title: Improvement of the green fluorescent protein reporter system in Leishmania spp. for the in vitro and in vivo screening of antileishmanial drugs.
abstract: Development of new therapeutic approaches for leishmaniasis treatment requires new high throughput screening methodologies for the antileishmanial activity of the new compounds both in vitro and in vivo. Reporter genes as the GFP have become one of the most promissory and widely used tools for drug screening in several models, since it offers live imaging, high sensibility, specificity and flexibility; additionally, the use of GFP as a reporter gene in screening assays eliminates all the drawbacks presented in conventional assays and also those technical problems found using other reporter genes. The utility of the GFP as a reporter gene in drug screening assays with Leishmania parasites depends on the homogeneity and stability of the GFP transfected strains. Stable expression of the GFP in the Old World Leishmania species has been demonstrated using integration vectors; however, no reports exist yet about the success of this methodology in the New World species. Here we report the generation of New World Leishmania strains expressing the GFP protein from an integration vector, which replaces one copy of the 18S RNA in the chromosome with the GFP coding sequence by homologous recombination. We also prove that the expression of the integrated GFP is stable and homogeneous in the transfected parasites after months in culture without selective pressure or during its use in hamster infection assays. The fluorescent strains are useful for in vitro, ex vivo and in vivo drug screening assays since no considerable variations in virulence or infectivity where seen attributable to the genetic manipulation during both in vitro and in vivo infection experiments. The platform described here for drug testing assays based on the use of stable fluorescent Leishmania strains coupled to flow cytometry and fluorescent microscopy is more sensitive, more specific and faster than conventional assays used normally for the evaluation of compounds with potential antileishmanial activity.
authors.affiliation: Program for Study and Control of Tropical Diseases, School of Medicine, University of Antioquia, Medellin, Colombia.
authors.forename: Sergio A
authors.initials: SA
authors.lastname: Pulido
authors.forename: Diana L
authors.initials: DL
authors.lastname: Muñoz
authors.forename: Adriana M
authors.initials: AM
authors.lastname: Restrepo
authors.forename: Carol V
authors.initials: CV
authors.lastname: Mesa
authors.forename: Juan F
authors.initials: JF
authors.lastname: Alzate
authors.forename: Iván D
authors.initials: ID
authors.lastname: Vélez
authors.forename: Sara M
authors.initials: SM
authors.lastname: Robledo
mesh_terms: D000818:Animals; D000981:Antiprotozoal Agents / Q000302:isolation & purification*; D006224:Cricetinae; D004195:Disease Models, Animal; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005434:Flow Cytometry / Q000379:methods; D015870:Gene Expression; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D007891:Leishmania / Q000187:drug effects* / Q000235:genetics; D007896:Leishmaniasis / Q000188:drug therapy* / Q000469:parasitology; D008647:Mesocricetus; D008856:Microscopy, Fluorescence / Q000379:methods; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods*
chemical_list: D000981:Antiprotozoal Agents; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins