Cell Transfection Wiki

What is Transfection?

Transfection is the process of introducing nucleic acids into eukaryotic cells, either chemically or mechanically. Chemical transfection can occur through endocytosis, liposome-mediated entry (termed lipofection), or a compound’s interaction with a cell surface marker. Mechanical transfection refers to methods of physically forcing the entry of nucleic acids into a cell, typically via electrical current in a process known as electroporation. Other methods for delivering nucleic acids into cells include viral delivery (commonly known as transduction), and transformation – using plasmids to deliver DNA into cells. In all cases, the process involves increasing the permeability of the recipient cell’s membrane to allow for exogenous genetic material to pass into the cytoplasm. Depending on design and destination of the payload, the delivered cargo may remain in the cytoplasm or continue on into the nucleus (if the cell has one). In the cytoplasm, nucleic acids can serve a number of purposes; genes can be expressed by cellular machinery, specific sequences can be used for transcriptional regulation of mRNA, and proteins can alter intracellular processes. In a nucleus, genes can be integrated into genomic DNA, thus ensuring their stable expression by a cell.

Transfection has been pivotal to scientific and medical research. The method has been used extensively to develop gene therapies for diseases ranging from AIDS to cancer, and it has been essential in our quest for understanding cellular processes. Genetically modified organisms are modified through transfection, and as such the technique has found wide applicability in the scientific community and the market. Transgenic mouse models are used to test novel drugs, while genetically modified pets are increasing in popularity. No matter the application, transfection has shown itself to be a powerful and versatile tool for biological advancement.

Transient vs. Stable Transfection

Transfection of nucleic acids can serve a variety of goals. For the most part, transfection is done to either limit protein expression or cause the expression of a protein encoded by the transfected nucleic acids. In both cases, the technical aspects of transfection significantly impact the final results. A transfected nucleic acid can be introduced into a cell and translated (known as transient transfection) or it can be incorporated into a cell’s genome (known as stable transfection). The details of the two types are listed below:

Transient transfection:

  • The transfected DNA enters the cell but does not integrate into the genome of the cell
  • The host cell translates the transfected molecule, resulting in large increase in the target protein expression
  • Due to lack of integration, the transfected molecule is only utilized until it is degraded by the cell, with typical effects only lasting several days
  • Transient transfection also applies to siRNA and miRNA that do not integrate into the cellular genome but can still impact gene expression
  • Transient transfection can be conducted through chemical and electroporation techniques

Stable transfection:

  • A small amount of transfected DNA particles integrates into the cellular genome
  • Integration of foreign DNA is not predictable and is usually rare
  • Linear DNA insertion is typically more successful that using supercoiled DNA
  • shRNA plasmid DNA can be integrated into genomic DNA in order to be transcribed as miRNA or siRNA; RNA molecules cannot be stably integrated into the genome
  • Viral or microinjection methods are commonly used
  • There is an inherent risk of non-specific integration into the cellular genome
  • Typically, the desired gene is coupled with a reporter gene (such as resistance to an antibiotic) to allow the elimination of cells that do not contain the desired gene post-transfection

Stable transfection is more difficult to achieve than transient transfection. However, in order to achieve long-term gene expression in gene therapies, long-term pharmacological experiments, large scale protein production, or studies of genetic regulation the extra effort is undertaken in order to produce stably transfected cell lines. These permanently modified cells can then be cultured, allowing for larger scale protein production or specific genetic studies that require such cells.

Stable cell lines that express larger than normal quantities of a desired protein are critical laboratory tools, performing roles such as producing therapeutic proteins (including recombinant antibodies), being the subjects of genetic studies, and being test environments for the screening of experimental drugs. Recombinant proteins are traditionally produced from stably transfected mammalian cells. Recent improvements in transfection and cell culture growth suspension methods have increased the ability of researchers to produce proteins from transiently transfected cells. Novel techniques have also contributed to public health, as current insulin production is largely derived from stably-transfected bacteria.

Many studies have used transfected cell lines for a variety of purposes. The following list includes the results of studies that were valuable contributions to the field of transfection, and which demonstrated the wide-ranging applicability of transfection techniques:

  • Transfection of protein-producing DNA with a lipid transfection reagent
    • Researchers used a cationic lipid transfection reagent to study transient transfection in HEK293 and CHO cells. Both types of cells produced acceptable titers of Green Fluorescent Protein (GFP) and secreted IgG antibodies. The same study also evaluated production of erythropoietin and factor IX in the subject cells.
  • Manufacturing high-titer helper-free retroviruses
    • Researchers used transient transfection in 293T cells in order to express retroviral packaging functions. Transient transfection avoided the time-consuming process of finding a stably-expressing clone and produced a high titer of retrovirus within 72 hours.
  • Transfection via laser
    • Cells were successfully transfected using femtosecond, high intensity, near-IR laser pulses that perforate the cell membrane. This method proved to have high efficiency, and left transfected cells largely intact.
  • Cationic polymer transfection
    • Researchers used a cationic polymer to transfect HEK293 cells (grown in suspension).  The cationic polymer (both branched and linear) was successful at transfection and is scalable.  The resultant proteins were produced at significant levels without the typical, undesired production properties such as medium-conditioning effects previously seen with the use of polymeric transfection reagents.

