Abstract:
Objectives: Several Leishmania strains with episomal expression of green fluorescent protein (GFP) require constant drug pressure for its continuous expression and hence limit its use for ex-vivo or in-vivo system. The aim of this study was to alleviate this problem by stably integrating the GFP gene into the parasite genome so as to use these transfectants for ex-vivo and in-vivo drug screening.
Methods: The GFP gene was integrated at downstream of 18S ribosomal promoter region. The GFP expressing parasites both Sodium Stibogluconate (SAG) susceptible (2001) and resistant isolates (2039) after initial selection were grown without adding G 418. The infectivity of these transfectants to macrophages (J 774.1) as well as to hamsters was checked. The ex-vivo screening assay was standardized using standard antileishmanial drugs.
Results: A constitutive and enhanced expression of GFP in promastigote and amastigote stages was achieved for about 12 months without any need of drug pressure. These were highly infective to macrophage cell lines as well as to hamsters as observed by fluorescence microscopy and flow cytometry (FACS). GFP tagged promastigotes as well as intracellular amastigotes were found to be highly susceptible to all antileishmanials viz. miltefosine, amphotericin B and pentamidine in a concentration-dependent manner except SAG which was inactive against the GFP-Promastigotes as well as SAG resistant intracellular amastigotes correlating well with earlier reports.
Conclusions: The GFP-transfectants were found suitable for FACS-based ex-vivo screening assays. These were also infective to hamsters upto day 60 p.i.
