Figure 3

Ldccys2 single allele gene replacement. (A) Schematic representation of the wild type and hyg/dhfr-ts targeted alleles of Ldccys2. The location of the Pst I sites used to characterize the hygromycin B resistant recombinants is shown by arrows. The bold lines labelled a, b and c represents the location of different probes used in Southern analyses. (B) Southern analyses of Ldccys2 heterozygous knockout mutants. Genomic DNA (2 μg/lane) from wild type L. (L.) chagasi (WT) and Ldccys2 heterozygous knockout mutants (KO) were digested with Pst I and separated on a 0.9% (w/v) agarose gel. The DNA was blotted on to Hybond N+ membrane and hybridized with probes a (i), b (ii) and c (iii). Asterisks: 1- represents wild type allele of Ldccys2 (3.5 kb), 2 and 3- represent disrupted allele (3.0 kb and 2.8 kb). The grey lines indicate the size of expected bands upon PstI digestion.