FIG. 1.
DNA constructs and analysis of integration into P. knowlesi CSP locus after transfection. (A) Linear DNA construct designed for integration into the P. knowlesi CSP locus. The 5-kb selectable marker cassette (TgDHFR cassette) contains P. berghei dhfr/ts flanking regions controlling expression of mutagenized T. gondii dhfr/ts. Relevant restriction sites are indicated: R, EcoRI; P, PstI; H, HindIII; B, BamHI. (C) The location of the PCR primers A and B and the specific probe used for Southern blotting are shown. ORF-containing sequences are marked with an open arrow, and sequences used for targeted integration are indicated. (B) PCR analysis of transfected parasites. Lanes 1 to 3 show PCR with CSP integration-specific primers A and B. Lanes: 1, P. knowlesi H-strain DNA; 2, PkCSPko clone DNA; 3, pDB.DTm.DB/CSPko vector DNA. PkCSPko and the transfection construct were all positive for PCR with the two T. gondii dhfr/ts-specific primers (not shown). (C) Southern blot analysis of transfected parasites. PstI-digested DNA from P. knowlesi H strain (lane 2), PkCSPko clone (lane 3), and the transfection vector pDB.DTm.DB/CSPko (lane 1) was used to prepare a Southern blot. The blot was probed with a P. knowlesi CSP-specific probe (see panel A).