Why cell transfection

The expression of a gene can be partially reduced gene knockdown or completely blocked gene knockout. Because any gene can potentially be targeted, gene silencing is a prevalent technique used to develop gene-based therapies to address monogenic pathologies, cancer and in immunotherapy strategies. How: Transfection of nucleic acids into cells cannot be achieved without the help of a transfection method.

There are several physical methods that exist such as electroporation, sonoporation or microinjection but these processes are complex and relatively toxic for mammalian cells. To solve these issues, chemical-mediated transfection offers a great alternative: easiness of use, high transfection efficiency and excellent cell viability.

Nucleic acids are negatively charged due to their polyphosphate backbone and are thus able to interact with positively charged transfection reagents polymers or lipids. This results in the formation of transfection complexes or nanoparticles, which protect nucleic acids from nuclease-mediated degradation.

Consequently it segregates into daughter cells, which enables sustainable transgene expression. The major drawbacks of virus-mediated transfection are immunogenicity and cytotoxicity. Introduction of a viral vector may cause an inflammatory reaction and an insertional mutation, because viral vectors integrate into the host genome randomly, which may disrupt tumor suppressor genes, activate oncogenes, or interrupt essential genes [ 9 ].

Another disadvantage of this method is that a virus package has limited space for a foreign gene to keep infectivity.

For these reasons, much effort has been made to develop non-viral transfection methods even though virus-mediated transfection is highly effective and easy to use.

Chemical transfection methods are the most widely used methods in contemporary research and were the first to be used to introduce foreign genes into mammalian cells [ 10 ]. Chemical methods commonly use cationic polymer one of the oldest chemicals used , calcium phosphate, cationic lipid the most popular method , and cationic amino acid [ 10 — 12 ].

The underlying principle of chemical methods is similar. Transfected DNA must be delivered to the nucleus to be expressed and again the translocation mechanism to the nucleus is not known.

However, these methods have merits of relatively low cytotoxicity, no mutagenesis, no extra-carrying DNA, and no size limitation on the packaged nucleic acid. Chemical transfection efficiency also varies depending on cell type. The physical transfection methods are the most recent methods and use diverse physical tools to deliver nucleic acids. The methods include direct micro injection, biolistic particle delivery, electroporation, and laser-based transfection [ 13 ].

In brief, the micro injection method directly injects nucleic acid into the cytoplasm or nucleus [ 14 , 15 ]. This method delivers nucleic acids into cells but demands skill, often causes cell death, and is very labor-intensive. Biolistic particle delivery employs gold particles that conjugate with nucleic acids [ 16 , 17 ]. This method is straightforward and reliable but it requires expensive instruments and causes physical damage to samples. Electroporation is the most widely used physical method.

The exact mechanism is unknown but it is supposed that a short electrical pulse disturbs cell membranes and makes holes in the membrane through which nucleic acids can pass [ 18 ]. Because electroporation is easy and rapid, it is able to transfect a large number of cells in a short time once optimum electroporation conditions are determined. Laser-mediated transfection also known as optoporation or phototransfection uses a pulse laser to irradiate a cell membrane to form a transient pore [ 19 — 22 ].

When the laser induces a pore in the membrane, nucleic acids in the medium are transferred into the cell because of the osmotic difference between the medium and the cytosol. The laser method enables one to observe the transfecting cell and to make pores at any location on the cell. This method can be applied to very small cells, because it uses a laser, but it requires an expensive laser-microscope system.

In addition to those mentioned above, there are other physical methods using ultrasound sonoporation and magnetic field magnetofection [ 23 — 25 ]. The merits include no risk of integration into the host genome, cell cycle-independent transfection efficiency, no need for immune inducible vectors, and adjustable and rapid expression. Using mRNA transfection, one can introduce any number of mRNAs into a cell, thereby overcoming overexpression of the genes. These advantages mostly originate from the fact that mRNA does not need to be located in a nucleus to be expressed.

Transfected DNA must carry a host cell or tissue-specific promoter to be transcribed to mRNA and the expression level is determined by strength of the promoter. Other strong advantages of mRNA transfection are:. Fluorescence images 2, 4, 6, and 8 h, respectively, after transfection.

Note the time-dependant increases in fluorescence. For these reasons, transfecting RNA is attracting interest for therapeutic purposes [ 29 ]. Therefore the plasmid used for in-vitro transcription must be designed with consideration of all factors affecting stability and translational efficiency. RNA interference RNAi is a powerful tool to knock-down specific genes and to observe consequent changes of phenotypes [ 6 , 32 ]. Despite the wide use of siRNA, large efforts are still being made to develop more effective, safe, and reliable methods to deliver siRNAs into cells, because of the great potential of RNAi in clinical use to treat diseases [ 33 ].

