Figure 3
Generation of a transgenic P. knowlesi line expressing SICA-HUVEC-Myc using monkey RBCs. (A) A schematic image of the SICA-HUVEC-Myc expression construct. Full-length of SICA-HUVEC was fused with Myc epitopes (Myc) at its C terminus and the P. falciparum CRT promoter region (PfCRT 5′) was used as a promoter. A human DHFR expression cassette was used for the drug selection. (B) Western blotting of the wild-type parental P. knowlesi line (WT) and the transgenic line expressing SICA-HUVEC-Myc in monkey RBCs. Proteins were sequentially extracted by freeze-thawing (FT), with 1% Triton X-100 (Tx), and then with 2% SDS for the transfectants; whereas they were extracted with 2% SDS only for the wild type parasites. Bands detected with anti-Myc antibody (α-Myc) around the expected size for the SICA-HUVEC-Myc are indicated with an arrowhead. (C) Trypsin treatment of the transgenic line expressing SICA-HUVEC-Myc. Trypsin-treated or untreated samples were sequentially extracted as described above and subjected to Western blot with α-Myc or a loading control anti-EXP2 antibody (α-EXP2; The original blot is presented in Fig. S10). To make the 24-kDa band (arrow) easier to see, the intensity of the upper image is increased. (D) IFA images of P. knowlesi expressing SICA-HUVEC-Myc in monkey RBCs stained with anti-Myc antibody (α-Myc, green). Signals detected with α-Myc were merged with DAPI nucleus signals (blue) and differential interference contrast (DIC) images (Merge). The bottom panels are images from a negative control reacted with normal mouse IgG. (E) Images of P. knowlesi expressing SICA-HUVEC-Myc in monkey RBCs stained with Giemsa's solution following IFA with α-Myc antibody. Signals detected with α-Myc were merged with Giemsa-stained images (Merge).
