Fig 6. Constitutive expression of a TS gene in CL-14.

The pROCKNeo vector used for transfection of CL-14 has the TS gene (Tc00.1047053509495.30) flanked by the ribosomal promoter and sequences containing signals for mRNA processing derived from the constitutively expressed housekeeping genes TcP2β (at the 5’ end) and gapdh (at the 3’ end) (A). Total RNA purified from epimastigotes from WT and transgenic parasites were subjected to northern blot and hybridized with a ³²P-labelled probe that contains sequences corresponding to the C-terminal SAPA repeats present in the TS gene. Lower panel shows ethidium bromide staining of rRNAs in the same gel before transferring to the membrane (B). Total protein extracts from epimastigotes from WT and transgenic parasites were evaluated for the expression of the transfected TS gene by western blotting with a monoclonal antibody anti-SAPA (C). The infection profiles of four cloned cell lines derived from CL-14 parasites transfected with the TS gene or with the empty pROCKNeo vector, were compared to WT CL-14 and CL Brener in in vitro infection assays of Vero cells. Equal numbers of tissue culture derived trypomastigotes from each parasite cultures were added to Vero cell monolayers and the total number of trypomastigotes released in the supernatant (D) or the numbers of trypomastigotes released in the supernatant each day post-infection were evaluated over 8 days (E). Five replicates for each infection experiment were performed.