Gene transfection of mammalian cells using membrane. Use a range of several ul of lipofectamine and ug dna to increase viability after transfection and efficiency. Chemical methods commonly use cationic polymer one of the oldest chemicals used, calcium phosphate, cationic lipid the most popular method, and cationic amino acid 1012. There are many different ways to transfect mammalian cells, depending on the cell line characteristics, desired effect, and downstream applications. Electroporationelectrotransfection mammalian cells. The gentle square wave pulse also allows for high cell viability of these cell types. The basic protocol describes the electroporation of mammalian cells, including es cells for the generation of transgenic and knockoutin mice. 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. We described 7 different buffers for nucleofection of different mammalian cells.
Transient transfection of suspension cells by electroporation 1. Transfection of mammalian cells with fluorescent protein fusions. The plasma membrane of cells is an effective barrier and, despite being 5 nm thin, impermeable to many ions and polar molecules. N4 cells electroporation protocol before you start culture jm8. It is preferred over the chemical method for transferring exogenous dna into bacteria, because of its 1 high transformation efficiency that is suitable for regular plasmid, ligated cdna, and genomic dna. Cells almost confluent change media on es cells 4 hours before electroporation.
Electroporation of antibodies into mammalian cells springerlink. Electroporation is a popular technique to introduce foreign dna into host cells. Generating mammalian stable cell lines by electroporation. For more information on procedures and optimization of transfection by electroporation, see dna transfection by electroporation sambrook and russell 2006a, electroporation nagy et al. Pulsed electrical fields can be used to introduce dna into a wide variety of animal cells 1,2.
These methods include chemical liposomemediated, nonliposomal lipids, polyamines, dendrimers, physical electroporation, microinjection or viralbased retrovirus, adenoassociated virus, lentivirus delivery systems. Highly efficient protein detection and purification from. Applications include the transfection of animal and human cell lines, embryonic stem cells, primary cells, oocytes and plant cells. An electroporation protocol for efficient dna transfection in pc12 cells.
The gene pulser mxcell electroporation system supports the convenient plate. Mammalian cell culture refers to the extraction of cells of a mammalian from specific tissues e. It should reach a threshold value at which the electroporation of. The gene pulser xcell electroporation system is warranted against defects in. Transient transfection of suspension cells by electroporation. After electroporation, selection steps must be followed to allow for cells containing only the target dna to grow. Electroporation of functional bacterial effectors into. We sought a means to directly introduce exogenous proteins into cells. Transfection by electroporation potter 2003 current.
No other manual has been so popular, or so influential. Factors that affect efficient electroporation of cells are waveform, pulse. In this protocol huvec transfection is shown as an example. We aim to optimize the electroporation settings for t lymphocytes transfection. Optimization experiments using different electroporation parameters for stimulated human pbls showed that the viability of transfected t cells was in the 6386% range 24 hours after electroporation with a transgene expression of 99% 3 days after electroporation. Foreign dna can be introduced into competent li using electroporation or chemical transformation. The effects of electroporation buffer composition on cell viability and. The current is thought to create momentary pores in the cell membranes, which forces the negatively charged dna into the cells by an electrophoresistype effect. The induced transmembrane potential is maximal at the poles of the cell in accordance with equation 1. Based on our previous results in successfully internalizing proteins into leishmania. Electroporation, or electropermeabilization, is a microbiology technique in which an electrical field is applied to cells in order to increase the permeability of the cell membrane, allowing chemicals, drugs, or dna to be introduced into the cell also called electrotransfer. For these, electroporation has become the method of choice, with researchers resorting to more involved methods, such as viral transduction, as a backup. Electroporation works well with cell lines that are refractive to other techniques, such as calcium. The electroporation procedures used in many laboratories could be optimized with limited effort.
T1 efficient electroporation of mammalian cells in culture. Be sure to use chilled pipette tips and keep all materials on ice. Efficient transfection of tcells using electroporation youtube. N2 expression of functional, recombinant mammalian proteins often requires expression in mammalian cells see single cell cloning of a stable mammalian cell line.
