The mse method is able to provide better gene confinement near the cell surface to facilitate gene transport into the cells and thus shows significant improvement over transgene expression of mammalian cells compared to current electroporation techniques. Gene transfection of mammalian cells using membrane sandwich. A wide variety of mammalian cell types is used in gene transfection studies. Transfection efficiency of rpe with this new technique exceeded that of standard electroporation by a factor 10,000. Haas k et al 2001 singlecell electroporation for gene transf. Transfection by electroporation using square wave pulses, as opposed to exponentially decaying pulses, was found to be significantly increased by repetitive pulses. Any cell types like mammalian cells, live cells, bacterial cells or any other cells can be effectively used in it. Strategy for increased efficiency of transfection in human cell. Numerous chemical and physical methods have been used to introduce dna expression vectors into mammalian cells both in vitro and in vivo. Highly efficient transfection of mammalian cells by electric. As an example for cells which can only be poorly transfected, we used hl60 a promyelocyte cell line. Here we present a method for the forward transfection of cas9 nuclease nls rnps into adherent mammalian cells using conventional lipofection reagents. D elivery solutions for immune cells thermo fisher scientific. Electroporation of functional bacterial effectors into mammalian cells.
Here we demonstrate an easy and inexpensive method to increase the efficiency of electroporation of mammalian cell lines up to 8fold. Size specific transfection to mammalian cells by micropillar. Finally, the use of gold nanoparticles gnps combined with electroporation has shown the enhanced dna transfection efficiency of mammalian cells 22. Electroporation using square wave pulses is performed with a relatively low voltage, but unlike exponential waveforms. Aug 01, 1989 analytical biochemistry 180,269275 1989 optimization of electroporation for transfection of mammalian cell lines grai l. Cell transfection introduction reference, mit opencourseware the following link provides an overview of two common transfection approaches electroporation and lipofection in addition to information about transient transfection and stable transfection of mammalian cells. The alternate protocol outlines modifications for preparation and transfection of plant. Transfection of primary human skin fibroblasts by electroporation.
Spontaneous transfection of mammalian cells with plasmid dna. Thus, rf electroporation represents an improved methodology for transfection of human cell lines. Optimizing electroporation conditions for highefficiency. Jan 01, 2015 transfection of mammalian cells cell transfection can be achieved through numerous methods. This protocol was adapted from dna transfection by electroporation in molecular cloning. A simple and reproducible procedure for the introduction of dna into mammalian cells by electroporation is described.
May 15, 2017 electroporation generates an electrical field across the cell membrane to induce pore opening. Transfection of mammalian cells by electroporation nature methods. The 24well optimization protocols were performed using the 10 l neon tip, and the cells were dispensed into. The mechanism of electroporation in mammalian cells is poorly understood. Cell transfection was performed using a single or dual pulse mode. The transfection efficiency for these was an average of 26. However, effective transformation by electroporation requires careful optimization of electric field strength and pulse characteristics. 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. Optimal electroporation condition for small interfering rna. But as with other transfection methods, the optimal conditions for electroporation of untested cell lines must be determined experimentally.
The parameters involving the cells, the dna, and the electric field are investigated. Pdf transfection is a powerful analytical tool enabling study of the function of genes and. Nanomaterialassisted lightinduced poration and transfection. Deaedextran enhances electroporation of mammalian cells. Lasermediated transfection also known as optoporation. Electroporation the use of highvoltage electric shocks to introduce dna into cells can be used with most cell types, yields a high frequency of both stable transformation and transient gene expression, and, because it requires fewer steps, can be easier than alternate techniques. Transfection of crisprcas9 nuclease nls ribonucleoprotein. The available protocols are established by trialanderror, instead of. Transfection by electroporation potter 2003 current. Optimizing electrotransfection of mammalian cells in vitro. The first pulse 6 kvcm, 10 as creates pores efficiently, whereas transfection efficiency te is low. These methods improve the ability to obtain high efficiency gene transfer into many mammalian cell types. When we used this approach for multigene targeting.
