title: Use of Leishmania donovani field isolates expressing the luciferase reporter gene in in vitro drug screening.
abstract: Currently available primary screens for the selection of candidate antileishmanial compounds are not ideal. These techniques are time-consuming, laborious, and difficult to scale and require macrophages, which limit their use for high-throughput screening. We have developed Leishmania donovani field isolates that constitutively express the firefly luciferase reporter gene (luc) as a part of an episomal vector. An excellent correlation between parasite number and luciferase activity was observed. luc expression was stable, even in the absence of drug selection, for 4 weeks. The transfectants were infective to macrophages, and intracellular amastigotes exhibited luciferase activity. The suitability of these recombinant field isolates for in vitro screening of antileishmanial drugs was established. The luciferase-expressing sodium stibogluconate-resistant cell lines offer a model for the screening of compounds for resistance. The system is in routine use at the Central Drug Research Institute, Lucknow, India, for high-throughput screening of newly synthesized compounds.
authors.affiliation: Division of Biochemistry, Central Drug Research Institute, Chatter Manzil Palace, M. G. Road, Lucknow 226 001 (UP), India.
authors.lastname: Ashutosh
authors.forename: Suman
authors.initials: S
authors.lastname: Gupta
authors.lastname: Ramesh
authors.forename: Shyam
authors.initials: S
authors.lastname: Sundar
authors.forename: Neena
authors.initials: N
authors.lastname: Goyal
mesh_terms: D000818:Animals; D000967:Antimony Sodium Gluconate / Q000494:pharmacology; D000981:Antiprotozoal Agents / Q000494:pharmacology*; D002478:Cells, Cultured; D016054:DNA, Protozoan / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004351:Drug Resistance; D017930:Genes, Reporter / Q000235:genetics*; D006801:Humans; D007893:Leishmania donovani / Q000187:drug effects* / Q000235:genetics*; D007898:Leishmaniasis, Visceral / Q000469:parasitology; D008156:Luciferases / Q000235:genetics*; D008264:Macrophages / Q000469:parasitology; D030781:Organisms, Genetically Modified; D015203:Reproducibility of Results; D014162:Transfection
chemical_list: D000981:Antiprotozoal Agents; D016054:DNA, Protozoan; D008156:Luciferases; D000967:Antimony Sodium Gluconate