Electroporation is an ionic restricted physical process that requires a cell suspension of high resistance and very low conductivity for a high degree of success dower et al. The dna enters the cells following a quick electric pulse that generates temporary openings in the cell membrane. The entire process of electroporation of mammalian cells will take electroporation of plant cells requires. Electroporation for the efficient transfection of mammalian cells with. Waveforms ac sine wave aligns cells by dielectrophoresis for electrofusion applications. Nucleofection, a modified electroporation method, has a number of limitations. Cells almost confluent change media on es cells 4 hours before electroporation media yellow. Electroporation is commonly used to introduce nucleic acids into cells, but has been more rarely applied to proteins although the biophysical basis is exactly the same. Spontaneous transfection, mammalian cells introduction dnamediated gene transfer into cultured mammalian cells is a routine approach for studying gene function in eukaryotes. However, the low expression levels of recombinant proteins in cultured mammalian cells. As with other transfection procedures, the experiment should be planned to allow for harvest or splitting of the cells 1 to 2 days after transfection.
Others, like stem cells, primary neurons and unstimulated t and b cells, not so much. Now available with pulse switcher for automatic polarity switching. Chicaybam l, barcelos c, peixoto b, carneiro m, limia cg, redondo p, lira c, paraguassubraga f, vasconcelos zf, barros l, bonamino mh front bioeng biotechnol. A cerebellar cells are optimally transfected at voltages above 340 v, with an approximately 30% transfection efficiency. After electroporation, selection steps must be followed to allow for. Analytical and bioanalytical chemistry 397, 31733178. T1 generating mammalian stable cell lines by electroporation.
Electrotransfection of mammalian cells using microchannel. Spontaneous transfection of mammalian cells with plasmid dna. Following the report by szybalska and szybalski 1962 on the ability of mammalian cells to take up exogenous dna, a great number of sophisticated methods. Does anyone have a recipe for electroporation buffer.
Jun 12, 2017 in electroporation, the cells are shocked electrically resulting in membranedistortion allowing for dna diffusion. Analytical biochemistry 180,269275 1989 optimization of electroporation for transfection of mammalian cell lines grai l. The cells are allowed to recover by a brief incubation at room temperature and then transferred to a tissue culture plate or tflask along with growth medium and serum. Jan 23, 2017 genetic modification of cell lines and primary cells is an expensive and cumbersome approach, often involving the use of viral vectors. Press the left arrow key to select yes, then press enter to overwrite the file. Suitable transfection methods for single particle tracing. Lasermediated transfection also known as optoporation. Transfection of mammalian cells by electroporation. Sep 05, 2012 mammalian cell transfection is a technique commonly used to express exogenous dna or rna in a host cell line for example, for generating rnai probes. Get the electroporator from the lin lab, and make sure no one is using it. Electroporation using squarewave generating devices, like lonzas nucleofector, is a widely used option, but the costs associated with the acquisition of electroporation kits and the transient transgene expression might hamper the utility of this methodology. The entire process of electroporation of mammalian cells will take cells in several ways, including permeabilization by sendai virus, trypsinization, osmotic shock, microinjection, electroporation, or after transfection of cells with expression vectors containing the genes of interest. The data shown exemplify how preset protocols can be used for optimizing electroporation conditions for the mammalian cell line of interest. Transfection of mammalian cells by electroporation nature.
Cell viability and electro transfection efficiency ete are dependent on various experimental factors, including pulse waveform, vector concentration, cell typedensity, and electroporation. Principles and mechanisms of mammalian cell transfection. Cultured mammalian cells offer an environment well suited for producing properly folded and functional mammalian proteins with appropriate post translational modifications1 2. Gene transfection of mammalian cells using membrane sandwich electroporation article in analytical chemistry 7915. The relevant parameters of vol electroporation of eukaryotic cells the multiporator in combination with the specially designed electroporation buffers is the optimum balanced system for the efficient and gentle electroporation of eukaryotic cells. A wide variety of mammalian cell types is used in gene transfection studies. Electroporation is a physical transfection method that uses an electrical pulse to create temporary pores in cell membranes through which substances like nucleic acids can pass into cells. Wash cells twice with optimem media without fbs life technologies. When using electroporation you apply an electric current across the cell. Square dc waveform provides the fusion pulse for electrofusion or is utilized in mammalian electroporation. In fusions between murine myeloma and t lymphoma lines, hybrid cell recoveries were typically one per 105 input myeloma cells. Highest transfection efficiency in cells at 90% confluency. Get a printable copy pdf file of the complete article 1.
