Generation of Tb<i>GAPDHL</i> and Tb<i>PGKL</i> procyclic null mutants.

<p>(<b>A</b>) Generation of Tb<i>GAPDHL</i> null mutants. (<b>B</b>) Generation of Tb<i>PGKL</i> null mutants. Cartoon schematics denote gene loci annotated with HindIII (H) restriction sites for (i) wild-type loci; (ii) loci following gene disruption and (iii) following endogenous gene-tagging with GFP (Tb<i>GAPDHL</i> only). Southern analysis of genomic DNA digested overnight at 37°C with HindIII shows blots probed sequentially with either coding sequence from the targeted gene (probe o) or sequence from the 3′ intergenic region (probe u). Relative positions of the probes are shown in the cartoon schematics. In (<b>A</b>) the order of lanes is 1, wild-type <i>GAPDHL</i>^+/+<i>T. brucei</i>; 2, heterozygous <i>GAPDHL</i>^+/− cells resistant to phleomycin; 3, heterozygous <i>GAPDHL</i>^+/− cells resistant to blasticidin/HCl; 4-5, <i>GAPDHL</i>^+/− heterozygotes from lanes 2 and 3, respectively, in which endogenous tagging of the remaining wild-type allele results in expression of a recombinant e<i>GFP:GAPDHL</i>; 6, a <i>GAPDHL^−/−</i> mutant obtained from the stable transformation of the phleomycin-resistant heterozygote cells from lane 2. In (<b>B</b>) the order of lanes is 1, wild-type <i>PGKL</i>^+/+, 2, heterozygous <i>GAPDHL</i>^+/− cells resistant to phleomycin; 3, heterozygous <i>GAPDHL</i>^+/− cells resistant to blasticidin/HCl; 4–5, independently obtained <i>PGKL</i>^−/− mutants derived from the stable transformation of heterozygous cell lines analyzed in lanes 2 and 3, respectively.