The Key to Gene Delivery to Cells —In Vitro Cell Transfection Techniques

Product Manager：Harrison Michael

Cell transfection techniques are the cornerstone of interdisciplinary research in molecular and cellular biology. They allow scientists to introduce foreign nucleic acids into cells to explore gene function, expression, and develop gene therapies or produce recombinant proteins. This article reviews the currently popular in vitro transfection techniques, including chemical transfection, physical transfection, and viral transduction, exploring their principles, methods, and applications.

Chemical Transfection Techniques: Chemical transfection involves using chemical reagents to facilitate the uptake of nucleic acids by cells. These reagents form complexes with nucleic acids, which are then internalized by the cells via endocytosis. The main chemical transfection methods include lipid-based transfection and calcium phosphate transfection.

Lipid-Based Transfection: Lipid-based transfection utilizes cationic lipids to form liposomes, which encapsulate nucleic acids and then fuse with the cell membrane, allowing nucleic acids to enter the cytoplasm. The steps include preparing the lipid-DNA complex, incubating to facilitate uptake, and post-transfection incubation to allow gene expression. This method is widely used for transient and stable transfection in various cell types, including primary cells and cell lines, for gene expression studies, protein production, and functional genomics.

Calcium Phosphate Transfection: Calcium phosphate transfection involves co-precipitating DNA with calcium phosphate, which is then taken up by cells via endocytosis. The steps include DNA precipitation, adding the precipitate to cells, and incubating to allow DNA uptake and expression. This method is suitable for both stable and transient transfection, especially in easily transfected cell lines such as HEK293, for gene expression and functional studies.

Physical Transfection Techniques: Physical transfection techniques use physical forces to introduce nucleic acids into cells. Electroporation and microinjection are the prominent methods.

Electroporation: Electroporation creates pores in the cell membrane by applying short electrical pulses, allowing nucleic acids to enter the cytoplasm. The steps include cell preparation, electroporation, and recovery. Electroporation is suitable for a wide range of cell types, including primary cells and stem cells, for gene editing, protein production, and vaccine development.

Microinjection: Microinjection involves directly injecting nucleic acids into the cytoplasm or nucleus of individual cells using a fine needle. The steps include preparation, injection, and incubation. Microinjection is ideal for single-cell studies, zygote injection in animal models, and precise gene editing applications.

Viral Transduction Techniques: Viral transduction uses engineered viruses to deliver nucleic acids into cells, providing high efficiency and stable gene expression.

Adenoviral Transduction: Adenoviral transduction uses replication-deficient adenoviruses to deliver DNA to the cell nucleus without integrating into the host genome. The steps include vector preparation, infection, and incubation. Due to its high efficiency and broad applicability to various cell types, adenoviral transduction is commonly used for transient gene expression, gene therapy research, and vaccine development.

Lentiviral Transduction: Lentiviral transduction, a subtype of retroviral transduction, integrates genetic material into the host genome, enabling stable gene expression. The steps include vector production, transduction, and selection of stably transduced cells. Lentiviral transduction is widely used for stable gene expression, creation of transgenic cell lines, and in vivo gene therapy due to its ability to infect non-dividing cells and provide long-term expression.

Transfection Technique	Principle	Key Steps	Related Biochemical Reagents	Applications
Chemical Transfection	Using chemical reagents to facilitate the uptake of nucleic acids by cells through endocytosis.	1. Select appropriate chemical transfection reagents 2. Prepare transfection complexes 3. Add to cell culture 4. Incubate to facilitate nucleic acid uptake and expression	Transfection reagents (e.g., liposomes, calcium phosphate)	Suitable for various cell types, used for gene function studies and protein production
Lipid-based Transfection	Cationic lipids form liposomes that encapsulate nucleic acids and fuse with the cell membrane.	1. Prepare lipid-DNA complexes 2. Incubate to promote complex binding to the cell membrane 3. Post-transfection incubation to allow gene expression	Lipofectamine, DOTAP, Lipofectamine 2000, FuGENE, LipoD293	Gene expression studies, protein production, functional genomics research
Calcium Phosphate Transfection	DNA co-precipitates with calcium phosphate, and is taken up by cells through endocytosis.	1. Mix DNA with calcium chloride to form a precipitate 2. Add phosphate buffer 3. Add precipitate to cell culture 4. Incubate to allow DNA uptake and expression	Calcium chloride, HEPES-buffered saline, DNA	Gene expression, stable transfection, functional studies
Physical Transfection	Using physical force to introduce nucleic acids into cells.	1. Select appropriate physical transfection methods 2. Prepare cells with nucleic acids 3. Apply physical force 4. Recovery and expression	Electroporation buffer, injection buffer	Suitable for cells that are difficult to transfect chemically or when higher transfection efficiency is required
Electroporation	Electrical pulses create transient pores in the cell membrane, allowing nucleic acids to enter cells.	1. Prepare cells 2. Electroporate to form cell membrane pores 3. Recover cells in fresh medium 4. Incubate to facilitate gene expression	Electroporation buffer, DNA/RNA, specialized electroporation equipment	Gene editing, protein production, vaccine development
Microinjection	Using fine needles to directly inject nucleic acids into individual cells.	1. Prepare cells and nucleic acid solution 2. Use microinjection equipment for injection 3. Post-injection cell incubation	Injection buffer, DNA/RNA, fine glass needles, microinjection equipment	Single-cell studies, zygote injection, precise gene editing
Viral Transduction	Using engineered viruses to introduce nucleic acids into cells.	1. Select appropriate viral vectors 2. Prepare and purify viral vectors 3. Infect or transfect cells 4. Incubate to achieve gene expression	Viral vectors, culture medium, virus preparation reagents	High-efficiency gene delivery, suitable for long-term or stable expression needs
Adenoviral Transduction	Adenoviruses infect cells and deliver DNA to the nucleus without integrating into the host genome.	1. Prepare adenoviral vectors with the gene of interest 2. Add viral vectors to cell culture 3. Incubate to allow virus entry and gene expression	Adenoviral vectors, culture medium, virus preparation reagents	Gene therapy research, vaccine development, transient expression
Lentiviral Transduction	Lentiviruses integrate genetic material into the host genome, enabling stable gene expression.	1. Produce lentiviral particles with the gene of interest 2. Infect target cells with lentiviral particles 3. Select stably transfected cells (if necessary)	Lentiviral vectors, culture medium, virus preparation reagents, selection antibiotics (e.g., puromycin)	Stable gene expression, creation of transgenic cell lines, in vivo gene therapy

Conclusion

Selecting the appropriate transfection technique depends on the experimental objectives, cell type, desired efficiency, and duration of gene expression. Chemical methods such as lipid-based and calcium phosphate transfection are easy to use and cost-effective, while physical methods such as electroporation and microinjection offer high efficiency and precision. Viral transduction techniques, particularly adenoviral and lentiviral transduction, provide high efficiency and stable gene expression capabilities. Each method has its unique advantages and limitations, making it essential to tailor the approach to specific research needs.

As transfection technologies advance, they not only deepen our understanding of cellular processes but also drive the development of novel therapeutic strategies, highlighting their central role in modern biological research. For more information on related experimental reagents for cell transfection, feel free to consult Aladdin to accelerate your research progress.

Aladdin：https://www.aladdinsci.com/