GFP expression in procyclic cells harbouring the p<i>Tb</i>FIX vectors.

A) Images of Lister 427 pTbFIX and pTbFIX-PARP cells induced for 24 h with 1 μg/mL of DOX. Scale bar 5 μm. B) Western blot probed with anti-tetR antibodies showing the levels of tetR expression in pTbFIX transfected cells (4x106 parasites per lane). For comparative purposes the parental strain (Lister 427) was included in the analysis. C) Doxycycline dose-dependent GFP expression in procyclic cells. GFP expression was measured by Western blot analysis 24 h post induction. D) Fixed induced (DOX) or non-induced parasites (NI) were analysed by flow cytometry. Approximately 50000 events were captured. Mean fluorescence intensity values can be found in S1 Table. E) PCF cultures were diluted to 1x106 cells/mL and induced with 1 μg/mL of DOX. After 48 h parasites were extensively washed with DOX free medium and incubated for additionally 96 h, followed by DOX re-addition. GFP expression levels were monitored daily by Western blot analysis in total cell extracts (4x106 cells per lane). Tubulin was used as a loading control. Cultures were diluted every two days to 1x106/mL to maintain cell density within a range that supports exponential growth.