Adhesion Is Not Linked to clag9 Deletion Figure 6. Generation and characterization of clag9 KO. A. Subtelomeric chromosome 9 deletion in D10. Schematic representation of the genes missing from the right arm of chromosome 9, as compared to 3D7. The break point open reading frame (BPORF) is present but none of the 25 genes between the BPORF and the telemores, including clag9 (red), were detected by PCR (data not shown). Each block corresponds to a single gene, clag9 and BPORF are larger for emphasis and the size of blocks does not illustrate gene size. The genes shown in yellow have been functionally identified. The identity of the remaining genes has not yet been elucidated. B. Plasmid pCC1 exon 1+8 knockout leading to the disruption of the clag9 gene by allelic exchange with insertion of the hDHFR selection marker. Primers (arrows) used for verification of KO and hDHFR integration, the restriction sites, and Southern blot expected fragment sizes are shown. C. PCR analysis of 3D7 (lanes 1–4), the clag9 E5KO (lanes 5–8), and F4KO (lanes 9–12). Lanes 1, 5, and 9: 59UTR-F - exon 1-R; lanes 2, 6, and 10: 59UTR-F- hDHFR 59 out; lanes 3, 7, and 11: hDHFR 39 out – exon 8-RC39; lanes 4, 8, and 12: exon 2-F - exon 6-R. D–F. Southern blot of clag9 knockout. Genomic DNA digested by EcoRI (lanes 1 and 4), SpeI (lanes 2 and 5) or EcoRI+SpeI (lanes 3 and 6) separated on 0.8% agarose gel, transferred onto Hybond N+ and hybridized with different probes. D. Exon 1, present in both 3D7 and the clag9 E5KO. E. Hybridization of Exon 6 only with 3D7. F. Integration of the hDHFR selectable marker in the E5KO. doi:10.1371/journal.pone.0029039.g006 PLoS ONE | www.plosone.org 8 December 2011 | Volume 6 | Issue 12 | e29039