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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.

PMC Copyright notice

A. Control integration strategy to recover one functional LplA1 gene copy. The targeting vector (b3D-LplA1-int2, II.) contains the endogenous stop codon and 3′ untranslated regions of the endogenous PbLplA1 but lacks the promoter and the start codon. Upon a single cross-over event, one functional LplA1 copy that is driven by the endogenous promoter and a 5′ truncated copy of the gene are generated (III.). Wild-type, plasmid, and integration-specific primer combinations are indicated by arrows and sizes of expected fragments are shown. B. Genotyping indicates successful integration of the control targeting construct. Both integration-specific PCRs (lane 1, 3′ specific integration using primer pair b3D-1 and Pb-11 and lane 2, 5′ specific integration using primer pair Pb-10 and b3D-2) amplified fragments of the expected sizes and their authenticity was verified by nucleotide sequencing. The population still contains episomal plasmid (lane 3; primer pair b3D-1 and b3D-2 amplifying a 2.35 kb band which was subcloned and sequenced) and residual wild-type parasites (lane 4; primer pair Pb-10 and Pb11 fragment was amplified and subcloned and sequenced).