<i>DIP13</i> Gene Deletion in <i>T. brucei</i> BSF.

<p>(A) Schematic diagram of the <i>TbDIP13</i> locus and the plasmid constructs used for deletion. Flanking sequences used to generate the targeting vectors are shown. The top panel shows the <i>TbDIP13</i> locus and the second panel shows the plasmid constructs used for targeted deletion of the locus by replacement with phleomycin/puromycin resistance genes (<i>BLE</i>/<i>PAC</i>) flanked by the actin 5′ UTR and either the actin 3′ UTR (<i>BLE</i>) or the aldolase 3′ UTR (<i>PAC</i>). Solid black bars represent fragments used as hybridisation probes. (B) Southern blot analysis of parental line Lister 427 (WT) and <i>DIP13</i> double replacement (DKO) parasite lines. Five micrograms of genomic DNA from each parasite line was digested with <i>Sac</i>I, size separated through 0.8% agarose, blotted and hybridised with DIG-labelled DNA probes (∼200 bp) as indicated. Corresponding DNA marker positions are shown (kbp). (C) Total cell lysates (5×10⁶ cells/lane) from cell lines as in (B) were immunoblotted and probed with purified anti-TbDIP13 and anti-EF1α to monitor equal sample loading. Corresponding protein marker positions are shown (kDa). (D) Growth of BSF cell lines as above monitored over a 96 hour time course. Mean values are shown (n = 3) +/− SD (not all error bars are visible). (E) Immunofluorescence analysis of cell lines as above, probed with mouse monoclonal antibodies against total α-tubulin, TAT1 (α-Tub), acetylated α-tubulin, 6-11B-1 (Acet-Tub) and paraflagellar rod proteins 1 and 2, L13D6 (PFR1/2), shown in green. The corresponding DIC image is presented for cells probed with anti-PFR1/2. All cells were co-stained with DAPI (blue). Bar, 5 µm. (F) Scanning electron micrographs of parental (WT) and <i>DIP13</i> double replacement cell line (DKO). Bar, 2 µm. (G, H) Cell body length (G) and paraflagellar rod length (H) in BSF parental line Lister 427 (WT) and <i>DIP13</i> double replacement (DKO) parasite lines. Mean values are shown as horizontal lines (+/− SD) (n = 100). Statistical analyses were performed using an unpaired t test. Asterisks represent statistical significance compared to the parental line (WT) (* = p<0.05, *** = p<0.001).