structure.
To assess the expression of the gene of interest, various molecular and cellular assays are employed. Messenger RNA (mRNA) expression can be quantified using real-time quantitative PCR (RT-qPCR). For protein expression, common methods include Western blot, which separates and detects specific proteins, and Enzyme-Linked Immunosorbent Assay (ELISA), a quantitative method for detecting and measuring proteins. Immunofluorescence microscopy can also be used to visualize the cellular localization of the expressed protein, while flow cytometry can quantify protein expression at the single-cell level.
Clonal isolation and expansion are also important steps to ensure a homogenous cell population expressing the gene of interest. This often involves performing limiting dilution, where cells are diluted to a concentration that allows for the growth of single colonies from individual cells. Each resulting colony, or clone, is then expanded and re-evaluated for gene and protein expression. Ongoing monitoring of gene and protein expression over multiple passages is also conducted to confirm the long-term stability of the generated cell line.