Cell transfection is a laboratory technique introducing foreign genetic material, like plasmid DNA, into eukaryotic cells. This modifies cell genetic makeup to study gene function, regulate expression, and produce specific proteins. Transfection plays a significant role in biomedical research, drug discovery, and gene therapy, enabling investigation of disease mechanisms, development of treatments, and generation of valuable proteins.
The Essential Ingredients
Introducing plasmid DNA into cells requires target cells and plasmid DNA. Cell types vary in suitability; immortalized cell lines, dividing indefinitely, are often easier to transfect than primary cells, isolated from tissues with a limited lifespan.
Cell line choice is important, as cell health, growth rate, and membrane composition affect DNA uptake and gene expression. Plasmid DNA, a small, circular piece separate from the cell’s main chromosome, serves as the vehicle for delivering the gene of interest, engineered to carry specific genetic instructions.
Key plasmid components include the gene of interest, coding for the desired protein or RNA molecule. A promoter sequence ensures active gene expression. Many plasmids also include a selectable marker gene, such as one conferring antibiotic resistance, allowing scientists to identify and select successfully transfected cells.
Methods of Delivering Plasmid DNA
Introducing plasmid DNA into eukaryotic cells, with their protective cell membranes, requires specialized methods. Non-viral delivery techniques, chemical or physical, temporarily disrupt the cell membrane or form DNA complexes for entry.
Chemical Methods
Chemical methods rely on reagents interacting with negatively charged DNA and the cell membrane. Liposomes, tiny spherical lipid vesicles, encapsulate DNA; these lipid-DNA complexes (lipoplexes) fuse with the cell membrane, allowing DNA entry. Cationic (positively charged) lipids are commonly used; their positive charge helps them bind to negatively charged DNA, forming stable complexes for cell interaction. Calcium phosphate is another chemical method, forming a precipitate with DNA cells take up via endocytosis.
Physical Methods
Physical methods directly create temporary openings for DNA to enter. Electroporation uses controlled electrical pulses to create transient membrane pores, temporarily increasing permeability for plasmid DNA entry. Microinjection uses a fine needle to directly inject plasmid DNA into individual cells’ cytoplasm or nucleus. While precise, microinjection is low-throughput. Other physical methods include sonoporation (ultrasound) and magnetofection (magnetic fields to guide DNA-magnetic particle complexes).
General Steps for Transfection
A cell transfection experiment follows generalized steps, beginning with careful cell preparation for optimal DNA uptake. Cells are cultured in appropriate growth media until desired confluence (70-90% for adherent cells), allowing sufficient surface area for DNA attachment and uptake, minimizing contact inhibition.
Next, plasmid DNA must be properly prepared by purifying it to remove impurities. The purified DNA is then diluted to a specific concentration, important for efficient delivery and minimal cellular toxicity; researchers test DNA concentrations to find the optimal amount.
For chemical methods, prepared DNA combines with a delivery reagent (e.g., lipid-based solution) to form a transfection mixture, then added to cells. For physical methods like electroporation, cells and DNA are mixed in a specialized cuvette and placed into an electroporator for precise electrical pulses; after DNA introduction, cells incubate (typically a few hours to overnight) for DNA uptake.
After incubation, the transfection solution is removed, and fresh growth medium added. This media change helps remove residual toxic reagents and provides nutrients for cell recovery and growth; cells are monitored for 24 to 96 hours for gene expression, with the timeline depending on experimental goals and whether transient or stable expression is desired.
Achieving Successful Transfection
Transfection success depends on several factors; researchers optimize conditions to maximize DNA delivery and gene expression. Healthy, actively dividing cells transfect more efficiently than stressed or overgrown ones; plasmid DNA quality and purity are important, as contaminants can reduce transfection rates and cause cell damage.
Transfection method choice and parameter optimization play a significant role. For chemical methods, this includes adjusting the DNA to reagent ratio and incubation time; for physical methods like electroporation, optimizing parameters (voltage, pulse duration, pulse number) is important to create temporary pores without excessive cell death. Optimizations are determined empirically.
Verification Methods
After transfection, verifying plasmid DNA entry and gene expression is important.
Reporter Genes: Reporter genes, like Green Fluorescent Protein (GFP), are common. If the plasmid contains the GFP gene, transfected cells glow green under a fluorescence microscope, confirming DNA uptake and expression.
Selectable Markers: Selectable markers, often genes conferring antibiotic resistance, are another technique. After transfection, cells grown in antibiotic-containing medium only survive if they have taken up and expressed the plasmid’s resistance gene, allowing for selection and expansion.
Functional Assays: Functional assays confirm the newly expressed gene produces its intended biological effect.