Figure 2
Integration of Pb CON into P. chabaudi ssu-rrna. Schematic representation of P. chabaudi ssu-rrna locus on contig827 (A). Schematic representation of linearized plasmid PbCON containing the Tgdhfr-ts cassette, conferring pyrimethamine resistance and gfp, flanked by P. berghei d-ssu-rrna target sequences for integration (B). Schematic representation of the contig827 ssu-rrna locus in Pc GFPCON clone 2.3, following integration of the PbCON plasmid (C). D: tgdhfr is present in Pc GFPCON (lane 1) but not wt (lane 2) DNA; amplification with L190/L191. Circular PbGFPCON plasmid is absent in Pc GFPCON (lane 3); circular Pb GFPCON (lane 4); amplification with PlF2/Pl5'R. Verification of the 5' integration site; Pc5'F/Pl5'R amplify a product in Pc GFPCON (lane 5) but not in wt (lane 6) DNA. Verification of the 3' integration site; Pl3'F/Pc3'R amplify a product in Pc GFPCON (lane 7) but not wt (lane 8) DNA. E: Tgdhfr is present in the genome of Pc GFPCON but not wt parasites. wt (lanes 1 and 2) and Pc GFPCON (lanes 3 and 4) genomic DNA digested with NheI (lanes 1 and 3) or HindIII (lanes 2 and 4). Integration of Pc GFP into the P. chabaudi ssu-rrna locus on contig827 results in restriction enzyme digestion products of 9.4 and 15.4 kb following NheI and HindIII digestion respectively. N, NheI; H, HindIII.