20] Uptake of DNA and RNA into cells mediated by electroporation

Abstract

Publisher Summary This chapter outlines simple methods that allow the uptake of plasmid DNA and RNA into plant protoplasts. The DNA uptake is mediated by a series of high-voltage, direct-current electrical pulses. Zimmerman described an electric shock method (electroporation) for the introduction of drugs into mammalian red blood cells. The transformation of Bacillus cereus spheroplasts with plasmid DNA was stimulated by high-voltage electrical pulses. High-voltage pulses have been used to mediate the uptake of DNA encoding herpes virus thymidine kinase (TK) into mouse cells. A substantial increase in frequency of DNA transformation was obtained when mouse lymphocytes were treated with colchicines prior to electroporation. Recently, experiments were described in which Agrobacterium Ti plasmid DNA, a chimeric antibiotic resistance marker gene, plant genomic DNA, and tobacco mosaic virus RNA were successfully introduced into plant protoplasts by electroporation. Two types of electrical pulses are presently used for creating pores in the membranes of plant and animal cells. One type of electroporation instrument generates a square wave field with pulse decay duration in the microsecond range. A second type of instrument generates an exponential wave field with a pulse decay time in the millisecond range. It is mentioned that electroporation may now be applied for the genetic modification of genes involved in photosynthesis, which are transcribed in the chloroplasts of economically important crop plants.