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. 2008 Jun 25;3(6):e2474. doi: 10.1371/journal.pone.0002474

Figure 1. Targeted deletion of the P. berghei plasmoredoxin gene.

Figure 1

(A) Replacement strategy for targeted gene disruption of PbPlrx. The wild-type Plrx locus (WT) is targeted with a KpnI (K)/ SacII (S)-linearized replacement plasmid (pPlrxRep) containing the 5′and 3′UTR of PbPlrx and the positive selection marker TgDHFR-TS. After double cross over homologous recombination, the Plrx open reading frame is substituted by the selection marker, resulting in the mutant Plrx(-) allele. Replacement- and WT-specific test primer combinations and expected fragments are shown as lines. (B) Replacement-specific PCR analysis. Confirmation of the predicted gene targeting is done by primer combinations that only amplify a signal in the recombinant locus (test). The absence of a WT-specific signal in the clonal Plrx(-) population confirms the purity of the mutant parasite line. (C) Depletion of Plrx transcripts in Plrx(-) parasites. cDNA from WT and Plrx(-) blood stages was used as template for Plrx-specific PCR reactions (upper panel). Amplification of glutathione reductase (GR) transcripts was used as a positive control (lower panel). (D) Western blot analysis of WT and Plrx(-) blood stages. Extracts from WT or Plrx(-) (16 µg total protein each) were separated on a 15% SDS gel and probed with the polyclonal anti-Plrx serum (upper panel) or a polyclonal anti-actin serum (lower panel). As a positive control 160 ng recombinantly expressed P. berghei plasmoredoxin (protein) was added.