FACS analysis Wild-type and DKO cells were grown to 1 ¥ 106 cells ml-1 in HMI9 medium, centrifuged (2500 g, 10 min, 25°C) and washed twice in HMI9 medium lacking thymidine. The cell pellets were re-suspended in thymidine-deﬁcient medium and incubated for a further 24 h. Cells were collected by centrifugation and washed twice in PBS before preparation for FACS analysis (Tu and Wang, 2005). Cells were ﬁnally adjusted to 5 ¥ 105 cells ml-1, re-suspended in 500 ml of PBS containing 50 mg ml-1 propidium iodide, 50 mg ml-1 RNase and 0.1% Triton X-100, incubated at room temperature for 20 min in the dark and analysed using FACSort analytical ﬂow cytometer using Cellquest software (BD Biosciences). Morphological analysis Cells were examined following ﬁxation with methanol or 4% (v/v) paraformaldehyde and stained with Giemsa or 4′,6- diamidino-2-phenylindole (DAPI), respectively, using a Carl Zeiss light and ﬂuorescent microscope. For electron micros- copy, WT and DKO cells (grown in the presence and absence of thymidine) were ﬁxed for 24 h in 2% (v/v) glutaraldehyde and 4% (v/v) paraformaldehyde in 0.2 M PIPES, pH 7.2. For transmission electron microscopy cells were prepared as described previously (Broadhead et al., 2006), post-ﬁxed for 1 h at 4°C with 1% (v/v) osmium tetroxide in 100 mM phos- phate buffer (pH 6.5) and rinsed brieﬂy in water prior to ﬁxing or staining en bloc with 3% (v/v) aqueous uranyl acetate. Cells were rinsed further in distilled water and subsequently dehydrated through a graded ethanol series with a ﬁnal wash in propylene oxide, prior to embedding in Durcupan resin. Sections were stained with 3% (v/v) aqueous uranyl acetate and Reynold’s lead citrate prior to examination using a JEOL- 1200 EX TEM. For scanning electron microscopy, the same ﬁxation and en block staining protocol was applied. After ethanol dehydration series the samples were critical-point dried and the membranes coated with gold-palladium by sputter staining and examined using a Philips XL 30 SEM. In vivo studies All cell lines were cultured for 24 h in the absence of select- able drugs before mice (ﬁve per group) were infected with a single intraperitoneal injection of 105 parasites in 0.2 ml of glucose saline. One set of animals were administered with 2.5 mg ml-1 doxycycline in their drinking water 3 days prior to infection with the cDKODHFR–TS and freshly prepared every second day for the duration of the experiment. Animals were inspected daily for clinical signs of infection (reduced activity, ruffled fur, increased respiration and prostration or collapse) and wet smears of tail blood were examined microscopically (¥40 lens and ¥10 objective) in 20 high-power ﬁelds for parasites. When more than ﬁve parasites were visible per high-power ﬁeld 1 ml of tail blood was drawn by capillary action into a Microcap glass capillary (Sigma), diluted 200- fold with glucose saline and parasite density was determined microscopically using a Neubauer haemocytometer. Any animal with a parasitaemia exceeding 1 ¥ 108 ml-1 was humanely killed as pilot experiments had established that Validation of DHFR–TS as a drug target 531 animals were unable to survive a further 24 h with WT (SM) strain. Acknowledgements We would like to thank Professor Dyann Wirth (Harvard Medical School) for stimulating our interest in folate metabo- lism as a drug target in African trypanosomes; Professor Steve Beverley (Washington University School of Medicine) for much useful advice and encouragement; Professor George Cross and Dr Simone Leal (Rockefeller University, New York, USA) for the provision of transgenic cell lines; Dr Lucia Güther, Dr Kirstee Martin, Dr Terry Smith and Dr Angela Mehlert for reagents and advice in transfection techniques; Martin Kierens and John James for help and advice with electron microscopy and Dr Rosemary Clark for assistance with the FACS analysis. This study was supported by the Wellcome Trust and the Drugs for Neglected Diseases Initiative. References Ackland, S.P., Beale, P., and Peters, G.J. (2003) Thymidylate synthase inhibitors. Cancer Chemother Biol Response Modif 21: 1–28. Ahmad, S.I., Kirk, S.H., and Eisenstark, A. 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