4. 2 Generation of Scd-3XHA transgenic parasites
For the 3XHA tagging of the stearoyl-CoA desaturase (Scd) (Pb ANKA_1110700) gene, two fragments, F1 (0.773 kb) and F2 (0.584 kb), were amplified from P. berghei genomic DNA using primers 1045/1055 and 1041/1042 and cloned into the pBC-3XHA-hDHFR vector at Xho I/Bgl II and Not I/Asc I, respectively. The construct was linearized with Xho I/Asc I and transfected into P. berghei ANKA schizonts as previously described (Janse et al. , 2006). The resistant parasites were selected using pyrimethamine (Sigma, USA), and genomic DNA was isolated using a genomic DNA isolation kit (Promega, USA). Correct 5’ and 3’ site-specific integration was confirmed by diagnostic PCR using primer pairs 1043/1218 and 1215/1044, respectively (primer sequences are given in Table S1). For the localization of Scd in the blood stage, transgenic Scd-3XHA-infected blood was spotted on 12-well slides (Thermo Fisher Scientific, USA) and fixed with 4% paraformaldehyde.