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. 2011 May 18;6(5):e19860. doi: 10.1371/journal.pone.0019860

Figure 2. Targeted gene deletion of Puf1/UIS9 and Puf2 in P. berghei.

Figure 2

(A–B) Replacement strategy to generate the puf1(-) and puf2(-) parasites. P. berghei PUF1 gene (A) consists of five exons encoding an 1183 amino-acid protein (PBANKA_123350), whereas PUF2 (B) consists of four exons encoding a 477 amino-acid protein (PBANKA_071920). The PUF domains are shown in blue. For each gene, the wild-type (WT) genomic locus was targeted with a replacement plasmid containing 5′ and 3′ regions of PUF1 or PUF2 and a positive selectable marker, Toxoplasma gondii dhfr/ts or human DHFR, respectively. Upon a double crossover event, the PUF1 or PUF2 gene is replaced by the selectable marker. Replacement- and wild type- specific test primer combinations and expected PCR fragments (WT, 5′ integration and 3′ integration) are indicated by arrows and lines, respectively. Restriction sites, Southern probes and expected restriction fragments are also shown. S, SpeI; X, XhoI; A, AfeI; E, EcoRV. (C) Puf1 replacement-specific PCR analysis. Confirmation of the predicted gene targeting is achieved by specific primer combinations (5′ and 3′ integration), which can only amplify a signal from the recombinant locus. A wild type-specific PCR reaction confirms the absence of residual wild-type parasites in the clonal puf1(-) population. (D) Southern blot analysis of genomic DNA isolated from WT, puf1(-) and puf2(-) parasites, using digoxigenin-labelled probes specific for Puf1. After digest with SpeI and XhoI, the Puf1 probe hybridizes to a 8.3 or a 6.9 kb fragment in WT and puf1(-) parasites, respectively. (E) Puf2 replacement-specific PCR analysis. Confirmation of the predicted gene targeting is achieved by specific primer combinations (5′ and 3′ integration), which can only amplify a signal from the recombinant locus. A wild type-specific PCR reaction confirms the absence of residual wild-type parasites in the clonal puf2(-) population. (F) Southern blot analysis of genomic DNA isolated from WT, puf1(-) and puf2(-) parasites, using digoxigenin-labelled probes specific for Puf2. After digest with AfeI and EcoRV, the Puf2 probe hybridizes to a 8.4 kb fragment in WT and a 4.0 kb fragment in puf2(-) parasites.