Skip to main content
. 2011 Nov 28;287(2):1306–1321. doi: 10.1074/jbc.M111.276865

FIGURE 6.

FIGURE 6.

Targeted gene disruption and HA tagging of the PfI3 locus. A, gene-targeting construct for gene disruption by single homologous recombination using the pCAM-BSD, and the locus resulting from integration of the knock-out construct. B, epitope tagging of PfI3 by knock-in strategy. Insertion of an HA epitope tag at the C terminus of PfI3 by single homologous recombination (knock-in). The locations of the primers (P29, P20, P21, P22, P27, P167, P168, and P639) used for PCR analysis are indicated as well as the blasticidin-resistance cassette (BSD). C, plasmid rescue experiments showing the presence of pCAM-PfI3 (lane 1) and pCAM-PfI3–2HA (lane 2) constructs in transfected parasite culture. D, analysis of pCAM-PfI3-transfected 3D7 culture by PCR; lanes 1–3 correspond to DNA extracted from transfected parasites; lanes 4–6 correspond to DNA extracted from wild type parasites. Lanes 1 and 4 represent the detection of a portion of the wild type locus (PCR with P29 and P20); lanes 2 and 5 represent the detection of episomal DNA (PCR with P167 and P168); and lanes 3 and 6 represent the detection of the integration at the 5′ end of the insert (PCR with P27 and P168). The absence of amplification of a PCR product using genomic DNA prepared from transfected parasite culture and using P27 and P168 as primers indicates the lack of homologous recombination (lane 3). E, analysis of pCAM-PfI3–2HA transfected 3D7 culture by PCR; lanes 1–3 correspond to DNA extracted from transfected parasites; lanes 4–6 correspond to DNA extracted from wild type parasites. Lanes 1 and 4 represent the detection of a portion of the wild type locus (PCR with P21 and P22); lanes 2 and 5 represent the detection of episomal DNA (PCR with P167 and P639), and lanes 3 and 6 represent the detection of the integration at the 3′ end of the insert (PCR with P29 and P639). The amplification of a PCR product at ∼600 bp using genomic DNA prepared from transfected parasites indicates the homologous recombination and integration of the 2-HA tag construct in endogenous PfI3 (lane 3). F, immunoblot analysis of total extracts of transfected 3D7 with pCAM-BSD-PfI3–2HA (lane 1) and wild 3D7 strain transfected (lane 2) 3D7 culture mAb anti-HA antibody.