Both relatively new transfection methods, mRNA and siRNA transfection, lead to new ways to execute cell research with their own distinctive advantages.

Each cell has distinct gene-expression patterns even when sharing morphological similarities. Because the functions of a cell are determined by its location and time, single-cell resolution of gene expression is important to elucidate gene function.

To achieve single-cell resolution of gene function, reliable single-cell transfection methods are needed. Some physical transfection methods have been applied to single-cell transfection with good results. Examples are:. All methods are performed under a microscope so that transfected cells can be trailed in real time. Micro-injection is straightforward and efficient but all the types of injectors actually perforate cell membranes resulting in physical damage to the cells.

Single-cell electroporation efficiently delivers nucleic acids into single cells and can easily be applied in vivo. Single-cell electroporation of enhanced green fluorescence protein EGFP plasmid has shown the morphology and growth characteristics of a single neuron in vivo [ 38 ]. Phototransfection is the most accurate means of delivering nucleic acids Fig.

Because the numbers and sizes of holes on the cell membrane can be adjusted, this method is the most suitable way of delivering population mRNAs.

The additional advantage of phototransfection is that we can dictate subcellular location through which nucleic acids pass e. Introducing nucleic acids into a subcellular location is important for study of single polarized cells in which different cellular domains perform distinct activities.

Neurons, especially, have soma, dendrites, and axons, each with a different function and localized gene expression. The data presented compares the relative effectiveness of plasmid delivery into pig fetal fibroblast P16 as well as primary human and pig tracheal epithelial cells HTE and PTE, respectively by chemical reagents and nucleofection.

HTE cells were transfected with the four reagents indicated below and by nucleofection Figure 2-C. The delivery of genes into primary and immortalized cell lines is an underpinning of mammalian molecular biology and has become increasingly important in biomedical research and therapeutic development.

Defining the parameters necessary for transfection optimization is, thus a critical element in further enhancing gene delivery efficacy in a wide range of cells.

While there has been significant work done in the development of chemical and viral reagents for the delivery of recombinant DNA, only limited improvements have been made in physical delivery systems [ 1 ]. The development of a novel electroporation system by AMAXA has shown considerable promise as a system for delivering DNA to a broad range of cell lines and cell systems that grow either as adherent monolayers or in suspension [ 7 , 31 — 34 ].

A number of cell lines from human and animals that have been particularly important for characterization of airway diseases such as cystic fibrosis and asthma, for somatic cell nuclear transfer, for the study of hematopoietic diseases, and for mutation analysis were evaluated and compared for their ability to be efficaciously transfected with the nucleofection system. With the exception of HEK cells, when compared to chemical DNA delivery vehicles, nucleofection appears to be, in general, more effective and less toxic.

The transfection efficiency and toxicity is equivalent following nucleofection or Lipofectamine transfection of HEK cells Figure 1. Transfection of two immortalized human airway epithelial cell lines, 16HBE14o- and CFB41o- and primary airway epithelial cells from pig and human PTE and HTE, respectively showed that nucleofection was more effective than the four chemical reagents tested with the exception of the HTE cells that were also effectively transfectable with Effectene.

While the reason for this difference is not certain, it is possible that cells at different passages or in different stages of differentiation will have varying responses to insult. Additional studies will need to be undertaken to determine whether the transfection efficiency and viability following nucleofection can be further enhanced. The development of somatic cell nuclear transfer using fetal fibroblast as donor cells has played a central role in the cloning of animals such as the pig and the rabbit [ 14 , 35 — 37 ].

In addition, differences in the age of the cultured cells, and cell density may also play a factor. These elements need to be considered when optimizing transfection conditions and should be addressed empirically. Suspension cultures of hematopoietic origin have been notoriously difficult to transfect with chemical reagents and have had to rely on viral vector systems to facilitate DNA delivery [ 1 ].

Thus, nucleofection may be an effective means of ex vivo genetic modification of hematopoietic stem cells that have multilineage potential.

Embryonic stem ES cells have become increasingly more important due their potential for organ regeneration and for the development of models to study disease. Mouse ES cells MESCs have been notoriously difficult to transfect with chemical reagents, and have thus been relegated to transfection by electroporation.

Standard electroporation protocols have resulted in high levels of cytotoxicity that have undermined the ability to transfer genes into the cells and the potential of the MESCs to produce viable embryos or differentiate in a lineage directed fashion. The nucleofection system has provided the opportunity to overcome some of these issues by enhancing transfection efficacy and MESC viability. These observations have important implications for the transfection of human ES cells and for their genetic modification and directed differentiation in that nucleofection has the potential of producing genetically modified cells that can be phenotypically manipulated without losing their pluripotency.

