Transformation electroporation

For the expression and maintenance of recombinant genes there is a need to introduce the recombinant vectors harbouring them into suitable hosts. The four main methods used to achieve this are transformation, electroporation, conjugation and transduction. 

Electroporation. When bacteria are exposed to an electric field a number of physical and biochemical changes occur. The bacterial membrane becomes polarized at low electric field. When the membrane potential reaches a critical value of 200—300 mV, areas of reversible local disorganization and transient breakdown occur resulting in a permeable membrane. This results in both molecular influx and efflux. The nature of the membrane disturbance is not clearly understood but bacteria, yeast, and fungi are capable of DNA uptake (see Yeasts). This method, called electroporation, has been used to transform a variety of bacterial and yeast strains that are recalcitrant to other methods (2). Apparatus for electroporation is commercially available, and constant improvements in the design are being made. 

One of the exciting features of the direct DNA delivery system is that it does not rely on an infection. The limited host range of other vector delivery systems is therefore irrelevant, and the way is opened for genetic engineering of cereals. Cereal protoplasts are equally amenable to uptake of foreign DNA after electroporation and the system therefore has potential for use with the major crop species. However, there is at present one drawback, namely that for cereals it has not yet proved possible to grow fertile whole plants from the genetically transformed cells. 

Transformation of plasmids pHispsaF and pCP47 into Thermosynechococcus elongatus (T. elongatus) cells was carried out by electroporation. 

The uptake of the plasmid into the bacterial cell is called transformation and in the laboratory can be induced in two ways. In one method the bacteria and DNA are placed in ice-cold CaCl2 (50 mmol l-1). This induces the DNA to stick to the outside of the bacteria. The temperature is then increased to 42 °C and the DNA enters the cell. The second method is by electroporation,... 

Therefore alternative techniques for gene transfer are necessary. While progress has been made in electroporation of Desulfovibrio (Rousset et al. 1991, 1998), the development of reliable and efficient procedures deserves further attention. Evidence has been obtained for the presence of a restriction barrier that limits transformation in D. vulgaris (Fu and Voordouw 1997). The efficiency of electroporation of plasmids into D. vulgaris was increased from an undetectable level to lO transformants... 

Because JC8679 competent cells for electroporation are not commercially available, we had to produce our own. We routinely prepare competent cells following the protocol reported previously (14). Although JC8679 competent cells for electroporation of 10 -10 cfii/pg pUC19 are routinely prepared, the apparent transformation efficiency for the ORF trap is much lower (typically 50-100 colonies per 30 pL of transformation culture). 

The calli used to generate cell lines can be transgenic and express the vaccine or therapeutic protein of interest. Transfer of foreign genes into calli takes place by Agrobacterium-va.Q6M.td transformation, particle bombardment, electroporation of protoplasts, or by viral vectors (Fischer... 

Available methods for carrying DNA into an animal cell vary in efficiency and convenience. Some success has been achieved with spontaneous uptake of DNA or electroporation, techniques roughly comparable to the common methods used to transform bacteria. They are inefficient in animal cells, however, transforming only 1 in 100 to 10,000 cells. Microiqjection—the injection of DNA directly into a nucleus, using a very fine needle—has a high success rate for skilled practitioners, but the total number of cells that can be treated is small, because each must be injected individually. 

Chuang, S.-E., Chen, A.-L., and Chao, C.-C. (1995) Growth of E coli at low temperature dramatically increases the transformation frequency by electroporation. Nucleic Acids Res. 23, 1641... 

Transformation is done by electroporation of 71-18 or DH5oi cells. According to our experience, the use of chemically competent cells is not recommended, because it gives much lower yields of transformants. 

Make a transformation test before creating the library, by electroporating 100 pL of competent cells with 1 pL of the ligation mix, and compare the transformation efficiency with that of a standard plasmid like pUC18. For reaching high library sizes, each transformation should yield between 106 and 107 transformants. Repeat electroporations to reach the desired diversity. 

Fig. 7.1. A planarian individual of a line transformed with a Hermes transposon-derived vector 12 months after microinjection and subsequent electroporation. Besides the transposon part, the vector contained the E(enhanced)GFP reporter gene under the control of an artificial promoter that responds to the transcription factor Pax6 (Gonzalez-Estevez et ai, 2003). Binding sites for this transcription factor are found in rhodopsin and other photoreceptor-specific genes. Reporter-gene activity was found in the eyes (arrows) of transformed individuals or in regenerated heads as a mosaic of EGFP-positive photoreceptor cells. Figure courtesy of Dr E. Salo.

For transformation experiments in general, biochemical or physical methods such as microinjection, electroporation or lipofection are available. These methods have been successfully applied for a variety of organisms including protozoan parasites (Clayton, 1999 De Koning-Ward et al., 2000) and were consequently tested for their ability to generate transiently transformed schistosomes by different laboratories. 

Salmenkallio, M., Hannus, R., Teeri, T.H. Kauppinen, V. (1990). Regulation of a-amylase promoter by gibberellic acid and abscisic acid in barley protoplasts transformed by electroporation. Plant Cell Reports 9, 352-5. 

Titomirov, A.V., Sukharev, S. and Kistanova, E. (1991) In vivo electroporation and stable transformation of skin cells of newborn mice by plasmid DNA. Biochim. Biophys. Acta., 1088,131-134. 

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