Getting The Most Out Of Transfection

Transfection is the process of introducing foreign nucleic acids, such as DNA, RNA, or oligonucleotides, into cells. This technique is widely used in molecular biology, biotechnology, and biomedical research for various purposes, including gene expression studies, functional genomics, protein production, and gene therapy.

Transfection methods can be broadly categorized into several main approaches:

Chemical Transfection

The chemical transfection methods are also described as reagent (chemical-based methods. These rely on creating a complex with an overall positive charge that enables it to interact with the negatively charged cell membrane that promotes uptake by endocytosis.

• Calcium Phosphate Transfection: This was one of the earliest methods used for transfection. Cells are typically treated with a calcium phosphate-DNA precipitate, which is taken up by endocytosis.
• Liposome-Mediated Transfection: Liposomes are lipid-based vesicles that can encapsulate and deliver nucleic acids into cells. Liposome-DNA complexes fuse with the cell membrane, releasing the DNA into the cytoplasm. The DNA will also be termed a transgene.  Commercially available lipid-based transfection reagents are commonly used for this purpose.
• Lipid Nanoparticles: A convenient method for delivering mRNA in particular into cells for protein translation. A successful method used for delivering mRNA vaccines .
• Polyethyleneimine (PEI): PEI is a cationic polymer that forms complexes with negatively charged nucleic acids, facilitating their entry into cells. PEI-DNA complexes can be taken up by cells via endocytosis and subsequently release the DNA into the cytoplasm.

Physical and Non-Chemical Transfection Methods

• Electroporation

In electroporation, cells are exposed to brief but strong electrical pulses that create temporary pores in the cell membrane, allowing the entry of nucleic acids as well as other molecules. This method is particularly effective for hard-to-transfect cell types but it is expensive because of high equipment costs and the need for a high-level of knowledge in operating said kit. The equipment is  not readily available either. 

In this method the cells and DNA are mixed together and placed into a cuvette between two electrode plates. So, a high DC voltage (500+ V) applied as a pulse. The DNA molecules ‘jump’ sideways into the cells because of their negative charge. Almost every cell treated this way is affected – the transfection rate is 80 to 90%. A square waveform works better than an exponential decay waveform for animal cells, the opposite is true for bacterial cells.

The process is optimised by exploring pulse length, voltage levels and types of waveform – these conditions are different for every cell type. In fact, it is surprising that individual kit types are needed for each cell type. It’s not a method either for large-scale transfection and practitioners recommend it for single small-scale experimentation. I’ve used it a couple of times and cells are easily killed. It’s also very sensitive to the presence of salt because it forms a current which causes the cells to lyse and breakup.

If the methodology is practiced and the appropriate conditions are chosen then transfection is efficient and produces stable transfected cells. The main issue is the expense of the kit along with the cuvettes used and then cleaning it properly. It can only work if the technique is practised and plenty of effort is spent optimizing conditions. It’s not a successful technology for adherent cells.

• Gene Gun/Biolistic Particle Delivery: In this approach, DNA-coated microprojectiles (such as gold or tungsten particles) are propelled at high velocity into target cells using a gene gun. The DNA is delivered directly into the cell nucleus.
• Microinjection: This method involves using a fine needle to directly inject nucleic acids into individual cells. It is highly precise but labor-intensive and typically used for specialized applications or difficult-to-transfect cells.

Viral Transduction

Viral vectors: Viral vectors derived from naturally occurring viruses (e.g., lentivirus, adenovirus, adeno-associated virus) are engineered to carry and deliver exogenous DNA into target cells. Viral transduction can be highly efficient and is often used in gene therapy and gene delivery applications.

Each transfection method has its advantages and limitations in terms of efficiency, cytotoxicity, ease of use, and applicability to different cell types and experimental requirements. Researchers choose the most suitable method based on factors such as the cell type, desired level of transfection efficiency, toxicity concerns, and the specific application of interest. Additionally, ongoing research and technological advancements continue to improve existing transfection methods and develop novel approaches for more efficient and precise nucleic acid delivery into cells.

