Introduction to Cell Transfection

• What is transfection and how to transfect cells
• Types of transfection
• Calcium Phosphate Transfection
• Liposome-mediated Transfection
• Electroporation
• Viral Transfection (Viral Transduction)
• Selecting a Transfection Reagent & Transfection Protocols

What is transfection and how to transfect cells

Transfection is the introduction of DNA, RNA, or proteins into eukaryotic cells and is used in research to study and modulate gene expression. Thus, transfection techniques serve as an analytical tool that facilitates the characterization of genetic functions, protein synthesis, cell growth and development. Transfection assays not only enable the advancement of cellular research, but also enhance drug discovery strategies. Similar strategies, such as viral transfection or viral transduction, utilize lentiviral particles to insert foreign material into eukaryotic cells. Whereas bacterial transformation is the process of horizontal gene transfer where bacteria uptake foreign genetic material.

Types of Transfection

There is a wide range of transfection methods that include physical, chemical and biological techniques. These techniques generally involve the use of transient or stable transfection methods to incorporate nucleic acids into cells.

Transient transfection techniques involve the introduction of DNA into cells, but in this method, the DNA does not integrate with the cellular chromosomes. This technique facilitates high transfection efficiencies and the gene transcripts can be analyzed after a period of 1-4 days. For large-scale transient gene expression (TGE) in mammalian cell cultures, transfection vehicles such as polyethylenimine (PEI) and calcium phosphate (CaPi) can be used. Furthermore, large-scale TGE methods have also been developed using Chinese hamster ovary (CHO) cells in the absence of serum¹.

Stable transfection techniques involve the integration of the transfected DNA into cellular chromosomes or the formation of episomes. The stably transfected cell can be subsequently identified using selectable markers such as dihydrofolate reductase (DHFR), hygromycin B phosphotransferase (HPH) and adenosine deaminase (ADA) among several others.

Some of the commonly used transfection techniques include calcium phosphate precipitation, lipofection, electroporation, and viral delivery. Additionally, these methods can be used in cotransfections. These techniques involve the simultaneous delivery of two distinct nucleic acids into the same cell and are often used to achieve stable transfections. Transfections methods have evolved to include several new methods such as the biolistic delivery systems that use high velocity microparticles to deliver nucleic acids into cells, and in vivo transfection protocols that facilitate systemic delivery of siRNA molecules.

Calcium Phosphate Transfection

The calcium phosphate transfection technique involves the precipitation of DNA and calcium phosphate. The precipitation is facilitated by mixing a HEPES-buffered saline solution, having sodium phosphate, with calcium chloride solution and DNA². Glycerol shock is often used to enhance the DNA uptake in certain cells.

While this technique is cost-efficient and can be used for transient or stable transfections in a wide range of cells, relatively small changes in pH (±0.1) can affect the efficiency of transformation. Furthermore, it is essential to maintain reagent consistency for reproducing the assay results. However, this transfection method does not work in RPMI, or other media with high phosphate concentrations.

Liposome-mediated Transfection

Liposome-mediated transfection (lipofection) techniques involve the use of liposome forming cationic lipids, or non-lipid polymers. Examples of lipofection transfection reagents may include DOTMA (N-[1-(2,3,-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride)and X-tremeGENE™ transfection reagents, suitable for transfecting a variety of DNA, small RNA, and CRISPR/Cas9 components into a diverse range of cell lines. Lipid transfections can also be adapted for cost-effective, as well as high-throughput systems;however, these transfections are usually cell-type specific.

Electroporation

This technique involves the exposure of cell membranes to high-intensity electric pulses which causes a temporary destabilization in certain areas of the cell. During this transient destabilization event, the cell membrane becomes highly permeable and allows the entry of various exogenous molecules including DNA⁴.

Electroporation is an easy, non-chemical technique that can yield high transformation efficiencies in various cell types. Although this technique does not alter target cell morphology and functions, the method can cause cell death if transfection is not performed under optimum conditions.

Viral Transfection (Viral Transduction)

This method involves the use of viral vectors to deliver nucleic acids into cells. Viral delivery systems such as lentiviral, adenoviral and oncoretroviral vectors can be used for transferring nucleic acids, even in hard-to-transfect cells.

