Figure 1. Generation of P. berghei CS^5M parasites.

A. Scheme of the strategy used for gene targeting of the replacement CS^5M molecule. Location of primers used for PCR verification of recombination is given below (primer sequences given in Table S1). Restriction sites are K – KpnI; Se – SexAI; Bs – BsmF1; X – XhoI; S – SacI. B. Verification of clones – i. genomic DNA from cloned parasites was amplified with the primers CS1 and S8R (giving a 1526 bp product) to verify recombination at the 5′ end, and the primers CS4 and PB106 (giving a 1001 bp product) to verify recombination at the 3′ end, genomic DNA from P. berghei ANKA was used as a control. ii. To verify that the parasite population was clonal, genomic DNA was amplified within the CS sequence with the primers F205 and R904 to give a 699 bp product. The PCR product was then digested with SexA1, which cuts in the P. berghei CS^5M product, but not the P. berghei ANKA product, to yield fragments of 510 and 186 bp. C. C57Bl/6 mice were immunized i.d. in the right ear with 5×10⁴ irradiated P. berghei ANKA or P. berghei CS^5M parasites. 10 days later the SIINFEKL-specific immune response in the spleen, draining lymph nodes and liver (pooled) was determined by ELISPOT (mean ± SEM; n = 3, data from one of 3 similar experiments; **  =  P<0.01). D. C57Bl/6 mice received 2×10⁶ SIINFEKL-specific effector CD8+ T cells 3 hours prior to challenge with 5×10³ P. berghei ANKA or P. berghei CS^5M sporozoites (grey bars); control mice did not receive effector cells (black bars). 40 hours later livers were taken and parasite rRNA concentration determined by real-time PCR (mean ± SEM; n = 4, data from one of 2 similar experiments, ns  =  not significant).