Reverse genetics of PfCK2β1. 3D7 parasites transfected with pCAM-BSD-KOPfCK2β1 with or without pCHD-PfCK2β1 were analyzed by PCR and Southern blotting. (A) Locations of the primers used for PCR screening, and the restriction enzymes used to cut the genomic DNA to give a diagnostic pattern of bands for analysis by Southern blotting. One truncated copy possesses its promoter and initiation codon, but lacks the C-terminal cysteine pair, a stop codon and a 3'UTR, whereas the other copy possesses both cysteine pairs but lacks a promoter and an initiation codon, and has an artificial stop codon introduced. (B) PCR screening of DNA from untransfected 3D7 parasites, two separate pCAM-BSD-KOPfCK2β1-transfected lines (KOCK2β1 1 and KOCK2β1 2), and parasites transfected with both the knockout plasmid and the complementation plasmid (KOCK2β1 + comp). (1) Amplification of the wild-type locus; (2) amplification over the 5' integration boundary; (3) amplification of the insert in the pCAM-BSD-KOPfCK2β1 plasmid. Evidence of integration was seen only in the DNA from the doubly transfected parasite culture (KOCK2β1 + comp; lane 2, faint band seen at 598 bp). (C) The parasite DNA was digested using the restriction enzymes EcoRI and ClaI, and analyzed by Southern blotting, using BSD and PfCK2β1 sequences as probes. (1) Untransfected 3D7; (2) KOCK2β1 1; (3) KOCK2β1 2; (4) KOCK2β1 + complement. See text for details.