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 markers. 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 donovani 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.
Ommen
Gabi
G
Leishmaniasis Research Group, Bernhard Nocht Institute for Tropical Medicine, Bernhard Nocht St 74, D-20359 Hamburg, Germany.
Lorenz
Sabine
S
Clos
Joachim
J
D000483:Alleles; D000818:Animals; D015153:Blotting, Western / Q000379:methods; D002478:Cells, Cultured; D016054:DNA, Protozoan / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics; D004352:Drug Resistance, Microbial / Q000235:genetics; D005240:Feasibility Studies; D018390:Gene Targeting / Q000379:methods; D017125:Genes, Protozoan; D007893:Leishmania donovani / Q000235:genetics* / Q000254:growth & development; D008264:Macrophages / Q000469:parasitology; D016254:Mutagenesis, Insertional / Q000379:methods; D015800:Protozoan Proteins / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D014162:Transfection / Q000379:methods*
D016054:DNA, Protozoan; D004274:DNA, Recombinant; D015800:Protozoan Proteins