530 N. Sienkiewicz, S. Jarosławski, S. Wyllie and A. H. Fairlamb Generation of bloodstream-form T. brucei knockout lines Knockout and recovery constructs were prepared using QIAprep Miniprep Plasmid Kit (Qiagen). DNA was digested with NotI, ethanol precipitated and redissolved in sterile water at a ﬁnal concentration of 1 mg ml-1. Trypanosomes were electroporated as described previously (Wirtz et al., 1999; Chang et al., 2002; Roper et al., 2002; Martin and Smith, 2006b). Transformed SKO cells containing the PAC or HYG genes were cultured in the presence of 0.1 mg ml-1 puromycin or 4.0 mg ml-1 hygromycin respectively. The SKO PAC line was used to produce a DKO line using the HYG knockout construct to replace the second endogenous copy of DHFR–TS by selection with hygromycin and puromycin. Transfection of the pLew82_EcTS and pLew100_ TbDHFR–TS recovery constructs into the DKO cells resulted in the selection of conditional DKO (cDKO) cells with 2.5 mg ml-1 phleomycin and blasticidin respectively. DNA and RNA analysis of KO cells Conﬁrmation of the correct integration of the knockout and recovery constructs was undertaken with restriction digests of 5 mg of gDNA with subsequent Southern blot analysis as described previously (Martin and Smith, 2006b) using DNA probes speciﬁc for DHFR–TS, PAC, HYG and ThyA. RNA was prepared from approximately 2 ¥ 108 cells (WT and SKO cells grown in HMI9 lacking thymidine and DKO cells in HMI9) using Qiagen RNeasy Mini Kit. The RNA samples (5 mg) were denatured in formaldehyde/formamide premix at 65°C for 15 min and run on a 1.2% agarose/2% formaldehyde/1¥ MOPS gel and transferred to Hybond-N nylon membrane (Amersham Biosciences). Blots were hybridized sequentially with [a-32P]-dCTP-labelled probes for TbPTR1, TbDHFR–TS and TbINO1 prepared with Rediprime (Amersham Biosciences). Western blot analysis of cell lysates Polyclonal antisera against T. brucei PTR1 were raised in adult male Wistar rats. An initial injection of 100 mg of puriﬁed antigen (Dawson et al., 2006), emulsiﬁed in complete Freund’s adjuvant, was followed by two identical booster injections of antigen emulsiﬁed in Freund’s incomplete adjuvant at 2-week intervals. Wild-type and SKO trypanosomes were grown in thymidine-deﬁcient HMI9 media while DKO cells were grown in HMI9 supplemented with thymidine. Mid-log cells (~1 ¥ 109 cells) were harvested by centrifugation (800 g, 10 min, 25°C), aspirated and pellets re-suspended in 1 ml of ice-cold PBS (137 mM NaCl, 8 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4) containing a cocktail of protease inhibitors (Calbio- chem, Protease Inhibitor Cocktail Set III). Following centrifu- gation (5000 g, 2 min, 25°C), cell pellets were further re-suspended in 500 ml of ice-cold PBS containing protease inhibitors, 1 mM EDTA and 1 mg ml-1 of both DNase and RNase. For biological safety, samples were freeze-thawed ﬁve times prior to mechanical disruption with sterile glass beads (Sigma, bore size 425–600 mm) in a micro pestle. Lysates were centrifuged (13 000 g, 20 min, 4°C), the result- ing supernatants were collected and protein concentration was determined. Cell extracts (20 mg) were then separated on SDS-PAGE and subsequently transferred onto nitrocellulose. After block- ing with 5% skimmed milk in PBS at room temperature for 1 h, the blot was incubated with T. brucei PTR1 polyclonal antiserum (1/1000 dilution) or T. brucei BiP (kindly supplied by Dr M.L. Güther, originally from Dr J. Bangs) at room temperature for 1 h, washed in PBS containing 0.1% (v/v) Tween 20 and then incubated with a secondary rabbit anti-rat (IgG) antibody (Dako; 1/10 000 dilution). The immunoblots were subsequently developed using the ECL® plus (enhanced chemiluminescence) system from Amersham Biosciences. Thymidylate synthase assays Thymidylate synthase activity in cell lysates was carried out as described (Nirmalan et al., 2004) with minor modiﬁcations. Approximately 2 ¥ 108 cells were prepared from WT, DKO, cDKODHFR–TS (with tetracycline induction), cDKOTS (with and without tetracycline induction), osmotically lysed with ice-cold distilled water, frozen and thawed three times in liquid nitro- gen and ﬁnally re-suspended in an equal volume of 2¥ TS assay buffer (0.4 M Tris-HCl pH 7.4 containing 2 mM EDTA, 40 mM 2-mercaptoethanol, 200 mM NaF and 30 mM CMP). The suspension was centrifuged for 10 min, at 12 000 g at 4°C and the resulting supernatant was used to determine the TS activity. This involves a tritium release assay using [5-3H]- dUMP (Moravek Biochemicals) and (6R,S)-5,10-methylene- 5,6,7,8-tetrahydrofolic acid (Schircks Laboratories). In brief the standard assay consisted of 200 ml of cell lysate prepared in TS assay buffer with the addition of 5 ml of 6.5 mM methylene tetrahydrofolate and 5 ml of [5-3H]-dUMP (19 Ci mmol-1). Assay mixtures were incubated at 37°C for 1 h and reactions stopped with the addition of 50 ml of ice- cold 100% (w/v) TCA. After incubation for 10 min on ice, residual [5-3H]-dUMP was removed by ﬁve extractions with activated charcoal suspension. After the ﬁnal extraction step, 200 ml of aqueous supernatant was counted in 3 ml of scin- tillation ﬂuid. Samples were analysed in triplicate and back- ground counts subtracted from negative control reaction mixtures containing heat-inactivated lysate. EC50 determination of inhibitors against WT and DKO cells Antifolates were tested against WT and DKO cells in HMI9 and FDM (both containing 160 mM thymidine). Serial doubling dilutions of drugs (10–20 dilutions from 10 mM stocks pre- pared in 10% DMSO) were added to ﬂat-bottomed 96-well plates containing 1 ¥ 104 cells ml-1 in a ﬁnal volume of 0.2 ml in either HMI9 or FDM. Plates were incubated for 72 h at 37°C/5% CO2 and cell densities were determined using Alamar Blue (Raz et al., 1997). EC50 values were calculated using GraFit software (Erithacus Software) with a non-linear 4-parameter robust curve ﬁt in triplicate. All inhibitors were from Sigma Chemical, except pemetrexed (International Laboratory USA), trimetrexate (Tocris) and raltitrexed (Tomudex, ZD1694, from Astra Zeneca). © 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd, Molecular Microbiology, 69, 520–533