Many transfection services are available commercially. Researchers that lack the laboratory equipment and conditions for proper transfection can still request specifically transfected cells from a variety of companies. Such transfection services are usually provided by GLP-certified research laboratories, examples of which can be found at Altogen Labs, WesternBlotServices, or Transfection.ws.

Transfection Techniques

Electroporation

Electroporation is a commonly used technique for transfecting cells. It consists of applying an electrical field to the cells, which allows for the cell membrane to accept charged DNA particles into the cell. Electroporation is more effective than chemical transfection, especially in cells that are generally tough to transfect, but the method is more toxic and requires careful handling of the cells and special healing buffers.  The success of electroporation depends on various factors including the electric field strength and duration of the applied voltage. Despite some setbacks, electroporation is highly successful in conducting transfection experiments and is versatile enough to be used in most cell types.

Liposomes

Liposomes are small, membrane-bound capsules made of polymers similar to those that make up the cellular membrane. When formulated to contain DNA, lipid transfection reagents fuse with the cell membrane and release their cargo into the cells.  This is often called “lipofection”.  Lipid-based transfection reagents take advantage of the semi-permeable feature of cell membranes to transport foreign nucleic acid molecules into a cell.  The lipid-nucleic acid complexes overcome the cell membrane typically by endocytosis, after which the nucleic acids are released into cytoplasm.

Chemical Transfection

Chemical transfection can be accomplished in a variety of ways. One of the most inexpensive methods is calcium phosphate precipitation, in which a calcium chloride solution containing the DNA cargo is mixed with HEPES-saline buffer with phosphate ions, causing precipitation of the insoluble calcium phosphate and binding the DNA on the precipitate surface.  The precipitate (in suspension) is then mixed with cells and the precipitate is taken up by the cells, including the DNA cargo.

Another method includes the use of dendrimers, which are highly branched organic compounds that bind DNA and deliver it into the cell. The method relies on the use of markers that can allow cargo to pass through the cell membrane.

Other Transfection Methods

A number of less common transfection methods exist, including the following:

  • Sonoporation: a high-intensity sound can be used to make the cell membrane permeable, which allows for transfection to take place.
  • Cell squeezing: the gentle squeezing of cells using a microfluidic device can induce transfection.
  • Impalefection: a nanofiber with DNA bound to its surface is physically inserted into the cell.
  • Optical transfection: a high-focus laser generates a temporary opening in the cell membrane of a single cell to allow DNA to penetrate into the cell.
  • Protoplast fusion: cells are treated with a lysozyme to remove cell walls, and then a secondary technique of fusion (using polymers or electroporation) fuses the protoplast along with foreign DNA into the cell.
  • Magnetofaction: magnetic nanoparticles combined with DNA are driven into the target cells using a magnetic field.
  • Gene gun (particle bombardment): DNA is coupled to a biocompatible solid nanoparticle and “shot” into the nucleus of the target cell.

 

Cell and Molecular Biology Research

Methods, Protocols and Lab Techniques

The goal of these pages is to collect standard protocols for a broad variety of microbiological and biochemical research techniques. Several of these websites cover techniques that include xenotransplantation for inflammatory, cancer, and other drug research, as well as gene silencing via RNAi effects, such as siRNA and shRNA. Also present are techniques specific to gene expression, cell signaling, transfection, protein production, the use of nanoparticles in biological research, and delivery of RNAi-species for therapeutic use. This specific portal is focused on molecular and cell biology research methods, experimental protocols, and laboratory techniques such as:

 

Future Developments

It is important to note that transfection is not defined by any one process. The technique has wide-ranging applicability due to its end result on cells, namely the functional modification of cellular processes. Although current in vitro techniques have been largely developed, efficient transfection of eukaryotic cells in organisms such as humans is still unsolved. Gene therapy is largely limited by the absence of an efficient process of genetically modifying cells. As such, current developments in transfection technology are aimed at potential methods by which stable transfection may be accomplished in larger organisms.