Transfection Reagents

The FreeStyle MAX Reagent

The FreeStyle MAX reagent is a transfection reagent developed by Thermo Fisher Scientific and is commonly used for high-efficiency transfection of plasmid DNA into mammalian cells, especially in HEK293 and CHO cells grown in suspension cultures. It’s part of the FreeStyle expression system, optimized for protein expression in large-scale applications like recombinant antibody or protein production.

Mechanism of Action:

1. Lipid-Based Formulation:
   FreeStyle MAX is a lipid nanoparticle formulation that encapsulates DNA and facilitates its delivery into cells. It is a cationic lipid-based reagent, meaning it has positively charged lipids.

2. DNA Complex Formation:
   When FreeStyle MAX reagent is mixed with plasmid DNA in a serum-free medium (usually OptiPRO SFM or similar), the positively charged lipids interact with the negatively charged phosphate backbone of DNA, forming lipoplexes (lipid-DNA complexes).

3. Endocytosis-Mediated Uptake:
   These lipoplexes interact with the cell membrane and are taken up by the cells through endocytosis. Once inside the cell, the complex escapes the endosome and releases DNA into the cytoplasm.

4. Nuclear Delivery:
   The DNA then travels into the nucleus, where it can be transcribed and expressed as protein.

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Advantages:

• Optimized for high-density suspension cultures (e.g., FreeStyle 293 and FreeStyle CHO cells).

• Serum-free, animal origin-free formulation – suitable for large-scale production.

• High transfection efficiency and low cytotoxicity.

• Compatible with large-scale bioreactor systems.

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Common Use Cases:

• Transient protein expression for research or preclinical development.

• Large-scale antibody production.

• Producing difficult-to-express proteins using HEK293 or CHO cells.

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 Example Protocol Summary:

1. Dilute DNA and FreeStyle MAX reagent separately in OptiPRO SFM.

2. Mix gently and incubate for ~10 minutes to allow complex formation.

3. Add the mixture to the cell culture (typically at ~1 million cells/mL density).

4. Incubate cells under standard conditions (37°C, 8% CO₂, shaking).

5. Harvest protein typically 48–96 hours post-transfection.

FuGENE HD Reagent

FuGENE® HD is a non-liposomal transfection reagent developed by Promega. It’s designed for high-efficiency transfection of plasmid DNA into a wide range of mammalian cell types, including hard-to-transfect or sensitive cells, with low cytotoxicity.

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What is FuGENE HD and How Does It Work?

Core Function:

FuGENE HD facilitates the delivery of plasmid DNA into eukaryotic cells by forming DNA–reagent complexes that enter the cells primarily via endocytosis. Once inside, the DNA is released and can be expressed by the host cell machinery.

Composition:

• Proprietary blend of lipids and other non-viral, non-liposomal components.

• It’s formulated to be gentle on cells while maintaining high transfection efficiency.

Mechanism of Action:

1. Complex Formation:

   • FuGENE HD reagent is mixed with plasmid DNA in a serum-free or serum-containing medium.

   • The reagent coats the DNA, forming stable, positively charged complexes.

2. Cellular Uptake:

   • These complexes attach to the negatively charged cell membrane and are internalized through endocytosis.

3. DNA Release:

   • Inside the cell, the DNA is released into the cytoplasm and eventually enters the nucleus, leading to gene expression.

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Key Features and Benefits:

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Common Applications:

• Transient expression of proteins

• Reporter gene assays (e.g., luciferase, GFP)

• RNA interference (when co-transfecting plasmids or siRNA)

• CRISPR/Cas9 plasmid delivery (in some cases)

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Example Protocol Overview:

1. Prepare Complexes:

   • Dilute plasmid DNA in serum-free medium (e.g., Opti-MEM).

   • Add FuGENE HD reagent (typically 3:1 ratio of FuGENE HD:DNA, but may require optimization).

   • Incubate for 10–15 minutes at room temperature.

2. Add to Cells:

   • Add the complex directly to cells in culture.

   • No need to change medium, even in serum-containing conditions.

3. Incubate and Analyze:

   • Incubate cells under normal conditions (e.g., 37°C, 5% CO₂).

   • Analyze protein expression typically 24–72 hours post-transfection.