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. 2005 Aug 8;102(33):11645–11650. doi: 10.1073/pnas.0504251102

Fig. 4.

Fig. 4.

Targeted gene replacement of TbALO. (A) Diagram of the TbALO allele and the effects of gene replacement. The 5′ and 3′ flanking sequences (solid bars) were amplified and cloned sequentially either side of cassettes containing the selectable marker [puromycin (PAC) or blasticidin (BLA)] plus tubulin intergenic elements required for processing of mRNA (hashed boxes). The dotted lines correspond to probes used to check integration. The position of the predicted XbaI sites plus the band sizes (in kbp) obtained after hybridization are shown. (B) Autoradiographs of XbaI-digested genomic DNA from T. brucei 221 wild type (lane 1), ALO::PAC single knockout (lane 2), ALO::BLA single knockout (lane 3), and three independent ALO::PAC+BLA double TbALO knockout (lanes 4–6) cell lines. Blots were hybridized with labeled TbALO, 3′ TbALO flank, PAC, and BLA probes. Sizes given are in kbp. Integration was also verified by PCR (data not shown). (C) Blots containing 15 μgofT. brucei total RNA from procyclic form (P), bloodstream form (B), and ALO::PAC+BLA double knockout (KO) cell lines were hybridized with a labeled TbALO probe. Sizes given are in kb. RNA loading was judged by hybridization by using the T. brucei β-tubulin gene.