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Introduction to Transfection


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Overview

Transfection involves the introduction of DNA or RNA 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 can not only enable the advancement of cellular research, but can also enhance drug discovery strategies.

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 in cell, and in vivo transfection protocols that facilitate systemic delivery of siRNA molecules.

Types of Transfection


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, such as DOTMA (N-[1-(2,3,-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride). Such lipids/polymers form liposomes that associate with DNA and merge with the cell membrane to enable the intracellular transfer of DNA3.

This technique can be used for efficient delivery of nucleic acids into cell cultures without much difficulty. Lipid transfections can also be adapted for cost-effective, as well as high-throughput systems. However, the 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 done under optimum conditions.

Viral Transfection

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

With the evolution of transfection protocols and the growing ease of transfections assays, it is very 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 such 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.


Sigma-Aldrich offers a complete line of transfection reagents with these benefits:

Transfection Reagents Benefits



Select your Sigma transfection reagent by technology, features, or application-specific protocol:

 

Product No. & Protocol
Name Description Features & Benefits Add to
Cart
XTG9-RO

Detailed Protocol
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

Detailed Protocol
 
X-tremeGENE™ HP DNA Transfection Reagent 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

Detailed
Protocol

(PDF)
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
T0956

Detailed
Protocol
Universal Transfection Reagent Unique formulation of a biodegradable, proprietary cationic polymer conjugated to a cationic lipid • Fast, easy protocol
• Suitable for stable and transient transfection
• High efficiency for a wide variety of cell types including primary cells
• Compatible with serum or serum-free transfection protocols
• Low toxicity
 
L3287

Detailed
Protocol

(PDF)
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

Detailed
Protocol

(PDF)
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

Detailed
Protocol
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

 

Select your Sigma transfection reagent by cell type or line.  We have developed a list of cell types successfully transfected with each of our reagents.  Protocols may still require optimization. Access the complete list here. 

Protocol Overviews

Generalized protocols for calcium phosphate transfections and lipofection techniques appear below for comparison. Please refer to product-specific protocols for detailed procedures (links in the table above).

 

 

 

 References

  1. Derouazi, M., Girard, P., Van Tilborgh, F., Iglesias, K., Muller, N., Bertschinger, M., & Wurm, F. M. (2004). Serum‐free large‐scale transient transfection of CHO cells. Biotechnology and Bioengineering, 87(4), 537-545.
  2. Conn, K. J., Degterev, A., Fontanilla, M. R., Rich, C. B., & Foster, J. A. (1998). Calcium phosphate transfection. DNA Transfer to Cultured Cells, 5, 111.
  3. Felgner, P. L., Gadek, T. R., Holm, M., Roman, R., Chan, H. W., Wenz, M., ... & Danielsen, M. (1987). Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proceedings of the National Academy of Sciences, 84(21), 7413-7417.
  4. Chang, D. C., Chassy, B. M., Saunders, J. A., & Sowers, A. E. (Eds.). (1992). Guide to electroporation and electrofusion. ISBN: 978-0-08-091727-6, Pages 1-5.
  5. Ausubel, F. et al (ed.) A Calcium Phosphate Transfection, Short Protocols in Molecular Biology, third edition, John Wiley & Sons, Inc., New York, p. 9.5-9.6, p. 9.39 (1995)

 

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