Fig 6.

The MARS complex in T. brucei contributes to parasite fitness. (a) Diagram of a conditional-null T. brucei MCP2 cell line in which the endogenous alleles were replaced with blasticidin (BSD-Ty1-HSVTK) and puromycin (PAC-Ty1-HSVTK) resistance markers and a tetracycline (Tet)-regulatable ectopic 3V5-tagged allele was inserted into the rDNA spacer. UTR, untranslated region. (b) TBE-agarose gel (1%) of PCR products from the conditional-null (C. null) cells diagrammed in panel a (see Materials and Methods for details). WT, wild type. (c) Western blot analysis (10% SDS-PAGE) of lysates of conditional-null cells diagrammed in panel a after 2 days of growth in the absence (lane −) or presence (lane +) of 250 ng/ml tetracycline using anti-V5 monoclonal antibodies. The blot was reprobed with monoclonal antibody MAb78 for HSP70 as a loading control. (d) Quantitative real-time PCR analysis of MCP2 mRNA in SM427 or conditional-null cells grown for 24 h without tetracycline or in the presence of 50 ng/ml or 250 ng/ml. β-Tubulin (β-tub) was used as an internal control. (e) Growth curve of conditional-null cells in presence or absence of 250 ng/ml of tetracycline. Results are averages of three experiments ± SDs. (f) Parasitemia in mice infected with the T. brucei MCP2 conditional-null line. Experiments were performed in the absence (−) or presence (+) of doxycycline (Dox). Mice received doxycycline (200 μg/ml) in drinking water starting 24 h before infection, and the doxycycline was maintained in the drinking water throughout infection (replaced daily). The data show the averages of three independent experiments ± SDs.