Figure 6.

Genotyping of P. berghei clones of knockout of fumarate hydratase. a, schematic representation of the selectable marker cassette inserted into the fh gene locus of P. berghei genome. Primers (P1–P8) used for diagnostic PCRs are indicated. b, schematic representation of the fh gene (PBANKA_0828100) flanked by 5′ UTR and 3′ UTR showing the location of primers P9 and P10. Shown is agarose gel electrophoresis of PCRs with genomic DNA from clones A–C (c, left panel) and clones H–M (c, right panel) for detection of 5′ integration; d, clones J and M for detection of 3′ integration (other clones did not answer for this PCR); e, clone A for the detection of fh gene; f, clones A–C for the presence of selectable marker cassette; g, clones C and O using primers P3 and P8. Shown in this figure is the genotyping of representative P. berghei clones, and the data on all 17 clones that were characterized are provided in Fig. S5. Clones C and M and O did not answer for 5′ integration, and only clones J, M, and Q answered for 3′ integration (c and d here and Fig. S5, c and d). All clones answered for the presence of the fh gene (e here and Fig. S5e). All clones except C and O answered by PCR with primers P2 and P6 indicating the integration of the entire selectable marker cassette into the genome (f here and Fig. S5f). Clones C and O answered for a shorter fragment of the selectable marker cassette covered by primers P3 and P8 (g here and Fig. S5g). hDHFR-yFCU, human DHFR-yeast cytosine and uridyl phosphoribosyltransferase; mr, molecular weight marker; numbers to the left of c–g are the sizes of the marker DNA fragments in kbp.