A transfection is a process that forces nucleic acids into a cell. Establishing transfection methods that enable highly efficient dna. Electroporation for the efficient transfection of mammalian. This protocol describes transfection of plasmid dna into mammalian cell lines using electroporation, a process whereby external application. Protocols are described for the use of electroporation in vivo to perform gene therapy for cancer therapy and dna vaccination. Transfection of mammalian cells by electroporation nature. The cell dna mixtures, in 1 cm transfection cuvettes, were nucleoporated according to aspecificpredefinedprogram. Following the electroporation, the cells were incubated in their respective culture medium preheated to 37c for 10 min, and then seeded into cell typespecific growth medium.
Feb 08, 2010 the cell dna mixtures, in 1 cm transfection cuvettes, were nucleoporated according to a specific predefined program. High transfection rates for adherent mammalian cell lines and primary cell cultures. Optimization of electroporation for transfection of mammalian cell. It is a highly efficient strategy for the introduction of foreign nucleic acids into many cell types, including bacteria and mammalian cells. In this chapter, in order to present these methods in a clear and organized way, we have classified them into three main categories. Transfection of mammalian cells by electroporation. Magneto transfection and laseropto transfection are relatively new techniques that use magnetic forcesoptical energy to permeabilize cells to deliver genetic material. Protein electroporation 2325 is a method to introduce proteins into living cells via electropermeabilization, also known as electrotransfection.
The eukaryotic cell membrane or cell wall is made up of lipids, phospholipids, proteins and chitin or pectin, mostly. Plasmid dna is commonly electroporated into mammalian cells to overexpress a gene of. When a cell is exposed to a pulse of high electric field, its cell membrane quickly becomes permeabilized. Spontaneous transfection, mammalian cells introduction dnamediated gene transfer into cultured mammalian cells is a routine approach for studying gene function in eukaryotes. Overall, we have presented a device and electrosonoporation method that meets or outperforms the transfection efficiency and cell viability standards for hela cells set by other reported electroporation and sonoporation methods. Analytical biochemistry 180,269275 1989 optimization of electroporation for transfection of mammalian cell lines grai l. Optimization of electroporation for transfection of human fibroblast cell lines with. Electroporation has proven to be a convenient method for transfection of many cell types.
Experimental strategies in efficient transfection of. Electroporation and electrophoretic dna transfer into cells. A number of cell lines including some neuron, t cell, fibroblast, and epithelial cell lines have demonstrated resistance to common cationic lipid transfection reagents 14. In this way, cell size specific electroporation is conveniently done and contributed to a 2. The basic protocol describes the electroporation of mammalian cells. Fundamental study on a gene transfection methodology for. Selection for permanently transfected cells and for cells carrying. Using these methods, we report nucleasemediated indel rates of up to 94% in jurkat t cells and 87% in induced pluripotent stem cells ipsc for a single target. We have used the transient expression of the firefly luciferase gene as a rapid and sensitive indicator of gene expression to describe the effects on transfection efficiency of altering. However, very little is known about the basic mechanisms of dna transfer and cell response to the electric pulse. Results show that the cell viability 98% three days after electroporation.
There are several ways in which to introduce cas9guide rna rnp complexes into cells. Increased transfection efficiency by the directed transport, especially for low amounts of nucleic acids high transfection rates for adherent mammalian cell lines and primary cell cultures suspension cells and cells from other organisms also successfully transfected but need to be immobilized mild treatment of cells. Optimization of electroporation for transfection of mammalian. Other methods for transfecting cells with dna are described in transfection mediated by deae. The longterm cell viability after electroporation was also evaluated without delivery of pi dye, because pi dye is cytotoxic for cells after 24 h. Electroporation efficiency in mammalian cells is increased by. Therefore, the condition of electroporation for other cells must be determined experimentally. The two most common waveforms for mammalian cell electroporation are exponential decay and square wave. Exponential decay waveform electroporation of mammalian cells is typically done using high capacitance and low voltage. Transfection of dna molecules into mammalian cells with electric pulsations, which is socalled electroporation, is a powerful and widely used method that can be directly applied to gene therapy.
