FIG. 2.

Integration of pHDGFP and its stable expression in P. falciparum. (A) PCR analysis of plasmid integration after transfection. Template genomic DNA from either the parent strain 3D7 (lanes 1 to 3) or transfected cells (lanes 4 to 6) was used to amplify (i) human DHFR by using primers KK5 and KK6 (lanes 1 and 4), (ii) HDGFP by using KK5 and G2 (5′-CTGCCTCGAGTTATAAATCTTCTTCAGATATTAATTTTTGTTC) (lanes 2 and 5), or (iii) the integrated fusion cassette by using the T7 promoter primer (5′-TAATACGACTCACTATAGGGAGA) and H1 (5′-GCGGATCCGTTATCTAACAAAAGTACGG; obtained from the coding region of HRP III) (lanes 3 and 6). Primer KK6 is indicated in Fig. 1A. The remaining primers are shown in panel C. (B) Southern analysis of parasite DNA from clones containing integrated or episomal pHDGFP. Genomic DNA from the parent strain 3D7 (lanes 1, 3, and 4) or a clone containing integrated pHDGFP (D8; lanes 2, 5, and 6) was digested with SnaBI (lanes 1 and 2) or NsiI (N; lanes 3 and 5) or double digested with KpnI-NsiI (KN; lanes 4 and 6) and probed with the full-length 5′hrp3 NsiI-KpnI fragment from pHDGFP (or a fragment of the gfp coding region; not shown). DNA from a clone containing episomal pHDGFP (C5) was double digested with KpnI-NsiI (lane 7) and probed as shown for lanes 1 to 6. Values to the left and right of panels A and B are in kilobases. (C) Physical map of the HRP III locus of clone P. falciparum D8. This was derived from Southern analysis of genomic DNA of clone D8 presented in panel B. (D) Green fluorescence detected in clone D8. Cells were imaged, and 0° projections of three-dimensional reconstructions of ring (r)- and trophozoite (t)-infected erythrocytes were obtained as described for Fig. 1B.