Figure 6.

Absence of Evidence for Multiple Integration Events

(A) Vector pools were transfected into marker-free lines with pre-existing barcodes in cdpk1, cdpk3, or cdpk4. New barcodes account for approximately half of the total, as would be expected if each parasite genome carried exactly one new barcode. The slight overrepresentation of background barcodes on day 4 probably comes from parasites that failed to integrate a vector and which were not yet completely eliminated after only 3 days of drug selection. All data points are supported by three experiments, and error bars show standard deviations. See Figure S3 for genotyping of marker-free lines.

(B) PCR genotyping was performed on parasite gDNA from six infected mice, each transfected with one of three final targeting vectors in the presence of a 20-fold excess of intermediate vectors (10 μg total DNA per transfection), which have the same homology arms but a zeocin resistance cassette that cannot be selected in P. berghei. Presence of intermediate vectors in the input cocktail but absence in the resistant parasite populations suggests that multiple integration events are rare or absent, since hitchhiking of marker free insertions would otherwise be observed.