Fig. 2

Genotype analysis of parasites surviving ZFN-induced DSB. a PCR analysis of the zfn (P1 P2) and egfp (P3 P4) loci from genomic DNA of blood-stage parasites. The generated transgenic clone (c1) and parasite populations from mice positive after sporozoite challenge were analyzed. Expected sizes of the PCR products are 4184 bp for SpZFN, 3475 bp for LsZFN and 837 bp for egfp. The PCR product size from the ZFN locus was smaller in SpZFN SI 5–11 and LsZFN SI 1, 3 and 4. The egfp product was slightly smaller in SpZFN SI 1–4 and two products are observed in SpZFN SI 8. b Schematic alignment of the genomic sequences obtained from all zfn loci with size varying from original clones. The boundaries of homology regions used for gene copy number reduction are depicted for both zfnL and zfnR. The range of perfect homology used for recombination is indicated. c Alignment of the sequenced egfp gene of all parasite lines. Binding sites of ZFNs are coloured in the first sequence and all others if present. Microhomology regions implicated in repair are highlighted in colour and with a red background in those sequences that have undergone repair. Note that SpZFN SI 8 was a mixed population that differed in the egfp gene from the other populations that underwent repair. d Overview of the detected genomic changes of all SI parasites. Note that all have either a modification of the zfn locus or of the egfp gene with the exception of LsZFN SI 2, which survived without any genetic changes. Positions of primers used for PCR are indicated