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.
For example, transfection of Elike protein 1 Elk-1 mRNA into dendrites of intact primary rat neurons induced cell death but introduction of Elk-1 mRNA in cell body did not cause cell death [ 20 ]. This experiment proved that localization of specific mRNA significantly altered the function of the mRNA, which was impossible to do using traditional transfection methods.
The experiment could not be performed without a combination of mRNA transfection and subcellular locational transfection. Therefore, the combination of mRNA transfection and phototransfection is a powerful tool for study of gene function in single cells by virtue of point-directed delivery and immediate action of mRNA.
An illustration of phototransfection. Laser beams green flashes create holes at specific regions of single cell subcellular locations and nucleic acids red dots are delivered into the local areas. Transfection methods are evolving rapidly. Even within a class, many new products and technologies are launched each year with improved efficiency and less cytotoxicity.
Future transfection technology should expand in two directions, being precise enough to transfect subcellular regions and up to whole-individual transfection. The ability to deliver foreign nucleic acids especially mRNA into subcellular locations e. In addition to the overall gene expression profiles of a cell, the location of expressed gene products plays a crucial role in determining the function of a cell [ 20 ].
Meanwhile, safe and reliable transfection methods that can be applicable to humans are needed to establish clinical therapeutics. In summary, transfection methodology has developed rapidly and diversely.
Consequently we now have plenty of options to choose from, fitting well into our experimental or clinical needs. However, as cell research progresses, more advanced transfection technologies are still in demand. This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author s and source are credited.
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Find Sales Contact. Contact Us Customer Support. 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. USA 92 , — Burkholder, J. Cappechi, M. Cell 22 , — Chan, C. Gene Med. Chuang, C. Bio-Protocol 7 , e Cullis, P. Ding, X. Dziegiel, N. Farhood, H. Acta , — Felgner, P. USA 84 , —7. Drug Deliv. NY Acad. Fraley, R. Gao, X. To increase the likelihood that recombination will occur in non-essential plasmid regions, such as the bacterial replicon or bacterial marker gene, linearize the plasmid with restriction enzyme s that cut within these non-essential regions.
Prior to transfection, purify the linearized DNA by ethanol precipitation, size exclusion or column purification. The above procedure works well for routinely transfected cell types. For hard-to-transfect cells, another method to generate stable cell transfectants is via lentivirus or retrovirus transduction. In this case, antibiotic resistance harboring virus particles generated after transfection of producer cell types such as HEKT are used to transduce cells that can then be selected for virus integration.
Details on virus production can be found here. In a transient transfection, gene expression changes can be studied in a window of 8 to 96 hours post-transfection. This method is useful for short-term expression of genes or gene products, gene knockdown, or small-scale protein production.
If performed with mRNA, which is only produced outside the nucleus in an unmodified cell, a transient transfection can deliver extremely rapid results. Stable transfection is a longer and more complex process, mainly reserved for protein production on a large scale, research on long-term genetic regulation, extended pharmacology research, or gene therapy. After insertion into a eukaryotic cell, exogenous nucleic acids may or may not become a part of the cellular genome.
If a segment integrates and becomes an inherent part of the host genome, the introduced DNA is subsequently replicated and expressed, even in daughter cells.
Expression of the foreign genetic code is continual and the transfection procedure is termed a stable transfection. If the host cell rejects the inserted DNA segment, the transfection becomes transient because expression of the introduced genetic material is lost with subsequent generations.
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