What is Transfection?

Transfection is a contemporary and effective technique to introduce foreign nucleic acids into eukaryotic cells. The nucleic acid introduction can facilitate applications in studying cellular processes, molecular mechanisms of diseases and the evaluation of gene therapy effects.

How are transformation and transduction different from transfection?

Transformation refers to the process of a bacterial cell taking up DNA from its environment. Transduction involves the introduction of DNA mediated by viral vectors.

Methods and Tools for Transfection

The selection of the most suitable transfection approach or strategy depends on various factors, such as the type and source of cells, the form of nucleic acids to be transfected, and considerations like experimental budget and the availability of required facilities.

There are two types of transfection methods:

Stable Transfection

Stable transfection involves either integrating foreign DNA into the nucleus of the host cell, becoming part of the host genome, or preserving an episomal vector within the host nucleus as an additional genetic element separate from the chromosomes.

Transient Transfection

Transient transfection does not involve the integration of nucleic acids into the genome of the host cell. Instead, nucleic acids such as plasmids or oligonucleotides can be introduced into the cell cytoplasm.

Choosing between Stable and Transient Transfection

The choice between transient or stable transfection depends on specific experimental goals and requirements. Transient transfection provides short-term gene expression, while stable transfection allows long-term or heritable gene expression.

Cell transfection with mRNA

Cell transfection with mRNA offers a promising alternative to plasmid transfection or using viral vectors for achieving protein expression. This is especially relevant in non-proliferative cells like primary human cells, as transfection eliminates the requirement for mRNA to enter the cell nucleus or integrate into the host genome, thereby providing an advantage.

Gene transfer approaches:

Viral-based transduction

Viral-based transduction utilizes viral vectors to transport a targeted nucleic acid sequence into a host cell. Viruses are an efficient vehicle to deliver genetic material into a target cell. The gene of interest is enclosed within a viral particle that is incapable of replicating. Commonly used viral vectors include retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, and herpes viruses.

Non-viral transfection

Non-viral transfection methods can be categorized into two main approaches: physical and chemical methods.

• Physical Methods: Electroporation is a widely utilized transfection technique for introducing foreign nucleic acids into cells by temporarily increasing the permeability of the cell membrane using electrical voltage. Sonoporation involves using microbubbles to create pores in the cell membrane, facilitating the transfer of genetic materials. Laser irradiation-assisted transfection utilizes a laser beam to generate small openings in the plasma membrane, enabling the entry of foreign genetic substances. Magnet-assisted transfection employs magnetic force to assist in the transfer of foreign genetic materials.
• Chemical methods: Cationic lipid transfection involves combining cationic lipids and nucleic acids in a solution, which is then introduced to cells. The cells take up the nucleic acid-lipid complex, resulting in the expression of the transfected genes within the cells. In calcium phosphate precipitation, DNA and calcium phosphate are mixed to form a precipitate, which is later added to cultured cells. The cells internalize the DNA, leading to the expression of the transfected genes. Cationic polymers can also serve as vehicles for delivering nucleic acids. DEAE-dextran is a cationic polymer widely used for nucleic acid transfection. The cationic DEAE-dextran molecule strongly associates with the negatively charged backbone of the nucleic acid. The resulting nucleic acid-DEAE-dextran complex carries a net positive charge, allowing it to adhere to the cell membrane. It can enter the cytoplasm through endocytosis or by inducing osmotic shock with substances like DMSO or glycerol. Recently, lipid nanoparticles (LNPs) have emerged as an attractive option for nucleic acid transfer. LNPs are engineerable to modulate their surface structure and charge which influences their cellular uptake.

Factors Influencing Transfection Efficiency

• Cell Type: Some cells are inherently more amenable to transfection and exhibit higher transfection efficiency, like actively dividing cells. Stem cells are more challenging to transfect because of their lower efficiency rates.
• Cell Confluence and Viability: Cells that are too confluent or have poor viability may be less receptive to transfection. It is essential to use an appropriate cell-specific medium to ensure optimal cell viability.
• Media and Serum: Choosing an appropriate cell culture medium and optimizing nutrients is essential to increase efficiency and post transfection recovery. The use of serum should be evaluated based on payload being delivered and stage of development where transfection is being employed.
• Antibiotics: Antibiotics help prevent bacterial contamination and maintain the sterility of the cell culture. However, use of antibiotics is not always an option and is influenced by intended use or development stage.
• Type of Molecule Transfected: Different types of molecules, such as plasmid DNA, mRNA, siRNA, viral vectors, or proteins, may have varying efficiencies in terms of delivery into cells. The size of the payload and quantity (single, double, or triple transfection) also plays a major role.
• Transfection Method: Cells can exhibit higher transfection efficiencies depending on the method of gene delivery. It is important to optimize your method if considering moving into GMP manufacturing. Efficiencies >95% are ideal but not always attainable.

Applications of Transfection in Research and Medicine

Generation of Stable Cell Lines

Transfection enables the generation of stable cell lines by introducing exogenous DNA into cells and selecting for those that have integrated the DNA. Stable cell lines can be used to manufacture biologics such as monoclonal antibodies and recombinant proteins.

Production of Viral Vectors

Transfection can be utilized to produce viral vectors by introducing required viral and accessory genes into host cells. These viral particles can be used as vehicles to deliver therapeutic genes during gene therapies.

Biologically Active Protein

Transient transfection has the potential to be employed in mammalian cell-culture systems to achieve substantial quantities of biologically active proteins, which can be utilized for biotherapeutic purposes.

Stem Cell Research

Transfection is utilized in stem cell research to introduce specific genetic material to reprogram and direct their differentiation into desired cell types.

Assays for Analyzing Transfection Effectiveness

Post transfection assays are commonly employed to study gene expression, protein function and cellular processes. Distinct transfection techniques exhibit varying degrees of effectiveness, gene expression levels and may also impact cell viability. It is essential to employ sensitive assays to assess transfection efficiency accurately and facilitate its optimization. Stable vs transient transfection influences the type and timing of the assays.

Trypan Blue Staining

Trypan blue staining is a simple assay that measures cell viability post transfection. It leverages a dye to permeate the membrane of dead cells to provide a quantifiable dead:live cell ratio in a sample.

ATP Assays

ATP assays are another type of assay that can help researchers quantify the number of viable cells.

Gene Regulation Assays

Gene regulation assays can be employed to measure down regulation of specific genes if the cargo was an oligonucleotide or siRNA.

Gene Expression Assays

Gene expression assays can be performed post transfection to determine attributes such as protein expression, titer, isoform and structure. Reporter genes are DNA sequences that code for proteins that can be readily detected and measured. They serve as powerful tools for assessing the effectiveness of gene delivery vehicles and the level of gene expression. Some commonly used reporter genes are green fluorescent protein, luciferase, β-galactosidase, chloramphenicol acetyltransferase, etc.

Future Direction: Advancements in Targeted Delivery

Future advancements in transfection techniques are expected to focus on enhancing targeted delivery and minimizing off-target effects, enabling more precise manipulation of cellular functions. Additionally, there will likely be a greater emphasis on developing non-viral transfection methods that are safer, more efficient, and capable of delivering larger cargo sizes, facilitating the advancement of gene therapy and other genetic engineering applications.

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