Animal Cell Electroporation and Electrofusion Protocols by Jac A. Nickoloff

By Jac A. Nickoloff

This fantastic number of well-tested protocols for the electroporation of proteins and DNA into insect, fish, and mammalian cells additionally comprises confirmed animal telephone electrofusion strategies for experiences of somatic telephone genetics and improvement, and for producing monoclonal antibodies. it really is distinct through its insurance of significant version phone varieties from many organisms and tissue kinds and its certain directions for the expansion and education of particular cells to accomplish optimal transfection. Its vast reference lists, citations of other move tools, recommendation on pitfalls to prevent, and outlines of anticipated effects determine simply reproducible effects even for newcomers.

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Similarly, DNA can enter cells for many minutes after electroporation, but probably not through the same mechanism by which small molecules enter cells, In our experience, the electroporation conditions described here for the introduction of restriction enzymes into cells do not result in successful transfection of DNA into the samecells. Generally, DNA is introduced more efficiently after a long pulse of fixed voltage, or after repeated short pulses, near the minimum voltage that causes permeabilization (21).

The fucosidase activity was assayed after 48 h. The transfection efficiency given by the enzyme activity of transfected cells is shown in Fig. 4. 4 ms. 5 kV m&m. Below this limit, the transfection efficiency is approximately linear with ET. 5 kV m&m. A single point (solid square) obtained using 4 kV/cm field strength pulses is exceptionally low in transfection efficiency, perhaps owing to too much cell death caused by irreversible membrane permeabilization. The effect of pulse strength and duration on the transfection of CHO cells was investigated by Wolf et al.

1990) High efficiency transformation of intact yeast cells by electric field pulses. Biotechnology S(3), 223-227. 4. , Shibata, A. (1991) Gene transfer into human leukemia cell lines by electroporation: experiments with exponentially decaying and square wave pulse. Leukemia Res. 15(6),507-513. 5. , Rhodes, S. , and Kaper, J. (1989) Effects of electroporatron profiles on the mcorporation of viral RNA into tobacco protoplasts. Biotechniques 7(10), 1124-1131. 6. Xie, T. and Tsong, T. (1992) Study of mechanisms of electric field-induced DNA transfection III, Electric parameters and other conditions for effective transfection Biophys.

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