Integration of pTREX-luc into the Dm28c genome . Southern blot of epimastigote genomic DNA from Dm28c stably transfected with pTREX, cloned Dm28c-luc, transfected with pTREX-luc, and Dm28c wild type were digested with EcoRI and probed with neo/gapdh sequence (A). PCR amplification of ribosomal promoter regions followed by the HX1 and luciferase sequences was performed in genomic DNA from Dm28c wild type (lanes 1, 4 and 7), as negative control; from cloned Dm28c-luc, (lanes 2, 5 and 8); and DNA from pTREX-luc plasmid, (lanes 3, 6 and 9), as positive control, using the specific primers: F-TSP1/R-LUC (lanes 1, 2 and 3) and; F-RS pTREX/R-LUC (lanes 4, 5 and 6). The luciferase gene was amplified by PCR, with F-LUC/R-LUC pair of primers (lanes 7, 8 and 9) (n = 3). All the PCR fragments were evaluated in an agarose gel and the fragments of 2.4 and 2.7 kb, amplified from Dm28-luc genomic DNA (lanes 2 and 5), were gel purified (B). The rescued PCR fragments of 2.4 and 2.7 kb and the pTREX-luc plasmid were sequenced with primers: F-TSP1; R-TSP1; F-RS pTREX and R-RS pTREX (n = 2). Ribosomal promoter sequences from rescued PCR fragments and from the pTREX-luc plasmid were aligned with the clustalw 2.1 multiple sequence alignment (C). Asterisks correspond to the identical region between the ribosomal promoter of Dm28c-luc strain and the pTREX-luc plasmid; TSP, transcription start point; RS, recombination site, underlined; BamHI, KpnI and SacII, restriction enzymes in the pTREX-luc plasmid.