Mutant/mutation
The mutant expresses a mutated form of merozoite surface protein 1, precursor (MSP1). The P. berghei MSP-1 19 kD C-terminal region is replaced with the P. falciparum MSP-1 19 kD C-terminal region.
An independent mutant in which the P. berghei MSP-1 19kD C-terminal is replaced with the P. falciparum  MSP-1 19kD C-terminal has been described (RMgm-201). Here, the  same strategy was applied to create the mutant. The primer sites used to amplify both the P. berghei target regions and the P. falciparum MSP-1 19kD C-terminal region are identical to the ones used to create the mutant described in RMgm-201. Therefore, there are no significant differences between these mutants with respectto the replaced P. berghei and P. falciparum MSP-1 19kD C-terminal regions.

Protein (function)
MSP1 is an attractive target for antibody therapy. Animals actively or passively immunized against MSP1 can be protected against parasite challenge, and mAbs to MSP1 can block parasite invasion of erythrocytes in vitro. MSP1 undergoes dual proteolytic processing. It is initially cleaved (primary processing) into multiple fragments that form a protein complex on the merozoite surface. Then, at erythrocyte invasion, the protein is cleaved again (secondary processing) and shed from the surface, except for a C-terminal 19-kDa polypeptide (MSP119) that comprises two epidermal growth factor (EGF)-like domains and which is carried into the newly invaded erythrocyte.

Phenotype
The growth rate of asexual blood stages of the mutant in mice was comparable to that of wild type parasites (see further 'Additional Information').

Additional information
A comparion of growth rates between wild-type and the PfMSP1-19Pb mutant parasites indicated that the replacement of the MSP1-19 region and the expression of the GFP protein were dis not affect the growth of the asexual blood stages of the mutant parasites. The mutant was used to evaluate the protective efficacy in vivo of specific IgG elicited by a PfCP-2.9 malaria vaccine that contains the PfMSP1-19. BALB/c mice passively transferred with purified rabbit IgG to the PfCP-2.9 survived a lethal challenge of the PfMSP1-19Pb mutant parasites, but not infections with wild-type P. berghei. Control mice passively transferred with purified IgG obtained from adjuvant only-immunized rabbits were vulnerable to both mutant and wild-type infections.

The PyrFlu/PbfMSP-1/ PbM3′ plasmid used to create the mutant is based on the transfection vector PyrFlu which contains the fusion gene dhfr-ts-GFPmut2 that confers pyrimethamine resistance and directs the GFP fluorescence signal. The plasmid and the P. berghei mutant created using this plasmid are described in more detail in RMgm-227.

To create the PbfMSP1 fusion gene, the 1,423-base pair (bp) P. berghei MSP1 targeting sequence and the 330-bp PfMSP1-19 fragment were PCR amplified from P. berghei ANKA genomic DNA and a P. falciparum FCC1/HN isolate, respectively, using primers PbF and PbR, and PfF and PfR. The two PCR products were fused in frame by using the primers PbF and PfR and resulted in PbfMSP1. The 860-bp PfHSP86 3′ untranslated region (UTR) was amplified from plasmid pHC1 (a gift from the Malaria Research and Reference Reagent Resource Center) using the primers HSPF (5′-CGGCTCGAGTTATATAATATATTTATGTAC-3′) and HSPR (5′-CGGGGATCCTATTTGATGAATTAACTACAC-3′) and ligated in the correct orientation to PbfMSP1 to create a 2.6 kb fragment that was cloned into the SacI/BamHI sites of pPyrFlu. The other 0.5 kb targeting sequence of PbMSP-1 3′UTR was also amplified from P. berghei ANKA genomic DNA using the primers Pb3′F and Pb3′R and then cloned into the ApaI/KpnI sites of pPyrFlu. The resulting vector containing PyrFlu/PbfMSP-1/ PbM3′ was linearized with KpnI and SacI before transfection.

An independent mutant in which the P. berghei MSP-1 19kD C-terminal is replaced with the P. falciparum  MSP-1 19kD C-terminal has been described (RMgm-201). Here, the  same strategy was applied to create the mutant. The primer sites used to amplify both the P. berghei target regions and the P. falciparum MSP-1 19kD C-terminal region are identical to the ones used to create the mutant described in RMgm-201. Therefore, there are no significant differences between these mutants with respectto the replaced P. berghei and P. falciparum MSP-1 19kD C-terminal regions.

Other mutants
RMgm-201: Another mutant in which the P. berghei MSP-1 19 kD C-terminal region is replaced with the P. falciparum MSP-1 19 kD C-terminal region.