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. 2017 Apr 18;7:46482. doi: 10.1038/srep46482

Figure 4. Cloning strategy for the development of two P. berghei parasite lines expressing P. vivax CSP VK210 or VK247.

Figure 4

(A) Schematic representation describing the generation of PbANKA-PvCSP(r)PbCSP (2196cl1 and 2199cl1) chimeric lines. The P. berghei csp (Pbcsp) gene coding sequence (CDS) was replaced by the GIMO deletion-construct (construct 1; pL1929) using the positive/negative selectable maker (SM; hdhfr::yfcu) cassette, resulting in the generation of the Pbcsp GIMO line (PbANKA-PbCSP GIMO; 2151cl1) after positive selection with pyrimethamine. Step 2: The GIMO insertion-construct (construct 2; pL1942 or pL1943) replaced the SM in the GIMO line with Pvcsp VK-210 or VK-247 CDS, respectively, after negative selection using 5-fluorocytosine (5-FC). (B) Schematic representation of the reporter PbANKA parasite line PbGFP-Luceef1α (676m1cl1), used to generate the replacement gene [RG] chimeric parasites. (C) Genotype analysis of Replacement Gene [RG] chimeric parasites and their intermediate GIMO mother-line knock out chimeric parasites using Southern analysis of chromosomes (chrs) separated by pulsed-field gel electrophoresis (PFGE). Left panel: PbANKA- ΔCSP GIMO: 2151 cl1. The correct integration of the SM in the right locus in Chr-4 and replacing the endogenous PbCSP gene (PBANKA_040320) was confirmed by using the 3′UTR Pbdhfr/ts probe. Right panel: PbANKA-PvCSP(r)PbCSP: 2196cl1 and 2199cl1. Confirmation of the correct integration of the PvCSP expression construct into the GIMO locus by Southern blot. (D) Genotype analysis by diagnostic PCR analysis of chimeric parasite lines to confirm the correct integration of both PvCSP VK210 and VK247 antigen expression cassettes. Primers sequences used are shown in Table S1, while the expected PCR product sizes and the primer numbers are listed in the table beside the Agarose gel image.