Although viral delivery methods are highly efficient, they can be quite laborious. Moreover, most viruses require containment and careful monitoring of biosafety levels. Before performing viral transfections, it is also important to consider several limiting factors such as the lytic nature of viral vectors, cell line packaging and host-cell specificity.

Selecting a Transfection Reagent & Transfection Protocols

With the evolution of transfection protocols and the growing ease of transfections assays, it is essential to select the appropriate transfection reagent to achieve optimum transfection efficiency.

While considering a suitable transfection reagent, it is important to identify the cell type and culture conditions for the assay. Rare cell cultures, neurons and primary cells are usually harder to transfect and hence will require reagents that can facilitate transfection even in hard-to-transfect cells.

Furthermore, the reagent levels and the cytotoxicity parameters should also be considered before selecting an appropriate transfection agent. An ideal reagent should have low cytotoxicity and high transfection efficiency for the required cell types.

Table 1 Features and benefits of our transfection reagent portfolio

Product No.	Name	Description	Features & Benefits
XTG360-RO	X-tremeGENE™ 360 DNA Transfection Reagent	Polymer transfection reagent for delivering DNA, siRNA, miRNA and CRISPR/RNP to many cell lines	• Efficient delivery of siRNA/miRNA, plasmid DNA, and CRISPR/Cas9 materials • Express recombinant proteins from plasmid DNA. • Minimal changes in cell morphology or induced cytotoxicity.
XTG9-RO	X-tremeGENE™ 9 DNA Transfection	A non-liposomal multi-component reagent	• Achieve new levels of transfection efficiency in primary cells and tumor cell lines • Generate physiologically relevant data using a reagent with low cytotoxic effects. • Increase experimental throughput and enable target evaluation using a simple and consistent protocol.
XTGHP-RO	X-tremeGENE™ HP DNA Transfection Reagent	A non-liposomal multi-component reagent, free of animal-derived components	• Achieve new levels of transfection efficiency in primary cells and tumor cell lines • Generate physiologically relevant data using a reagent with low cytotoxic effects. • Increase experimental throughput and enable target evaluation using a simple and consistent protocol.
SITRAN-RO	X-tremeGENE™ siRNA Transfection Reagent	A proprietary blend of lipids and other components, free of animal products.	• Knock down gene expression over 90% in many different cell types. • Maximize experimental flexibility with a single reagent for siRNA- and cotransfection-based gene-knockdown experiments. • Low cytotoxicity • Work with or without serum, avoiding medium changes
NPT01	NeuroPorter™ Transfection Kit	Unique formulation of a proprietary cationic lipid optimized for delivery of DNA into primary neurons, glial cells, and cultured neuronal cell lines with high efficiency and low toxicity Addresses problems such as poor cell viability, low transfection efficiency and neuro-degeneration	• Optimized for primary neurons, glial cells, and cultured neural cell lines • Very low toxicity with no neuro-degeneration or dendrite withdrawal • Efficient DNA delivery primary neurons, glial cells, and cultured neural cell lines • Fast and easy to use • Compatible with both serum and serum-free transfection protocols
L3287	Escort™ IV Transfection Reagent	Unique formulation of a proprietary polycationic lipid and a neutral non-transfecting lipid.	• Suitable for stable and transient transfection • Optimized for a wide variety of cell lines • Low toxicity • Compatible with both serum and serum-free transfection protocols • Ideal for insect cells
L3037	Escort™ III Transfection Reagent	Unique formulation of a proprietary polycationic lipid and a neutral non-transfecting lipid	• Suitable for stable and transient transfection • Optimized for a wide variety of primary cells • Low toxicity • Compatible with both serum and serum-free transfection protocols • Ideal for PC-12 cells
CAPHOS	Calcium Phosphate Transfection Kit	Commonly used method for the introduction of DNA into eukaryotic cells	• Suitable for transient and stable transfection • Reproducible for a wide range of cell types • Widely referenced • Inexpensive
