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. 2015 Jun 19;10(6):e0130356. doi: 10.1371/journal.pone.0130356

Fig 7. The U1 mediated knockdown strategy is not functional in P. falciparum.

Fig 7

(A) Schematic showing the strategy used to replace the endogenous pfsub1 3’ UTR with a floxed heterologous PbDT 3’ UTR with a downstream sequence comprising 10 U1 recognition sequences. The single cross-over homologous recombination event also results in the fusion of a HA3 epitope tag to PfSUB1. The predicted results of DiCre mediated recombination induced by treatment with rapamycin are shown. The positions of hybridization of PCR primers designed to specifically amplify sequences from the intact and excised locus are indicated with red and blue arrows. (B) IFA of mature schizonts of integrant clone D3, probed with mAb NIMP.M7 (anti PfSUB1) and mAb 3F10 (anti HA3). (C) Efficient DiCre-mediated recombination between the integrated loxP sites, as shown by diagnostic PCR analysis of parasite genomic DNA extracted 44 h following rapamycin treatment of two independent parasite clones D3 and B11. The blue and red arrows correspond to primer pairs marked in (A). (D) Upper panel: Western blot analysis with mAb 3F10 shows no down-regulation of PfSUB1 protein expression protein 44 h or 6 days after rapamycin treatment. Lower panel: loading controls, showing total protein SDS PAGE gels detected by UV fluorescence on the BioRad Chemidoc XRS system prior to Western transfer.