Creation of Tetracycline Regulatable Knockouts of ORFF Gene from the LD1 Locus of Leishmania donovani

Abstract

The tetracycline repressor/operator system was adapted for use in Leishmania donovani. In order to investigate the possible importance and function of open reading frame F (ORFF) in Leishmania donovani strain AG83 that has a single copy of ORFF in the LD1 locus on chromosome 35, we have attempted to generate tetracycline regulatable single allele mutants of ORFF. Leishmania cells expressing the tetracycline repressor were transfected with tetracycline inducible vector expressing ORFF gene and having hygromycin as the selectable marker. The first endogenous genomic copy of ORFF was knocked out by homologous replacement with an inducible copy. Single ORFF gene disruption having an inducible copy of ORFF did not affect the growth of the parasites. This study, even though not conclusive, shows the possibility of generating functional tetracycline regulatable single knock out mutants of Leishmania donovani. The present study also demonstrates that ORFF gene might be an important gene and may be involved in various functions like attachment of Leishmania to the macrophages.