Fig. 1.

Generation and analysis of luminescent Trypanosoma cruzi. (A) Construct and cloning strategy used to generate luminescent T. cruzi. pBS:THT-x-T (obtained from Wesley Van Voorhis, University of Washington), a plasmid with a pBluescript backbone and a hygromycin-resistance gene, was modified by insertion of the firefly luciferase gene. After linearization with SalI, the plasmid was transfected into T. cruzi epimastigotes and the Hyg and Luc genes integrated into the tubulin locus by homologous recombination. (B) Serial dilutions of antibiotic-resistant, T. cruzi epimastigote transfectants and wild-type epimastigotes were examined for luciferase activity. A black, 96-well plate containing 50 μL suspensions of parasites, mixed with 50 μL of Steady Glo reagent was read by a SpectraMax Gemini XS Microplate Spectrofluorometer and analyzed with SoftmaxPro 4.8 software. (C) Luminescent trypomastigotes and amastigotes were examined for luciferase activity in active, in vitro rat myoblast infections using parasite:myoblast ratios of 40:1, 20:1, 10:1, 5:1, 2:1 or uninfected (NI). Five days p.i., D-luciferin was added to each well and the 6-well plate was imaged with the Xenogen IVIS system after a 5-min incubation (see Materials and methods for detailed description). In the pseudocolor image the luciferase activity or photon intensity ranges from the lowest intensity (blue) to highest intensity (red). Abbreviations: RLU, relative light units; min, minimum.