DOTAP	DOTAP Liposomal Transfection Reagent	Cationic liposome-forming compound for transfection of DNA, RNA and other negatively charged molecules into eukaryotic cells	• Up to 100x more efficient than calcium phosphate and DEAE transfection • Lower toxicity than calcium phosphate and DEAE based transfection • Compatible with both serum and serum-free transfection protocols • Compatible with in-vivo transfection protocols
XTG360-RO	X-tremeGENE™ 360 DNA Transfection Reagent	Universal polymer reagent for delivering DNA, siRNA, miRNA and CRISPR/RNP to many cell lines	• Efficient delivery of siRNA/miRNA, plasmid DNA, and CRISPR/Cas9 materials •  Express recombinant proteins from plasmid DNA. •  Minimal changes in cell morphology or induced cytotoxicity.
70967	GeneJuice® Transfection Reagent	Non-lipid based chemical transfection reagent optimized for maximum transfection efficiency, ease-of-use, and minimal cytotoxicity on a wide variety of mammallian cells.	• Highly efficient DNA transfer for both stable and transient transfections • Minimal cellular toxicity • Compatibility with both serum-containing and serum-free media • Simple protocol—no need for media changes • Ideal for high-throughput transfection in a multi-well plate format
72181	293-Free™ Transfection Reagent	Animal-free polycationic liposomal transfection reagent optimized for the transfection of HEK293 cells grown in suspension culture.	• Optimized for transient transfections of HEK 293 suspension cultures • Derived from non-animal sources • Minimal cellular toxicity • Protocol easily scales up for production • Compatible with both serum-containing and serum-free media • Eliminates the need for media changes
72622-M	NovaCHOice® Transfection Kit	Optimized transfection reagent specifically developed for mammalian protein production in chinese hamster ovary (CHO) cells.	• Enhances expression in certain media formulations • Comprised of animal-origin free components • Compatible with many chemically defined media formulations • Eliminates the need for a culture medium change post-transfection • Suitable for both transient and stable transfection
71115	RiboJuice™ siRNA Transfection Reagent	Optimized, easy-to-use siRNA transfection reagent formulated for minimal toxicity.	• Efficiently delivers siRNA into a wide range of mammaliancell lines for targeted gene suppression • Compatible with GeneJuice® Transfection Reagent for cotransfection of siRNA and plasmid DNA • Used for gene suppression in a variety of cell lines
71259	Insect GeneJuice® Transfection Reagent	A proprietary liposome formulation optimized for maximal transfection efficiency of Sf9 insect cells.	• Extremely low toxicity to the cells • Used for both transient and stable transfections in serum-containing or serum-free media • Used for cotransfection of transfer plasmids with linearized virus DNA for the production of recombinant baculoviruses • Ideal for large-scale protein expression in suspension-culture transfections
TR-1003	Polybrene Infection / Transfection Reagent	A highly efficient method of gene transfer into mammalian cells leveraging infection with retroviral vectors.	• Enhanced efficiency of retroviral infection • Used to mediate DNA transfer into a variety of cell types

Generalized Protocols

Generalized protocols for calcium phosphate transfections and lipofection techniques appear below for comparison. Please refer to product-specific protocols for detailed procedures.

References

1. Derouazi M, Girard P, Van Tilborgh F, Iglesias K, Muller N, Bertschinger M, Wurm FM. 2004. Serum-free large-scale transient transfection of CHO cells. Biotechnol. Bioeng.. 87(4):537-545. https://doi.org/10.1002/bit.20161

2. Ravid K, Freshney I. 1998. DNA Transfer to Cultured Cells. Wiley-Liss Inc..

3. Felgner PL, Gadek TR, Holm M, Roman R, Chan HW, Wenz M, Northrop JP, Ringold GM, Danielsen M. 1987. Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure.. Proceedings of the National Academy of Sciences. 84(21):7413-7417. https://doi.org/10.1073/pnas.84.21.7413

4. Chang DC, Saunders JA, Chassy BM, Sowers AE. 1992. Overview of Electroporation and Electrofusion.1-6. https://doi.org/10.1016/b978-0-08-091727-6.50004-6

5. Ausubel FM. 1995. Short Protocols in Molecular Biology. 3. New York: John Wiley & Sons.