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. 2009 May 12;4(5):e5510. doi: 10.1371/journal.pone.0005510

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Gunther et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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A. Schematic representation of endogenous PfLplA1 gene locus (I.), transfection plasmid pHH1-LplA1-KOkon (II.) and PfLplA1 gene locus after single cross-over recombination (III.). The restriction enzyme used for diagnostic digest is shown (NdeI) and the expected sizes of diagnostic bands after hybridization with PfLplA1 or hDHFR are indicated. B. Southern blot analyses of transfected parasite lines. Genomic DNA of wild-type and LplA1-KOkon parasites was digested with NdeI and probed with the PfLplA1 ORF (left panel). In all parasite lines analysed, the endogenous gene is present (1.9 kb band) and in lanes 3 and 4 two additional DNA fragments are detected by the probe (in lane 2 less DNA was loaded on the gel so that the plasmid band is hardly visible). The faint 6 kb band is diagnostic for the presence of the transfection plasmid (see scheme), but the band of ∼9 kb (*) cannot be assigned to any specific integration event. Lane 1, wild-type D10; lane 2, PfLplA-KOkon, cycle 1; lane 3, PfLplA-KOkon, cycle 2; lane 4, PfLplA-KOkon, cycle 3. The same blot was stripped and re-probed with the probe specifically detecting the selectable marker hDHFR and apart from faint plasmid bands at 6 kb in lanes 2, 3 and 4 two additional bands of ∼9 kb (*) and 0.5 kb (*) are detected. Lane 1, wild-type D10; lane 2, PfLplA-KOkon, cycle 1; lane 3, PfLplA-KOkon, cycle 2; lane 4, PfLplA-KOkon, cycle 3. C. Genotyping by pulsed field gel electrophoreses. Chromosomes of wild-type P. falciparum D10 (lane 1) and parasites transfected with the pHH1-LplA1-KOkon construct in cycle 0 (lane 2) and cycle 3 (lane 3) were analysed by PFGE and the blots were probed with the PfLplA1 open reading frame (left panel) or with the hDHFR open reading frame (right panel) present in the transfection plasmid. The PfLplA1 probe detected the endogenous PfLplA1 gene locus on chromosome 13 in wild-type and transfected parasites. However, the probe also detected a strong signal at the bottom of the blot where the non-separated chromosomes <10 are running. The hDHFR probe similarly generated signals on chromosome <10, but there is no sign of integration into the LplA1 gene locus on chromosome 13.