To replace the PbRCC-PIP gene by double homologous recombination, a pBS-DHFR vector was used (Guttery et al., 2012 (link)). Fragments corresponding to the 5′ upstream and 3′ downstream sequence of the PbRCC-PIP gene (PBANKA_0820800) were amplified using primers p56-p57 and p58-p59, respectively, and inserted in the pBS-DHFR vector. The construct was linearized before transfection as described (Guttery et al., 2012 (link)).
For HA-tagging of PbPP1, promoter and coding regions of PBANKA_1028300, obtained using primers p38-p39 and p40-p41, respectively, were cloned into the p-TRAD4Ty-TetO7-HA-hDHFR plasmid (Pino et al., 2012 (link)). Before transfection, the sequence was linearized using BglII.
P. berghei ANKA transfections were performed by electroporation of schizont stages according to Janse et al. (2006) (link), using 10 μg of linearized constructs for each transfection. The schizont stages were obtained from 6 week-old infected Fisher rats (Charles River) and separated on a 55% Nycodenz gradient after 16 h culture at 37°C in RPMI1640 culture medium supplemented with 0.4% AlbuMAXTM II Lipid-Rich BSA (Life Technologies).
Genotyping was performed in parallel on total DNA from parental and transfected parasites extracted from schizont pellets using the KAPA Express Extract Kit (KAPABioSystem). Primers used for genotyping are indicated in Supplementary Figures2 , 4 and Table 1 .
For HA-tagging of PbPP1, promoter and coding regions of PBANKA_1028300, obtained using primers p38-p39 and p40-p41, respectively, were cloned into the p-TRAD4Ty-TetO7-HA-hDHFR plasmid (Pino et al., 2012 (link)). Before transfection, the sequence was linearized using BglII.
P. berghei ANKA transfections were performed by electroporation of schizont stages according to Janse et al. (2006) (link), using 10 μg of linearized constructs for each transfection. The schizont stages were obtained from 6 week-old infected Fisher rats (Charles River) and separated on a 55% Nycodenz gradient after 16 h culture at 37°C in RPMI1640 culture medium supplemented with 0.4% AlbuMAXTM II Lipid-Rich BSA (Life Technologies).
Genotyping was performed in parallel on total DNA from parental and transfected parasites extracted from schizont pellets using the KAPA Express Extract Kit (KAPABioSystem). Primers used for genotyping are indicated in Supplementary Figures
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