Transfection by electroporation

As an alternative to the chemical transfection systems, the primary cells can be effectively transfected by electroporation (Appendix 5). Amaxa has developed a nucleofection (electroporation) system that is very effective for delivering expression vectors into cells at high efficiency and low cellular mortality. The step-by-step conditions for the transfection of numerous primary cell types, including human airway epithelial cells, can be found at http //www.amaxa.com/primary-cells.html. 

Teshigawara K, Katsura Y (1992), A simple and efficient mammalian gene expression system using an EBV-based vector transfected by electroporation in G2/M phase, Nucleic Acids Res. 20 2607-2611. 

If human embryo kidney (HEK) cells are to be transfected by electroporation, proceed as follows Confluent monolayers of cells are harvested by trypsinization as described above and resuspended in IX HBS at 2 x 107 cells/mL and kept on ice. Add 30 [xg of plasmid DNA to 0.5 mL of cells and transfer to a chilled, 0.4 cm gap electroporation cuvet. Pulse at 960 xF and 260 V. Replace the cuvet on ice for a further 5-10 min then transfer the cells to a 15 cm diameter Petri dish containing 15-20 mL of pre warmed complete medium. Incubate overnight at 37°C. Change the medium the next morning. 

Transfection by Electroporation. Materials 15 mg of plasmid DNA, 0.4 cm Electroporation cuvettes (BioRad, Hercules, CA, 165-2088), Electro-porator (BioRad Gene Pulser II) and Electroporation media (EM) (20% FBS/SF RPMI), 6-well tissue culture plates, SF RPMI, and complete RPMI. 

Human fibroblasts can be transfected by electroporation. For transfection, 4 X 10 cells are resuspended in 500 //I PBS containing 20 /itg DNA. The suspension is placed in an electroporation cuvette (Bio-Rad Laboratories, UK) with a gap of 0.4 cm, and kept in ice for 10 min before electroporation. For the electroporation, a Gene-Pulser Electroporator (Bio-Rad Laboratories, UK) can be used with the following working parameters voltage, 380 V capacitance, 850 fjF. After the pulse, the cells are resuspended in normal culture medium and plated onto cover-slips in 24-weU plates at a density of 1.5 x 10 cellsAvell (Bonazzi et al., 2005). 

In a similar study by Yang and Huang (1996), melanoma tumors in mice were in vivo transfected by the direct intratumoral administration of naked pDNA, resulting in reporter gene expression for up to ten days. In another study, Walker 256 carcinoma tumors in rats were found to express CAT after intratumoral injection of naked pDNA encoding CAT (Nomura et al., 1997). More recently electroporation has been used to deliver naked pDNA to tumors (described further in the following section). 

The mechanism by which electroporation enhances i.m. pDNA expression and efficacy requires further study. For muscle transfection, it has been demonstrated that electroporation appears to increase the number of muscle fibers transfected by as much as ten-fold (Dupuis et al., 2000). In addition, the number of muscle nuclei containing transfected pDNA is higher after electroporation. The mechanism by which electroporation enhances transfection of tumor tissue is unclear at this time. 

The linearized vector is transfected into an appropriate embryonic stem cell by electroporation. 

Ex vivo therapy is the transfection of cells outside the body. Typically, a small amount of tissue is removed from the patient and the cells within that tissue are put into the culture, which allows clonal expansion of the cells. The approach simplifies the delivery of the genes and allows for post-transfection manipulation of the cells. The genetically modified cells, typically blood, bone marrow, or others, are then returned back to the patient, usually by blood transfusion or direct engraftment. Ex vivo transfection of cells by electroporation can be done using either a discontinuous or a continuous process. 

Fig. 8. Replication assay. Recircularized viral genomes were transfected into C127 cells by electroporation, and low molecular weight DNA was isolated every day for 5 d posttransfection. The DNA was cleaved by Hindlll and Dpnl, and replicated viral genomes were detected by Southern blot analysis as described in the text.

Driver EC, Kelley MW (2010) Transfection of mouse cochlear explants by electroporation. Curr Protoc Neurosci Chapter 4 Unit 4.34.1-10... 

In addition to calcium phosphate transfection, methods for DNA transfer into mammalian cells by electroporation [31, 32] and by transfection mediated through cationic lipids, liposome [33-35], biohstics [36] and polymers [37, 38] have been developed. Most of these techniques have been reported to mediate higher transfection efficiencies as compared to calcium phosphate-mediated DNA transfer. Such claims must be regarded with some caution, as all DNA transfer techniques estabhshed so far suffer from high variabihty due to technical difficulties. Other factors to cause major variations in transfection efficiency are the type of cells used and the condition of the cells prior to transfection. 

Zheng, Q.A., Chang, D.C., 1991. High-efficiency gene transfection by in situ electroporation of cultured cells. Biochim. Biophys. Acta 1088, 104—110. 

Satyabhama, S., and Estonia, A. L. (1988) Short-term efficient expression of transfected DNA in human hematopoietic cells by electroporation Definition of parameters and use of chemical stimulators. DNA 7, 203-209. 

Fountain, J.W., Lockwood, W.K. Collins, F.S. (1988). Gene 68 167-172. Transfection of primary human skin fibroblasts by electroporation. 

To test the ability of the artificial mRNA to support GLP-1 expression in whole cells, Chinese hamster ovary cells were transfected with artificial mRNA by electroporation. Twelve hours after transfection, cells were harvested and GLP-1 was determined by sandwich ELISA. mRNA derived from both the 130-nt and the 170-nt RNA supported GLP-1 expression, albeit weakly, whereas control RNA with no cap 1 or poly(A) tail did not. 

Procedures for transfection by calcium phosphate-DNA coprecipitation and by electroporation are given in our earlier reviews (Cherbas et al. 1994 Cherbas and Cherbas 1998). The electroporation protocol for Kc cells (Cherbas et al. 1994) can be modified for S2 cells by increasing the voltage from 440 V/cm (Kc) to 715 V/cm (S2) (Cherbas and Cherbas 1998) the higher voltage also works well for S3 cells, for the shibire line EH34A3, and for the haploid line D (K. Klueg and M.A.T. Muskavitch, pers. comm.). 

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