Frontiers an efficient electroporation protocol for the. An efficient electroporation protocol for the genetic modification of mammalian cells. Mammalian cell fusion in an electroporation device. Electroporation for the efficient transfection of mammalian cells with dna. Despite many cells being easily transfected with this approach, other cells are rather difficult or hard to transfect, including protist cells of the genus leishmania. Transfection of dna or rna molecules into cultured mammalian cells can be accomplished using various methods and reagents. Establishing transfection methods that enable highly efficient dna. Optimization of electroporation for transfection of mammalian.
Flowthrough electroporation of mammalian cells in decoupled. Purification of total rna from mammalian cells and tissues. Electroporation protocol for tcells transfection protocol protocol no. Preset protocols were created to allow many factors that affect electroporation to be tested simultaneously. Change media on es cells night before electroporation. Yet, in many fields, including drug discovery, gene therapy, antibody production, and regenerative medicine, there has been a growing demand to deliver into cells membraneimpermeable exogenous agents, such as small drugs, proteins, and nucleic acids.
Transfer 50 l electrocompetent cells into a prechilled cuvette labeled controlcells on ice. Gene transfection of mammalian cells using membrane sandwich. Volker briese, universitatsfrauenklinik rostock electroporation can be used for both transient and stable transfection of mammalian cells. Efficient electroporation of mammalian cells in culture. Electroporation was carried out when the cells were at passage 5.
Cells are placed in suspension in an appropriate electroporation buffer and put into an electroporation cuvette. Electroporation is the cost effective method for introducing foreign dna into the primary and other hard to transfect cells. Mammalian cell culture was developed to provide vehicle for viral vaccine production and to produce monoclonal antibodies and other recombinant proteins. Electroporation is an attractive method for transfection because it is more rapid and involves fewer manipulations than other methods and can be used for many types of adherent. These will be a negative control no plasmid dna added 3. Transfection system the neon transfection system is a novel, benchtop electroporation device that employs an electroporation technology by using the pipette tip as an electroporation chamber to efficiently transfect mammalian cells including primary and immortalized hematopoietic cells, stem cells, and primary cells. The cells should be in the exponential phase of growth. B the viability of the neurons in each transfection well decreases as the voltage increases, thus the lowest voltage that produces reasonable transfection efficiency is optimal.
In the course of our studies in this area, we have developed a simple and economical method of fusing mammalian cells, using an electroporation device. Two hours after protoplast isolation, cells were transfected by electroporation. Im doing electroporation in mammalian cells, ive been reading some protocols, and now i know that the electroporation has to be done in a 1x106 cellml concentration, but how many cells should i seed per well in a 24well plate. Electroporated area is presented with dashed line voltage imposed by external electric field should reach a critical value to trigger. It is a highly efficient strategy for the introduction of foreign nucleic acids into many cell types, including bacteria and mammalian cells. Some cells, like hela cells, are easy to transfect with lipid reagents.
Es cell suspension and 50 ul of dna the concentration can be variable. Check confluency and differentiation of cells prewarm m10g medium, pbs and trypsin in 37oc water bath prepare cuvette for electroporation and place in hood things youll need. Search for user documents, sdss, vector maps and sequences, application notes. The transfection efficiency of electroporation can be affected. N4 c57bl6n es cells see separate protocol prepare dna see separate protocol for all medium and reagents see separate protocol change media on es cells night before electroporation. Molecular cloning, also known as maniatis, has served as the foundation of technical expertise in labs worldwide for 30 years. A standard electroporator was used to introduce affinitytagged bacterial effectors into mammalian cells. The day before plate cells at 45x106p100 to get the following day 8090% confluency. The introduction into mammalian cells of antibodies with specificities for endogenous cellular factors permits the functional assessment of such factors in the context of living cells.
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