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. 2022 Dec 30;24(1):656. doi: 10.3390/ijms24010656

Figure 1.

Figure 1

Generation of T. b. rhodesiense ICP mutants. (A) Schematic representation of the ICP locus in wild-type (WT) T. b. rhodesiense IL1852 and the constructs containing the same 5′ and 3′ flanking regions (FR), and either a blasticidin (BSD) or hygromycin (HYG) resistance cassette, for the homologous recombination to generate an ICP knock-out line (Δicp). The oligonucleotides used to generate the FR for construction of recombination cassettes are listed in Table 1 (methods). Open reading frames (ORFs) of ICP and of flanking genes are shown as arrows. DHFR, dihydrofolate reductase. The predicted DNA fragment sizes after digestion with StuI and SphI are shown. (B) Schematic representation of the construct used, containing the phleomycin (BLE) resistance cassette, for the re-integration of ICP in the tubulin locus of the Δicp line to generate an ICP re-expressing line (Δicp:ICP). The predicted DNA fragment size after digestion with StuI and SphI is shown. (C,D) Genomic DNA isolated from the T. b. rhodesiense WT line, a Δicp clone, and a Δicp:ICP clone was digested with StuI and SphI, separated on a 0.8% agarose gel, transferred to a nylon membrane and probed with alkaline phosphatase labeled 5′ FR of ICP (C) or ICP ORF (D). Lane 1, WT; Lane 2, Δicp; Lane 3, Δicp:ICP.