Introduction to Transfection

Transfection generally refers to the introduction of foreign DNA into bacterial and/or mammalian cells. Transfection is an important tool used in studies investigating gene function and the modulation of gene expression, thus contributing to the advancement of basic cellular research, drug discovery, and target validation. This section provides an overview of different transfection methods, transfection workflow, factors affecting transfection efficient, and protocols.

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Transfection Methods

Transfection can be accomplished using chemical, biological, or physical methods. Common methods include electroporation, the use of a virus vector, lipofection, and biolistics. Many types of genetic material, including plasmid DNA, siRNA, proteins, dyes, and antibodies, may be transfected using any of these methods. However, a single method cannot be applied to all types of cells; transfection efficiencies and cytotoxicity may vary dramatically and depend on the method, cell type being utilized, and types of experiments being performed. Therefore, to obtain high efficiencies, all relevant factors should be considered for planning and selecting the appropriate transfection method.

Method	Function	Recommended Cells	Products
Electroporation	Nucleic acids or other molecules are introduced into cells by creating transient pores in the plasma membrane using an electric pulse	Eukaryotic cells (primary, stem cells), prokaryotic cells (bacteria, yeast), plant protoplasts	Gene Pulser Xcell™ electroporation system Gene Pulser MXcell™ electroporation system MicroPulser™ electroporator
Lipid-mediated	Uses lipids to cause a cell to absorb nucleic acids; transfer of genetic material into the cell takes place via liposomes, which are vesicles that can merge with the cell membrane	Immortal cells, adherent (attached), or suspension cells	TransFectin lipid reagent SiLentFect lipid reagent
Biolistic particle delivery	Delivery of nucleic acids into cells via high velocity nucleic acid-coated microparticles	Plant, primary cells, tissue, and in vivo applications	Helios™ gene gun PDS-1000/He™ biolistic particle delivery system
Viral vector (for example, retrovirus, lentivirus, adenovirus, or adeno-associated viruses)	Uses viral vectors to deliver nucleic acids into cells	Attached adherent cells, stem cells, primary cells

Transfection Efficiency

Successful transfection is usually measured in terms of transfection efficiency and cell viability — the higher the efficiency and viability, the better the transfection. Several important factors, such as the DNA quantity and quality, cell type, cell health, and transfection method (as stated above) affect transfection results. Transfection efficiency, for example, varies greatly with the cell type and its physiological condition prior to transfection. Ideally, the cells should be actively growing, healthy, and free of contamination.

Another way to present factors impacting transfection results is to consider the entire transfection workflow and the series of key sub-experiments it comprises. Each sub-experiment can significantly affect efficiency and viability. The typical workflow for a transfection experiment is as follows:

Transfection Workflow

Typical workflow for a transfection experiment.

The following table summarizes the factors to consider for efficient transfection:

Cell Health	Cells should be grown in medium appropriate for the cell line, supplemented with serum or growth factors as needed for viability Contaminated cells and media (e.g., contaminated with yeast or Mycoplasma) should never be used for transfection Make sure the medium is fresh if any components are chemically unstable, e.g., thiamine Medium lacking necessary factors, such as serum, can negatively affect cell growth Incubate cells at 37°C supplied with CO₂ at the correct percentage (5–10%) and kept at 100% relative humidity
Confluency	Transfect cells at 40–80% confluency (cell type dependent) Too few cells will cause cell cultures to grow poorly without cell-to-cell contact Too many cells results in contact inhibition, making cells resistant to the uptake of DNA and other macromolecules Actively dividing cells take up DNA better than quiescent cells
Passages of DNA	Number of Passages (cell type dependent) Number of passages should be low (<50) Number of passages for cells used in a variety of experiments should be consistent Cell characteristics can change over time with immortalized cell lines and cells may not respond to the same transfection conditions. Cells may not respond to the same transfection conditions after repeated passages DNA Quality and Quantity Use high-quality plasmid DNA for transfections that is free of proteins, RNA, and chemicals DNA is typically suspended in sterile water or TE buffer to a final concentration of 0.2–1 mg/ml The optimal amount of DNA to use in the transfection will vary widely and depend on the type of DNA, transfection reagent/method, target cell line, and number of cells
Time/Serum	Time Optimal transfection time depends on the cell line, transfection method, and molecule transfected Transfection times vary from 30 min to 4 hr (or may require overnight incubation based on the reagent used); some reagents do not require either media changes or additions Cell morphology is monitored during the transfection interval because some cell lines lose viability during this period, e.g., cells maintained in serum-free medium In addition to saving time, a shortened transfection time may significantly reduce the risk of cell death during the transfection procedure Serum Transfection protocols often require serum-free conditions for optimal performance because serum can interfere with many commercially available transfection reagents

Transfection Protocol Library

The transfection protocol online library contains protocols obtained from the literature, developed by Bio-Rad scientists, or submitted by scientists like you. Browse protocols to view our library and find your starting point or submit a protocol by clicking the proper technology.

Videos

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation
This video protocol describes the preparation of primary hematopoietic cell cultures from murine bone marrow for electroporation.

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

Preparation of Gene Gun Bullets and Biolistic Transfection of Neurons in Slice Culture

Gene Pulser Xcell™ Electroporation System: Components, Application, and Troubleshooting
This tutorial highlights the main components and features of the Gene Pulser Xcell system. It provides information about system installation and the setup of electroporation experiments, including important troubleshooting tips and answers to frequently asked questions. Ordering information for system components and accessories is also provided.

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