Conditional mutagenesis of PbCDPK1.

(A) Schematic representation of the conditional mutagenesis of the PbCDPK1. (B) Southern hybridization demonstrating modification of the PbCDPK1 locus through the addition of FRT sites in FlpL/TRAP parasites. (C) PCR analysis using oligos p1 and p2, demonstrates excision of PbCDPK1 in genomic DNA obtained from (1) WT erythrocytic stages (2) FlpL/TRAP erythrocytic stages used for mosquito feeding (3) FlpL/TRAP erythrocytic stages recovered through bite infection from infected mosquitoes (4) CDPK1 cKO erythrocytic stages used for mosquito feeding (5) CDPK1 cKO erythrocytic stages recovered through bite infection from infected mosquitoes. (D) Intrahepatic development of CDPK1 cKO and control FlpL/TRAP sporozoites in HepG2 cells. Intracellular liver stages were quantified at 40–44 h p.i. (D) Parasite egress from infected HepG2 cells was monitored by quantifying the number of extracellular merosomes 66–70 h p.i. (E) In vivo infection of CDPK1 cKO and control FlpL/TRAP sporozoites was monitored by the appearance of erythrocytic stage parasites in mice infected through sporozoite injection.