Transfection is a foundational technique in molecular biology, representing the process of introducing foreign genetic material, such as DNA or RNA, into eukaryotic cells. This cellular engineering allows scientists to manipulate cell function and study gene expression outside of the organism. It serves as a fundamental method for understanding biological processes and developing new biotechnological applications.
The Core Concept of Cotransfection
Cotransfection expands on this fundamental process by introducing two or more distinct nucleic acid molecules into a single cell simultaneously. The most frequent application involves delivering a specific “gene of interest” alongside a separate “selectable marker gene,” both typically carried on individual plasmid vectors.
The purpose of the marker gene is to provide a clear method for identifying and isolating only those cells that have successfully internalized both desired genetic elements. This simultaneous delivery ensures that cells acquiring the gene of interest are also likely to have acquired the marker gene, simplifying the subsequent identification and selection steps. This strategy is particularly effective because the efficiency of DNA uptake by cells is often low, making direct identification of cells with the gene of interest challenging without a co-delivered marker.
Methods of Introducing Genetic Material
Introducing genetic material into cells requires overcoming the cell’s natural membrane barrier, and scientists employ various techniques to achieve this.
Chemical methods facilitate the entry of DNA by altering the cell membrane’s permeability or by complexing with the DNA itself. One common approach is calcium phosphate precipitation, where DNA is mixed with calcium chloride and phosphate buffer, forming a fine precipitate that adheres to the cell surface and is subsequently taken up by endocytosis.
Another widely used chemical method is lipofection, which utilizes lipid-based reagents, such as Lipofectamine, to form complexes with the DNA. These lipid-DNA complexes, often referred to as lipoplexes, can readily fuse with the lipid bilayer of the cell membrane, allowing the DNA to enter the cytoplasm. The positive charge of the lipid reagents helps bind the negatively charged DNA and facilitates interaction with the cell membrane.
Physical methods directly create transient openings in the cell membrane to allow DNA entry. Electroporation is a popular physical technique that applies a brief, high-voltage electrical pulse to a cell suspension. This pulse temporarily disrupts the cell membrane, creating microscopic pores through which the genetic material can diffuse into the cell. Once the electrical pulse ceases, these pores reseal, trapping the newly introduced DNA inside the cell.
Applications in Scientific Research
Cotransfection has diverse applications across various fields of scientific research.
One use is in the production of recombinant proteins, where one plasmid carries the gene encoding a desired protein, such as human insulin or specific monoclonal antibodies, while a second plasmid contains a selectable marker. This allows for the creation of “cell factories” that continuously produce valuable therapeutic or research-grade proteins.
The technique is also used to study gene function and how different genes or their protein products interact within a cellular environment. Scientists can introduce a specific gene and observe its impact on cell growth, differentiation, or disease progression. Cotransfection can be employed to investigate protein-protein interactions by introducing genes for two different proteins and then analyzing if and how they bind to each other.
Another application is the creation of stable cell lines, which are cell populations that have permanently incorporated the foreign DNA into their own genome. While transient transfection leads to temporary gene expression, cotransfection with a selection marker helps isolate the rare cells that have undergone stable integration. These stable cell lines are useful for long-term studies, drug screening, and large-scale protein production because the introduced genetic material is passed on to subsequent generations of cells.
Verifying Successful Cotransfection
After introducing genetic material into cells, scientists must confirm which cells have successfully undergone cotransfection.
Selectable markers are a primary method for this verification. These genes confer resistance to a specific cytotoxic agent, such as an antibiotic like G418 or puromycin. When the appropriate antibiotic is added to the cell culture medium, only the cells that have successfully incorporated and expressed the resistance gene, and by extension the gene of interest, will survive and proliferate, while untransfected cells will die.
Reporter genes offer a visual and often non-destructive way to identify successfully transfected cells. A common reporter is the gene for Green Fluorescent Protein (GFP), originally isolated from jellyfish. When the GFP gene is cotransfected with the gene of interest, cells that have taken up the DNA will produce GFP and emit a bright green fluorescence when viewed under a specialized microscope equipped with the correct light filters. This allows for easy visualization and quantification of transfection efficiency, as well as the ability to sort fluorescent cells using flow cytometry.