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Cell Transfection

Updated January 2012 - Edited by K.G.S., M.D, Ph.D., M.Sc. Hon.B.Sc..

This Transfection site is your portal for all things related to the transfection of cells with DNA plasmid or vector, RNA whether siRNA or RNAi, or even recombinant protein. We provide you with all the background information, protocols for cell transfection, optimization and analysis steps, and cell transfection troubleshooting information to become the ultimate expert!

Transfection Table of Contents

What is Transfection?

Transfection describes the introduction of foreign material into eukaryotic cells using a virus vector or other means of transfer.

transfection

 

The term transfection for non-viral methods is most often used in reference to mammalian cells, while the term transformation is preferred to describe non-viral DNA transfer in bacteria and non-animal eukaryotic cells such as fungi, algae and plants.

Transfection of animal cells typically involves opening transient pores or 'holes' in the cell plasma membrane, to allow the uptake of material. Genetic material (such as supercoiled plasmid DNA or siRNA constructs), or even proteins such as antibodies, may be transfected. In addition to electroporation, transfection can be carried out by mixing a cationic lipid with the material to produce liposomes, which fuse with the cell plasma membrane and deposit their cargo inside.

The original meaning of transfection was 'infection by transformation', i.e. introduction of DNA (or RNA) from a eukaryote virus or bacteriophage into cells, resulting in an infection. Because the term transformation had another sense in animal cell biology (a genetic change allowing long-term propagation in culture, or acquisition of properties typical of cancer cells), the term transfection acquired, for animal cells, its present meaning of a change in cell properties caused by introduction of DNA.

Transfection is a method by which experimental DNA may be put into a cultured mammalian cell. Such experiments are usually performed using cloned DNA containing coding sequences and control regions (promoters, etc) in order to test whether the DNA will be expressed. Since the cloned DNA may have been extensively modified (for example, protein binding sites on the promoter may have been altered or removed), transfection is often used to test whether a particular modification affects the function of a gene.

Transfection Diagram:

Transfection Applications

The ability to synthesize RNA in the laboratory is critical to many techniques. Radiolabeled and non-radiolabeled RNA probes are required for many protocols, and they can be synthesized easily in small scale in vitro transcription reactions allowing their use in blot hybridizations and nuclease protection assays.

 

Requirements for Transfection?

  • Vector or DNA
  • Cells
  • Transfection Reagent

Optimizing Transfection

In vitro transcription requires a purified linear DNA template containing a promoter, ribonucleotide triphosphates, a buffer system that includes DTT and magnesium

Which Transfection Reagent to Use?

In vitro transcription requires a purified linear DNA template containing a promoter, ribonucleotide triphosphates, a buffer system that includes DTT and magnesium

Transfection Methods

There are various methods of introducing foreign DNA into a eukaryotic cell. Many materials have been used as carriers for transfection, which can be divided into three kinds: (cationic) polymers, liposomes and nanoparticles.

One of the cheapest (and least reliable) methods is transfection by calcium phosphate, originally discovered by F. L. Graham and A. J. van der Eb in 1973[citation needed] (see also [1]). HEPES-buffered saline solution (HeBS) containing phosphate ions is combined with a calcium chloride solution containing the DNA to be transfected. When the two are combined, a fine precipitate of the positively charged calcium and the negatively charged phosphate will form, binding the DNA to be transfected on its surface. The suspension of the precipitate is then added to the cells to be transfected (usually a cell culture grown in a monolayer). By a process not entirely understood, the cells take up some of the precipitate, and with it, the DNA.

Other methods use highly branched organic compounds, so-called dendrimers, to bind the DNA and get it into the cell. A very efficient method is the inclusion of the DNA to be transfected in liposomes, i.e. small, membrane-bounded bodies that are in some ways similar to the structure of a cell and can actually fuse with the cell membrane, releasing the DNA into the cell. For eukaryotic cells, lipid-cation based transfection is more typically used, because the cells are more sensitive.

Another method is the use of cationic polymers such as DEAE-dextran or polyethylenimine. The negatively charged DNA binds to the polycation and the complex is taken up by the cell via endocytosis.

A direct approach to transfection is the gene gun, where the DNA is coupled to a nanoparticle of an inert solid (commonly gold) which is then "shot" directly into the target cell's nucleus. DNA can also be introduced into cells using viruses as a carrier. In such cases, the technique is called viral transduction, and, the cells are said to be transduced.

Other methods of transfection include nucleofection, electroporation, heat shock, magnetofection and proprietary transfection reagents such as Lipofectamine, Dojindo Hilymax, Fugene, jetPEI, Effectene or DreamFect.

 

Stable vs Transient Transfection Methods

For most applications of transfection, it is sufficient if the transfected gene is only transiently expressed. Since the DNA introduced in the transfection process is usually not inserted into the nuclear genome, the foreign DNA is lost at the later stage when the cells undergo mitosis. If it is desired that the transfected gene actually remains in the genome of the cell and its daughter cells, a stable transfection must occur.

To accomplish this, another gene is co-transfected, which gives the cell some selection advantage, such as resistance towards a certain toxin. Some (very few) of the transfected cells will, by chance, have inserted the foreign genetic material into their genome. If the toxin, towards which the co-transfected gene offers resistance, is then added to the cell culture, only those few cells with the foreign genes inserted into their genome will be able to proliferate, while other cells will die. After applying this selection pressure for some time, only the cells with a stable transfection remain and can be cultivated further.

A common agent for stable transfection is Geneticin, also known as G418, which is a toxin that can be neutralized by the product of the neomycin resistant gene.

 

 

Which Transfection Reagent or Method to Use?

In vitro transcription requires a purified linear DNA template containing a promoter, ribonucleotide triphosphates, a buffer system that includes DTT and magnesium

 

Tips for Transfection

  • Use RNAse inhibitors always!
  • Saving Money with T7 RNA Phage Polymerase: Clones with an N-terminal His-6 tag are available. If you obtain this clone, you can purify large amounts of T7 polymerase with high activity (ref: He B, Rong M, Lyakhov D, Gartenstein H, Diaz G, Castagna R, McAllister WT, Durbin RK. Protein Expr Purif. 1997, 9, 142–151).
  • To remove the DNA template, use an RNAse-free DNAse. We have used RQ1 DNase (Promega) added for 15 minutes and it works well. 

Storing Trasfection Reagent

  • RNA is best stored at a neutral pH with EDTA.
  • TE buffer is recommended (10 mM Tris-HCl, pH 7.5, 1 mM EDTA), however this should be prepared with RNAse free purified (preferablly Millipore water).

Troubleshooting Transfection Problems

Failed Transfections

Causes include:
  • Old transfection reagent
  • Improperly stored transfection agent
  • Poor quality DNA or vector
  • Insufficient DNA amount
  • Insufficient transfection reagent
  • Too much DNA
  • Too much transfection reagent
 

References on Transfection

  1. Bacchetti S, Graham F (1977). "Transfer of the gene for thymidine kinase to thymidine kinase-deficient human cells by purified herpes simplex viral DNA". Proc Natl Acad Sci U S A 74 (4): 1590-4. PMID 193108.

Cell Transfection Forum Topics

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Transfection Troubleshooting

Transfection Troubleshooting

Transfection Products & Services

Transfection Articles

Stable Transfection

Transient Transfection

Related Transfection Articles & Methods

Cleaning Glassware

 

 

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