Fig. 6.

Integration PCR of parasite lines generated by detached cell transfection using a single or b double crossover integration. a P. berghei parasites were transfected with pL0017-NLuc [18], a construct that allows integration by single crossover into the P. berghei c-ssu-rRNA or d-ssu-rRNA locus. PCR amplification was used to verify integration of the plasmid into the P. berghei genome. The schematic shows the possible annealing regions of the primer pairs for the different parasite genotypes that could result. If the construct did not integrate into the parasite genome the primer pairs 1 + 4 for the c-ssu-rRNA or 2 + 4 for the d-ssu-rRNA locus generated a 3 kb PCR product. If the construct was successfully integrated, the primer pairs are too far apart (>14 kb) to generate a PCR product. Primer pairs 1 + 3 and 2 + 3 are designed to confirm integration, a PCR product of 3 kb is generated only if the construct has integrated. PCR results of the PbNLuc parasites show integration into the c-ssu-rRNA locus, as the band for primer pair 1 + 4 is absent and the primer pair 1 + 3 gives a 3 kb band, there is no integration into the d-ssu-rRNA locus, which shows the same PCR products as for the WT parasites. b P. berghei parasites were transfected with PbGEM-336027, the construct facilitates integration of the hdhfr/yfcu cassette via double crossover into the PBANKA_1145300 gene locus, resulting in a knock-out (KO). The primer pair QCR2 + QCR1 results in a 650 bp fragment, if parasites have a PBANKA_1145300 WT genotype. The primer pair QCR2 + GW2 is designed to show integration. A 990 bp PCR product is generated only, if the construct has integrated at the correct site of the parasite genome. The PCR results show integration of the hdhfr/yfcu cassette into the PBANKA_1145300 gene locus. Transfected parasites are present in a mixed population of parasites having a PBANKA_1145300 KO genotype and PBANKA_1145300 WT genotype