Mutant/mutation
In the 'double chimeric' mutant the endogenous P. berghei cs gene has been replaced by the P. vivax VK210 cs gene (PVP01_0835600) and the P. berghei trap gene has been replaced by the P. vivax trap gene (PVP01_1218700; XP_001614147.1).This line is drug-selectable marker free.

(the PvCSP insert consisted on the N- and C- terminal region from the Salvador I strain (NCBI Reference Sequence XP_001613068.1) flanking the central repeat regions of the circumsporozoite (CSP) VK210 of the Belem strain (GenBank accession number P08677).)

(the Pvtrap insert consisted of a synthetic allele composed of the protein-coding sequence of P. vivax TRAP (NCBI accession number XM_001614097) from the Salvador I strain

Protein (function)

Phenotype
Normal development throughout the complete life cycle showing complementation of P. berghei CS by P. vivax CS VK210 and P. berghei TRAP by P. vivax TRAP

Additional information
This chimeric parasite line has been used for analysing (protective) immune responses in mice immunized with different vaccines targeting P. vivax CS and P. vivax TRAP. Immunized mice were challenged with chimeric sporozoites expressing P. vivax CS and TRAP.

The double chimeric line PvCSP/PvTRAP in which both the csp (PBANKA_040320) and the trap (PBANKA_134980) gene of P. berghei were replaced with csp (PVP01_0835600; PVX_119355; XP_001613068.1) and trap (PVP01_1218700; XM_001614097) genes of P. vivax was generated by replacing the Pbtrap with the Pvtrap in a single chimeric line in which the Pbcsp had been previously replaced by the VK210 allele of Pvcsp. The single chimeric line, PbANKA-PvCS VK210(r)PbCS (line 2196cl1), had been generated using the gene insertion/marker out (GIMO) based transfection technology (RMgm-4136) and is free of a drug-selectable marker. In addition, it contains the GFP-Luciferase fusion gene as a reporter. The absence of a drug-selectable marker allowed for replacing the Pbtrap gene with the Pvtrap gene in parasites of the PbANKA-PvCS VK210(r)PbCS line in a single transfection experiment using the trap-replacement construct described by Bauza et al. (2014) (RMgm-1103). This construct contains a synthetic trap allele composed of the protein-coding sequence of P. vivax TRAP (accession number XM_001614097) from the Salvador I strain, which was codon optimized for expression in P. berghei using the GeneOptimizer software. In addition, this construct contains a positive/negative selectable marker (SM) cassette with  the hdfhr::yfcu fusion gene. Transfection of PbANKA-PvCS VK210(r)PbCS parasites with the trap-replacement construct and positive selection with pyrimethamine was performed using standard methods for transfection of P. berghei. This resulted in selection of double chimeric parasites (line 2207). Selected double chimeric parasites were cloned by the method of limiting dilution, resulting in the line PbANKA-PvCSP-VK210+PvTRAP+SM line (2207cl3). Subsequently,  the positive-negative SM cassette was recycled from the parasites of line 2207cl3 by applying negative selection by providing 5-fluorocytosine (5-FC) in the drinking water of mice. Negative selection and removal of the hdfhr::yfcu SM cassette resulted in selection of PbANKA-PvCSP-VK210+PvTRAP parasites (2207cl3m0). Selected parasites were cloned by the method of limiting dilution resulting in parasite line 2207cl3m0cl1. This double chimeric line PbANKA-PvCS VK210(r)PbCS-PvTRAP(r)Pbtrap, is referred to as PvCSP/PvTRAP parasites and does not contain a SM.


Other mutants