Figure 4. Pbvit knockout affects blood- and liver- stage growth of P. berghei.

(a) Double crossover strategy for pbvit knockout and genotyping of the Pbvit− transgenic clonal line by PCR. Lane 1, detection of knockout construct integration at the 5′ end (primers a+b, 1.38 kb); lane 2, knockout construct integration detection at the 3′ end (primers c+d, 1.23 kb); lane 3, wt pbvit locus (primers e+d, 1.45 kb). (b) Parasitemia of C57Bl/6J mice following infection (i.v.) with 10⁴ wt P. berghei or Pbvit⁻ iRBCs, determined by counting of iRBC in Giemsa-stained blood smears (N=10 for wt P. berghei-infected mice and N=5 for mice infected with Pbvit⁻ A2 or D1). (c) Survival of C57Bl/6 J mice infected i.v. with 10⁴ wt or Pbvit⁻ iRBC (N=20 for wt P. berghei-infected mice and N=10 for mice infected with Pbvit⁻ A2 or D1). Median survival was 7 and 9 days for wt and Pbvit⁻, respectively, P<0.01, log-rank Mantel–Cox test. (d) Parasite liver load 6 and 45 h after i.v. injection of wt or Pbvit⁻ sporozoites, assessed by reverse transcription PCR measurement of parasite 18s RNA expression, normalized to mouse hypoxanthine-guanine phosphoribosyltransferase, shown are fold expressions relative to the average of controls–wt P. berghei (shown is the pool of two independent experiments). (e) Number of EEFs per mm² of livers 45 h after infection with 50,000 wt and Pbvit⁻ sporozoites. Each point represents an average number of EEFs per mm² per mouse liver by counting the number of EEFs in 5–8 slices per liver (shown is a pool of two independent experiments, in total 795 wt EEFs and 210 Pbvit⁻ EEFs were counted). (f) Size of liver EEFs determined by measuring the PbUIS4 surrounded area by ImageJ in confocal images of liver sections. The mean±s.e.m. size of wt and Pbvit⁻ EEFs was 728±25 (N=58) and 629±23 (N=46) μm², respectively. In b,d,e and f error bars represent s.e.m. and the asterisks denote significant differences using the two-tailed, unpaired Student's t-test: *P<0.05; **P <0.01 and ***P<0.001.