title: Development and validation of four Leishmania species constitutively expressing GFP protein. A model for drug discovery and disease pathogenesis studies.
abstract: Green fluorescent protein (GFP)-parasite transfectants have been widely used as a tool for studying disease pathogenesis in several protozoan models and their application in drug screening assays has increased rapidly. In the past decade, the expression of GFP has been established in several Leishmania species, mostly for in vitro studies. The current work reports generation of four transgenic parasites constitutively expressing GFP (Leishmania mexicana, Leishmania aethiopica, Leishmania tropica and Leishmania major) and their validation as a representative model of infection. This is the first report where stable expression of GFP has been achieved in L. aethiopica and L. tropica. Integration of GFP was accomplished through homologous recombination of the expression construct, pRib1.2αNEOαGFP downstream of the 18S rRNA promoter in all species. A homogeneous and high level expression of GFP was detected in both the promastigote and the intracellular amastigote stages. All transgenic species showed the same growth pattern, ability to infect mammalian host cells and sensitivity to reference drugs as their wild type counterparts. All four transgenic Leishmania are confirmed as models for in vitro and possibly in vivo infections and represent an ideal tool for medium throughput testing of compound libraries.
authors.affiliation: School of Science, University of Greenwich at Medway, Chatham ME4 4TB, UK.
authors.forename: Asha Parbhu
authors.initials: AP
authors.lastname: Patel
authors.affiliation: School of Science, University of Greenwich at Medway, Chatham ME4 4TB, UK.
authors.forename: Andrew
authors.initials: A
authors.lastname: Deacon
authors.affiliation: School of Science, University of Greenwich at Medway, Chatham ME4 4TB, UK.
authors.forename: Giulia
authors.initials: G
authors.lastname: Getti
mesh_terms: D000981:Antiprotozoal Agents / Q000494:pharmacology*; D045744:Cell Line, Tumor; D055808:Drug Discovery / Q000379:methods*; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007891:Leishmania / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D007896:Leishmaniasis / Q000188:drug therapy / Q000473:pathology*; D030781:Organisms, Genetically Modified; D012680:Sensitivity and Specificity; D013997:Time Factors
chemical_list: D000981:Antiprotozoal Agents; D049452:Green Fluorescent Proteins