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Spirochetes have complex life cycles and are associated with a number of diseases in humans and animals. Despite their significance as pathogens, spirochete genetics are in their early stages. However, gene inactivation has been achieved in Borrelia burgdorferi, Brachyspira hyodysenteriae, and Treponema denticola. Here, we review methods that have been used in spirochetes for gene inactivation and DNA exchange, with a primary focus on B. burgdorferi. We also describe factors influencing electrotransformation in B. burgdorferi. In summary, optimal transformation frequencies are obtained with log phase bacteria, large amounts of DNA (up to 50 microg per transformation), and high field strength (12.5-37.5 kV/cm). Infectious B. burgdorferi isolates transform with frequencies 100-fold lower than those found for high passage, non-infectious strains. Surface characteristics of the bacteria, which often correlate with infectivity, are among the obstacles to effective transformation by electroporation.