FIG. 3.

Optimization and efficiency of gene tagging. (A) Agarose gel showing plasmids in the forms supercoiled (SC), linearized within the targeting sequence (LN), or nicked/open circle (OC). (B) Linearization of the vector is necessary for efficient integration. Only the LN construct integrated into the homologous loci, whereas the SC and OC constructs did not integrate during the 30-day experiment. (C) Linearization site and efficiency of integration. Two restriction enzyme digestion sites were used, cutting 350 or 750 into the 1-kb targeting sequence. Both linearized constructs were shown to be equally effective in integration into the endogenous ACP locus. (D) Δku80 favors homologous recombination. pCNA-YFP, ACP-YFP, and MIC3-YFP constructs with either HXGPRT or DHFR-TS selectable markers were transfected into RH or Δku80, and YFP-positive parasites were enumerated over time and are expressed as a percentage of the total population. Note that the y-axis scale of the MIC3-YFP graph is smaller than for the pCNA-YFP and ACP-YFP graphs.