One-Step Generation Of Double-Allele Gene Replacement Mutants In Leishmania Donovani

Due to the apparent lack of sexual recombination in Leishmania spp., gene replacement strategies in this diploid organism necessitate the subsequent targeting of two gene alleles by using two constructs, bearing different antibiotic resistance markets. This approach is time consuming and often gives rise to spontaneous amplification of the targeted gene, nullifying efforts to create functional null mutants. Here, we show that by using two homologous recombination constructs in a co-transfection of Leishmania donovard promastigotes, we can obtain double-allele gene replacement mutants. This approach reduces the time required for the generation of functional null mutants and the number of in vitro passage cycles, potentially limiting culture-associated artefacts. (C) 2008 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.