Stable transfection is a laboratory technique used to introduce foreign genetic material, such as DNA, into a cell’s genome, leading to its long-term, heritable expression. This process incorporates new DNA into the host cell’s chromosomes, ensuring that as the cell divides, the introduced genetic information passes to all subsequent generations. The goal of stable transfection is to create a cell line that continuously produces a specific protein or expresses a particular gene of interest. This differs from transient transfection, where the foreign DNA remains outside the cell’s chromosomes and is expressed only for a short period before being degraded or lost.
Applications of Stable Transfection
Stable cell lines, created through stable transfection, offer benefits in biological research and biotechnology due to the consistent and long-term expression of introduced genetic material. These cell lines are employed in studies understanding gene function, to observe the effects of overexpressing or silencing a gene. For instance, a stable cell line might produce a fluorescent protein, to track cellular processes or structures.
Stable cell lines are also used for producing recombinant proteins, including therapeutic antibodies, hormones, and enzymes, on a large scale. These cells manufacture the desired protein, harvested and purified for medical or industrial applications. They also serve as platforms for drug screening and discovery, to test the efficacy and toxicity of potential drug candidates in a consistent cellular environment. Stable cell lines can be engineered to mimic human diseases, providing models for disease mechanisms and new treatments.
Overview of the Stable Transfection Process
Generating stable cell lines begins with preparing the DNA construct, typically a plasmid, which contains the gene of interest and a selectable marker gene. Plasmid DNA needs high purity and concentration for efficient delivery. Next, this foreign DNA is introduced into host cells using various molecular biology techniques.
After DNA introduction, cells undergo selection to isolate those that have integrated the foreign DNA into their genome. Selection involves exposing cells to a specific agent, like an antibiotic, to which the selectable marker gene confers resistance. Unmodified cells, lacking the integrated DNA and resistance gene, perish, while transfected cells survive and proliferate. The final stage involves isolating individual cell clones, expanding them, and verifying consistent gene expression. This ensures a homogeneous and genetically stable cell line.
Methods for DNA Delivery and Cell Selection
Introducing foreign DNA into host cells for stable transfection can be achieved through several methods, each with its own advantages for different cell types.
Chemical Methods
Chemical methods use positively charged lipids or polymers to form complexes with DNA. These complexes fuse with the cell membrane, allowing DNA entry through endocytosis. This approach is widely used for its simplicity and effectiveness.
Physical Methods
Physical methods deliver DNA by directly bypassing the cell membrane. Electroporation exposes cells to brief, high-voltage electrical pulses, temporarily creating small pores for DNA entry. Microinjection directly injects DNA into individual cells using a fine needle, offering high efficiency but being labor-intensive.
Viral Methods
Viral methods, especially lentiviral vectors, efficiently deliver and integrate DNA into various cell types, including primary and non-dividing cells. These modified viruses carry the gene of interest and integrate it into the host cell’s genome reliably.
Cell Selection
After DNA delivery, cell selection ensures only cells with integrated foreign DNA survive and proliferate. This is accomplished by including a selectable marker gene in the DNA construct, conferring resistance to a specific cytotoxic agent. For example, the neomycin phosphotransferase gene (neoR) confers resistance to G418, and the hygromycin phosphotransferase gene confers resistance to hygromycin B. When exposed to the selective agent, only cells expressing the resistance gene survive, forming colonies that can be expanded into stable cell lines.
Characterizing and Maintaining Stable Cell Lines
After selection, stable cell lines are characterized to confirm foreign gene integration and expression. Molecular techniques verify integrated DNA presence, such as PCR to detect the specific gene sequence. Western blotting confirms desired protein production by detecting its presence and size in cell lysates. Immunofluorescence microscopy visualizes the protein within cells, providing localization and expression pattern information.
Clonal isolation ensures stable cell line homogeneity, as initial selection may yield a mixed population with varying integration sites or foreign DNA copy numbers. This involves diluting cells and plating them at low density so individual cells grow into distinct colonies, each from a single cell. Each isolated clone is expanded and re-characterized to identify the most stable and consistent gene expression. Long-term maintenance involves proper cell culture techniques: regular media changes, appropriate cell passage, and cryopreservation in liquid nitrogen to ensure viability and genetic integrity.