Cell Transfection Wiki
Transfection Background, Stable vs Transient Transfection and Transfection Protocols
What is Transfection?
The method of introducing nucleic acids into cells, either chemically or mechanically, is called transfection. Chemical transfection relies on the uptake of the test reagent either through endocytosis, liposome binding to the cell membrane or interaction with a cell surface marker. Mechanical transfection is the physical movement of nucleic acids into the cell, typically via electrical current. Other methods exist, such as viral delivery of DNA into cells, which is termed transduction. A similar process of delivering plasmid DNA into bacteria or non-animal eukaryotic cells is called transformation.
The term transfection is used in reference to transferring a purified nucleic acid (DNA or RNA) into mammalian, eukaryotic cells. When the same process is performed in plant cell lines, fungi, bacteria or algae, it is referred to as transformation. Without transfection or transformation, the scientific community would be decades behind current understanding of genetics and gene therapies.
Whether it is chemical or mechanical transfection, the process is to create an opening in the recipient cell membrane. These openings allow exogenous genetic material to pass into the cytoplasm. Dependent on the design and destination of the payload (siRNA, miRNA, plasmid DNA), the delivered cargo either remains in the cytoplasm for transcriptional regulation of mRNA or continues into the nucleus for integration into genomic DNA and stable expression (plasmid DNA).
The transfection method is used in the development of genetic therapies for diseases such as AIDS, obesity, cancer and cardiac issues. This method is also utilized to deliver nucleic acids in the creation of transgenic mouse models, genetically modified plants, genetically modified flowers and even pets.
Transient vs. Stable Transfection
The two main methods acids can be manipulated to enter a cell is either in a transient or a stable transfection.
- The transfected DNA enters the cell nucleus but the foreign DNA 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 whose actions in the cell results in transient effects
- Transient transfection is resultant of chemical or electroporation methods
- A small amount of transfected DNA particles integrates into the cellular genome
- Integration of foreign DNA is not predictable and is a rare event
- 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 or a fluorescence expressing gene (e.g. GFP, RFP)
Stable transfection is more complicated than a transient transfection. However, in order to achieve long-term gene expression in cases such as gene therapy, 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.
Stable cell lines over-expressing a protein of interest are critical laboratory tools, performing roles such as producing therapeutic proteins (including recombinant antibodies), studying gene functions and screening experimental drugs. Recombinant proteins are traditionally produced from stably transfected mammalian cells. Recent improvements in transfection and cell culture growth suspension methods has increased the ability of researchers to produce proteins from transiently transfected cells.
Examples of studies that utilize cell transfection:
- 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. Also, they evaluated production of erythropoietin and factor IX in the cells.
- Manufacturing high-titer helper-free retroviruses
- Transfection via laser
- Cells were successfully transfected using femtosecond high intensity, near-IR laser pulses that perforate the cell membrane. This is a high efficiency and high rate of cell survival method that leaves the cell 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.
- There are commercially available transfection services provided by GLP-certified research laboratories: Altogen Labs, WesternBlotServices, or Transfection.ws.
Electroporation is another commonly used technique for transfecting cells. It consists of applying an electrical field to the cells, which permeabilizes the cell membrane and drives the charged DNA into the cell. Electroporation is more effective than chemical transfection, especially in hard to chemically transfect cells, but it is more toxic and requires careful handling of the cells and special healing buffers. The success of the electroporation depends on various factors including amount, length and type of electrical charge applied to the cells. Electroporation is highly successful in conducting transformation and transfection experiments and is versatile enough to be used to transfect/transform most types of cells.
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 to transfer nucleic acids into a cell is accomplished in various 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, thus, 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. Cationic polymers can also be used as a chemical transfection method, as they bind the negatively-charged DNA and the entire polymer is endocytosed by the cell.
Other Transfection Methods
A number of less common transfection methods exist, including the following:
- Sonoporation: Utilizes high-intensity sound to transfect cells instead of electric energy like electroporation.
- Cell squeezing: Robert Langer’s group at MIT invented this a gentle squeezing of cells using a microfluidic device which induces 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 the cell.
- Protoplast fusion: Cells are treated with lysozyme to remove cell walls, and then a secondary technique of fusion (e.g. polymers or electroporation) fuses the protoplast containing the DNA into the cell.
- Magnetofaction: Magnetic nanoparticles associated 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. Expert scientists will 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 discussed is inducible gene expression,cell signaling, transfection, protein production, the use of nanoparticles in biological research and delivery of RNAi-species for therapeutic use. This portal is focused on molecular and cell biology research methods, experimental protocols and laboratory techniques such as:
- gene silencing and RNA Interference (RNAi)
- stem cells and cancer cell lines
- in vitro protein expression
- cell banking
- plasmid DNA cloning
- in vivo transfection kits
- Transient and Stable Transfection
- mRNA expression and qRT-PCR
- siRNA/shRNA/microRNA transfection expression
- antibody production
- liposome encapsulation
- development of stable cell lines
- electroporation and DNA fusion
- in vivo siRNA transfection
- contract laboratory service (CROs)
- cell-based assay development
- xenograft animal model services
Discussion Topics and Articles
Molecular Biology is the field of Biology studying molecules and molecular processes that form the underlying basis of life. The research focus of Molecular Biology is proteins and nucleic acids (DNA and RNA). This field includes and is closely related to Biochemistry, Genetics, Gene Expression, Cell Biology, Microbiology, Pharmaceuticals, Bioengineering and Biotechnology. Also, in conjunction with computers and big data analytics, has given rise to such fields as Computational Biology, Bioinformatics and Molecular Genetics.
Transfection is the process of introducing a segment of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) into a suitable eukaryotic cell. Transfection is a term typically used when the process is performed in vitro using cultured cells. The same procedure in plants, fungi, bacteria and algae is referred to as transformation. Transfection is a primary technology used for studying genetics and devising gene therapy strategies. After the transfection step, the exogenous oligonucleotide may or may not become a part of the host cellular genome. If it integrates into the cell’s genome then the gene is said to be stably expressed. On the other hand, a temporary expression or efficacy from the transfection said to be a transient transfection.
Just as all living beings will eventually die, cells in multicellular organisms are also programmed to die. This process of programmed cell death is referred to as apoptosis. However, stem cells are undifferentiated cells that maintain the capability to self-divide without end. This is not only for renewal of its own cell type but also to differentiate into cellular subtypes and become specialized.
Gene silencing, also called RNA interference (RNAi), is a technique through which the expression of an expressed genes expression levels are turned off or knocked down. Typically consisting of 20-25 nucleotides of double stranded RNA (dsRNA), siRNA is extensively used in RNA interference (RNAi) particularly in mammalian cells. Although a gene may be silenced at either the transcriptional or post-transcriptional stages, RNAi is specifically a post-transcriptional gene silencing mechanism achieved by introducing a double stranded RNA (dsRNA).