Figure 1

Production of cell lines stably expressing tetR and T7 RNA polymerase. (A) Simplified map of pLEW13 indicating the relative locations of the three transgenes [7]. (B) CHEFE analysis of chromosomal DNA isolated from CL-Brener [pLEW13] epimastigotes showing aberrant migration of the pLEW13 DNA. Lanes 1–3, a 0.8% PFC agarose CHEFE gel (auto-algorithm set for 300 kb-3 Mb separation). Lanes 1 (Saccharomyces cerevisiae size standards (Bio-Rad)) and 2 (CL-Brener [pLEW13]), the ethidium bromide stained gel. Lane 3, an autoradiograph obtained with the T7 RNA polymerase probe. Lanes 4–6, a 1.0% PFC agarose CHEFE gel (auto-algorithm set for 300 kb-1 Mb). Lanes 4 (S. cerevisiae size standards) and 5 (CL-Brener [pLEW13]), the ethidium bromide stained gel. Lane 6 is an autoradiograph obtained with the T7 RNA polymerase probe. Molecular sizes are given in kb. (C) Expression of the transgenes for T7 RNA polymerase and tetR in pLEW13 transformed epimastigotes. 10 μg total RNA was blotted and hybridised with either the T7 RNA polymerase (T7 POL) or tetR probes. (D) Splice acceptor sites used by T. cruzi to process the transcripts as mapped by RT-PCR. The AG dinucleotide sites of spliced leader addition identified following sequencing of the RT-PCR products are red and underlined. The numbers adjacent to the boxes indicate the distance in nucleotides between the sequence shown and the start codon of each gene. The T7 RNA polymerase is flanked by the T. brucei procyclin spliced leader acceptor site, whereas both neo and tetR are flanked by T. brucei actin spliced leader acceptor sites. In the case of the T7 RNA polymerase and tetR transcripts, only one addition site was identified; in the case of the neo transcript, three were found.