Electroporation for transfection and differentiation of. Hippocampal neurons were transfected with an average efficiency of 17. Introduction of foreign dna into the nucleus of eukaryotic cells. Transfection by electroporation current protocols wiley. Safe and effective nonviral dna delivery to the mammalian retina may help to materialize the enormous potential of the ocular gene therapy. The procedure has been applied to a broad range of animal cells.
Cell viability was greater than 95% after the delivery figure 3b. Transfection of cochlear explants by electroporation. Cell synchronization effect on mammalian cell permeabilization. Pc12 is an established rat pheochromocytoma cell line, which responds to exposure to ngf with cessation of growth, expression of cytoplasmic processes, and. 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. Efficient in situ electroporation of mammalian cells grown on. Thus, electroporation is employed for in vitro transfection experiments. One drawback has been that it requires large amounts of dna.
Optimization of transfection methods for huh7 and vero cells. In addition, electroporation often requires more cells than chemical methods because of substantial cell death, and extensive optimization often is required to balance transfection efficiency and cell viability. An electroporation protocol for efficient dna transfection in. Recent advances in mammalian cell transfection techniques. By introducing wellpatterned micropillar array on the electrode surface, the number of pillars each cell faces varies with its cell membrane surface area, despite their large population and random locations. Keywords pc12 cells cell culture dna transfection dna electroporation ngf neural differentiation introduction pc12 cells are a cell line originating from pheochromocytoma in the rat adrenal medulla schaefer et al. Request pdf gene transfection of mammalian cells using membrane sandwich electroporation to avoid safety issues such as immune response and cytotoxicity associated with viruses and liposomes. Mariepierre rols, mechanism by which electroporation mediates dna migration and entry into cells and targeted tissues, electroporation protocols, 10. Electroporation of mammalian cells using microchanneltype electroporation chip. Small pores could be made in the cell membrane during the shorts, allowing the gene electrotransfer.
Mammalian cell penetration,sirna transfection, and dna. The use of electric fields to facilitate transfer of small molecules, dyes, or dna into living cells was first demonstrated in the early 1980s neumann et al. Atcc ccl240 and tr146 cells squamous cell carcinoma cell line. An electroporation protocol for efficient dna transfection in pc12 cells.
Knutson and yee 1987 alluded to successful transfection of hsfs by electroporation but did not provide any confirming data. Electroporation as a technique for the transfer of macromolecules. Alternative transfection approaches including electroporation 5 and virusmediated sirna. The protocols are preprogrammed with the optimal parameters for some cells. Aug 11, 1995 efficient electroporation can be achieved under conditions of minimal cell killing, and can be performed with quiescent cells as well as with confluent epithelial sheets. Transfection of mammalian cells with fluorescent protein fusions. Investigation of plasmid dna delivery and cell viability dynamics.
Electroporation is a physical transfection method, in which short highvoltage electrical pulses are used to transiently permeabilize the cell membrane and allow transgenetic material into the cells. Mammalian cell penetration, sirna transfection, and dna. Comparative transfection of dna into primary and transformed. Establishing transfection methods that enable highly efficient dna uptake has become increasingly important. Electroporation can be a highly efficient method for introducing dna molecules into cultured cells for transient expression of genes or for permanent genetic modification. Electrotransfection of mammalian cells using microchannel. During this permeabilized state, macromolecules from the external medium can enter the cell. Electroporation has been used to successfully transfect primary human fetal fibroblasts and a large number of immortalized mammalian cultures chu et al. Pdf gold nanoparticles enhanced electroporation for. Dna transfection by electroporation sambrook and russell 2006a, electroporation nagy et al. Genetic materials can then enter the cells when the pores are transiently open. The method is a useful extension of electroporation technology, and will allow the application of electroporation to a wider spectrum of biological systems. The neon transfection system efficiently delivers nucleic acids, proteins, and sirna into all mammalian cell types including primary and stem cellswith a high cell survival rate.
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