4 × 104 m p , c 3 × 104 d e s a le e 2 × 104 r r e t ]-aw 104 3H [ 0 WT cD+KOEcTS– + DKO -TS cDKOEcTS cDKODHFR Cell line Fig. 8. TS activity in WT and transgenic T. brucei. TS activity was determined by release of [3H]-water from [3H]-dUMP as described in Experimental procedures. Error bars represent the standard error of triplicate assays. The minus and plus signs refer to growth in HMI9 medium in the absence or presence of tetracycline respectively. into the DKO and found to be correctly targeted to the ribosomal DNA locus resulting in cDKODHFR–TS cells (Fig. 7E). cDKODHFR–TS cells were able to grow in the absence of thymidine, with or without induction by tetra- cycline (generation times for both are 12 h; Fig. 7F). Enzymatic activity approaching WT levels was found in this cell line (Fig. 8), conﬁrming that TS was functional. Loss of tetracycline control is a frequently observed phe- nomenon with the pLew family of vectors (Krieger et al., 2000; Milne et al., 2001; Chang et al., 2002; Martin and Smith, 2006a). Loss of expression of the TETR protein or mutation or ablation of the tet operator site has been proposed as possible mechanisms of escape from tetra- cycline repression. However, only one study has demon- strated that this results from deletion of the TETR gene (Roper et al., 2002). This is not the case here, as PCR conﬁrmed the presence of TETR (not shown). Thus, loss of tetracycline control in our experiments must be due to another mechanism. Validation of DHFR–TS as a drug target 527 Virulence in mice The nutritional environment in vivo is considerably differ- ent from in vitro culture conditions underlining the need for target validation studies to be carried out in animal models, where possible (Frearson et al., 2007). To conﬁrm the essentiality of DHFR–TS, mice were inocu- lated with WT or genetically modiﬁed parasites and the course of the infection monitored over a 30-day period. A survival curve for the various infections is shown in Fig. 9. Mice infected with WT cells were unable to survive beyond 3–4 days, whereas DKO-infected mice remained completely free of parasites and survived beyond 30 days. This indicates that the thymidine level in mouse plasma (1 mM; Clarke et al., 2000) is not sufficient to sustain DKO cell viability in vivo, consistent with the in vitro data presented in Fig. 4. Virulence was restored in cDKODHFR–TS cells indicating that the selectable markers PAC and HYG had not suppressed the virulence phenotype. SKO infections were also lethal. In one experi- ment with SKO (PAC), survival times were extended to up to 16 days, but this was not reproduced in a second experiment. Virulence of a second single-knockout line (SKO HYG) was also similar to WT, except for one mouse that remained aparasitaemic throughout the 30-day experiment. These results demonstrate deﬁnitively that DHFR–TS is absolutely essential for parasite survival in the host. They also suggest that sustained inhibition of DHFR–TS by >50% would be required for a chemothera- peutic effect. Discussion The genetic studies presented here clearly indicate that DHFR–TS is essential for growth and survival of the African trypanosome in vitro and in vivo. Nutritional rescue of the DHFR–TS is speciﬁc for thymidine and cannot be replaced by other pyrimidines, consistent with a speciﬁc requirement of both enzymes in the thymidylate cycle (Fig. 1). The ﬁnding that PTR1 levels are unchanged Fig. 9. Virulence of WT, SKO and DKO T. brucei infections in mice. Each data set represents the combined results of two independent experiments presented as a Kaplan–Meier survival plot. The ﬁgures in the legend are the numbers of survivors per infected group. As the cDKODHFR–TS was not responsive to tetracycline induction these data sets have been combined. Symbols: WT, dark blue; SKO (PAC), green; SKO (HYG), light blue; DKO, black; cDKO, red. © 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd, Molecular Microbiology, 69, 520–533