This study demonstrates the nucleofection system is effective for a broad range of cell lines and cell types, resulting in high levels of transgene expression and low toxicity. Not only is it superior when compared to various commercially available chemical DNA delivery vehicles in terms of transfection efficacy and viability, it also has potential therapeutic applications in ex vivo gene delivery. CAS Google Scholar.

Adv Genet. Article Google Scholar. Mol Ther. Zimmermann U: Electric field-mediated fusion and related electrical phenomena. Biochim Biophys Acta.

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View Transfection Products. An Introduction to Transfection Transfection is the process of introducing nucleic acids into eukaryotic cells by nonviral methods. Chemical Reagents DEAE-dextran, a cationic polymer, was one of the first chemical reagents used to transfect cultured mammalian cells Vaheri and Pagano, ; McCutchan and Pagano, Nonliposomal Reagents Although liposomal transfection reagents have a wide range of applications, they may not be efficient in all cell types.

Physical Methods Physical methods for gene transfer were developed beginning in the early s. General Considerations Reagent Selection. Table 1. Comparison of Transfection Reagents. Transient Expression Cells are typically harvested 24—72 hours after transfection for studies designed to analyze transient expression of transfected genes.

Stable Transfection The goal of stable, long-term transfection is to isolate and propagate individual clones containing transfected DNA that has integrated into the cellular genome. Type of Molecule Transfected Plasmid DNA is most commonly transfected into cells, but other macromolecules can be transferred as well. Assays for Transfection After cells are transfected, how will you determine success? Factors Influencing Transfection Efficiency With any transfection reagent or method, cell health, degree of confluency, number of passages, contamination, and DNA quality and quantity are important parameters that can greatly influence transfection efficiency.

Cell Health Cells should be grown in medium appropriate for the cell line and supplemented with serum or growth factors as needed for viability.

Optimization of Transfection Efficiency You will need to optimize specific transfection conditions to achieve the desired transfection efficiencies. Charge Ratio of Cationic Transfection Reagent to DNA The amount of positive charge contributed by the cationic lipid component of the transfection reagent should equal or exceed the amount of negative charge contributed by the phosphates on the DNA backbone, resulting in a net neutral or positive charge on the multilamellar vesicles associating with the DNA.

DNA Amount The optimal amount of DNA will vary depending on the type of nucleic acid, number of cells, culture dish size and target cell line used. Table 2. General Transfection Protocol Preparing Cells for Transfection Removing Adherent Cells Using Trypsin Trypsinizing cells prior to subculturing or cell counting is an important technique for successful cell culture.

The 1X solution can be frozen and thawed for future use, but trypsin activity will decline with each freeze-thaw cycle. Remove medium from the tissue culture dish. Rock the plates to distribute the solution evenly. Remove and repeat the wash. Remove the final wash. Add enough trypsin solution to cover the cell monolayer. Remove the flask from the incubator. Strike the bottom and sides of the culture vessel sharply with the palm of your hand to help dislodge the remaining adherent cells.

View the cells under a microscope to check whether all cells have detached from the growth surface. If necessary, cells may be returned to the incubator for an additional 1—2 minutes.

When all cells have detached, add medium containing serum to cells to inactivate the trypsin. Gently pipet cells to break up cell clumps. Cells may be counted using a hemocytometer, distributed to fresh plates for subculturing, or both.

Table 3. Area of Culture Plates for Cell Growth. Real-Time Assays For some types of transfection experiments, especially those examining the changes in gene expression levels associated with pathological mechanisms, monitoring reporter activity in living cells is desirable. Stable Transfection Selecting Stably Transfected Cells Optimization for stable transfection begins with successful transient transfection.

Prior to transfection, determine the selective drug concentration required to kill nontransfected cells. Forty-eight hours after transfection, trypsinize adherent cells and replate at several different dilutions e. For effective selection, cells should be subconfluent since confluent, nongrowing cells are resistant to the effects of antibiotics like G For the next 14 days, replace the drug-containing medium every 3 to 4 days.

Drug-resistant clones can appear in 2—5 weeks, depending on the cell type. Cell death should occur after 3—9 days in cultures transfected with the negative control plasmid. Transfer individual clones by standard techniques e. Table 4. Antibiotics Used to Select Stable Transfectants. Promega Transfection Products. References An, H. Bockamp, E. Genomics 11 , — Boussif, O.

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