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Figure 4 | Malaria Journal

Figure 4

From: Plasmodium serine hydroxymethyltransferase: indispensability and display of distinct localization

Figure 4

Targeted deletion of Pbcshmt . (A) Schematic diagram depicting the genomic organization of Pbcshmt locus following disruption or allelic replacement with Pvshmt coding sequence. Enzyme restriction sites, along with fragment sizes and their specific probes are indicated. (B) PCR diagram of molecular characterization of transfected parasites. Lanes 1–5: (1) water control; (2) gDNA of P. berghei wild type; (3) pL0017_(Pv)Δshmt; (4) and (5) gDNA of P. berghei transfected with pL0017_Δshmt and pL0017_(Pv)Δshmt, respectively. Primer pairs a & b, c & d, and e & f (sequences reported in Additional file1) are used to amplify (i) endogenous Pbcshmt, (iii) 5 integrated fragment, and (iv) 3 integrated fragment, respectively. Amplification of putative Pbmshmt (ii) was performed as a control. (C) Southern blot hybridized with 5 or 3UTR probe to confirm Pvcshmt allelic replacement at Pbcshmt locus. DNA was digested with Eco RV and Bgl II. Lanes are: (1) pL0017_(Pv)Δshmt plasmid, (2) gDNA of P. berghei wild type, and (3) gDNA of transgenic ΔPbPvcshmt P. berghei, respectively. P, I, and WT indicate expected band size for pL0017_(Pv)Δshmt plasmid, integrated Pvcshmt, and endogenous Pbcshmt, respectively. (D) RT-PCR diagram for detection of shmt transcript. Lanes are: (M) 1kb ladder, (−) water control, (RT-) no RT control, (RT+) cDNA, (W) P. berghei wild type, and (V) transgenic P. berghei harbouring Pvcshmt. Pbmshmt and Pbα-tubulin-2 were amplified as control genes.

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