Use of Leishmania donovani field isolates expressing the luciferase reporter gene in in vitro drug screening.
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.
Ashutosh
Division of Biochemistry, Central Drug Research Institute, Chatter Manzil Palace, M. G. Road, Lucknow 226 001 (UP), India.
Gupta
Suman
S
Ramesh
Sundar
Shyam
S
Goyal
Neena
N
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
D000981:Antiprotozoal Agents; D016054:DNA, Protozoan; D008156:Luciferases; D000967:Antimony Sodium Gluconate