Generation of Trypanosoma cruzi Colombiana parasites expressing nanoluciferase.

(A) Schematic representation of pBEX-NLuc and its integration into the T. cruzi ribosomal RNA gene locus by homologous recombination. 18SA and 18SB: T. cruzi 18S ribosomal RNA; IR1-3: Strigomonas culicis tubulin intergenic regions [29]; NLUC: nanoluciferase gene; NEO: neomycin phospotransferase gene. (B) PCR amplification of the 18SA fragment followed by the IR1 and NLuc sequences was performed using genomic DNA extracted from TcCOL-NLuc and primers 18SF and NLuc R (lane 2). Genomic DNA extracted from TcCOL-wt and DNA from pBEX-NLuc with identical set of primers, were included as negative controls (lanes 3 and 4, respectively). The NLuc gene was amplified from pBEX-NLuc using primers NLuc F and NLuc R and used as positive control (lane 5). PCR products were evaluated in a 1% agarose gel stained with ethidium bromide. 1 Kb ladder (lane 1); non-template PCR control (lane 6).