Mutant/mutation
The mutant is a 'Flp/FRT conditional knock-out mutant' of MSP-1 (merozoite surface protein 1, precursor). The mutant expresses the yeast Flp recombinase under the control of the uis4 promoter  and contains a FRTed 3'UTR of the msp-1 locus. In addition it expresses GFP under the control of the hsp70 promoter. The gfp gene is stably integrated into the genome. This mutant has been generated by replacement of the endogenous msp-1 gene by an msp-1 gene with a FRTed 3'UTR in the mutant RMgm-269 that expresses Flp and GFP.
The 3'UTR of msp-1 locus is removed from the genome by using the Flp/FRT site-specific recombination (SSR) system (see RMgm-269). Removal of the FRTed 3'UTR of msp-1  has been achieved by  transmission of the mutant through mosquitoes, thereby activating expression of the Flp recombinase in sporozoites that resulted in the excision of the 3'UTR of msp1 which was flanked by FRT sequences.

Protein (function)
Merozoite attachment to the erythrocyte surface is mediated by a family of proteins called merozoite surface proteins (MSPs). MSP-1, is a glycosylphosphatidylinositol (GPI)-anchored protein that covers the entire surface of free merozoites. Antibody inhibition experiments and and molecular genetic studies have shown that MSP-1 is essential for merozoite adhesion to erythrocytes.  In addition, expression of MSP-1 is switched on during the first half and peaks during the second half of the liver stage developmental process. This process, which follows hepatocyte invasion by sporozoites, consists in karyokinesis without cytokinesis before budding off of thousands of uninucleate merozoites and lasts 50–70 hr in rodents.

Phenotype
The Flp/FRT site-specific recombination (SSR) system of yeast has been used to silence MSP-1 expression specifically in liver stages. Removal of the FRTed 3'UTR of msp-1  has been achieved by  transmission of the mutant through mosquitoes, thereby activating expression of the Flp recombinase in sporozoites that resulted in the excision of the FRTed 3'UTR of msp-1. Removal of the 3'UTR region of msp-1 resulted in 'down-regulation' of expression of MSP-1. Quantification of MSP-1 expression in mature liver stages showed that mutant parasites produced 6.5% of the amounts of MSP-1 synthesized by control parasites (see 'Additional information'). This indicated that MSP-1 was silenced in the 'excised' MSP1/Cond mutant liver stages. Because the proportion of non-excised parasites (5%) was similar to the proportion of residual MSP-1 (6.5%) in the parasite population, the excised parasites appear to produce virtually no MSP-1.
The liver stages of the mutant yielded on average 30-fold fewer merozoites than wild type liver stages and mature liver stages showed aberrant merozoite formation (see also 'Additional remarks'), indicating that MSP-1 plays a crucial role in the formation of merozoites by the liver stage of the parasite.

Additional information
In the paper unsuccessful attempts  to inactivate MSP-1 in erythrocytic stages by disruption of msp-1 using standard transfection technology  are mentioned (see RMgm-326), indicating the essential nature of MSP-1 for P. berghei blood stage multiplication.

To verify that the excised MSP1/Cond liver stages did not produce MSP1, fluorescent HepG2 cells containing MSP1/Cond or control parasites were sorted by FACS after 58 hr incubation, and the cell extracts were analyzed by western blotting using anti-MSP-1 antibodies. Quantification of the MSP-1 signal showed that mutant parasites produced 6.5% of the amounts of MSP1 synthesized by control parasites. This indicated that MSP-1 was silenced in excised MSP1/Cond mutant liver stages. Because the proportion of non-excised parasites (5%) was similar to the proportion of residual MSP-1 (6.5%) in the parasite population, the excised parasites appear to produce virtually no MSP1.

Liver stage development was investigated by using transmission electron microscopy (TEM). HepG2 cells were incubated with mutant and control sporozoites, and after 58 hr incubation, fluorescent cells were sorted by FACS and processed for TEM. The multinucleated control parasites generated merozoites of homogenous size and regular shape that budded off from the periphery of the parasite mass, progressively filling the parasitophorous vacuole and ultimately the host cell cytoplasm. By contrast, the majority of MSP1/Cond parasites contained numerous vacuoles of various sizes and generated abnormal progeny. Merozoite buds emerged within the intraparasitic vacuoles, rather than at the parasite periphery, and the internal buds displayed abnormal shapes, frequently having an enlarged base or an orientation tangential rather than perpendicular to the plane of the parasite membrane. these results indicate that MSP-1 appears to play a crucial role in the formation of merozoites by the liver stage of the parasite.

In the Flp/FRT site-specific recombination (SSR) system of yeast, the Flp recombinase recognizes the 34 bp FRT sites and excises any DNA located between two directly oriented sites (referred to as the FRTed sequence). The activity of Flp recombinase is  reduced at 37°C and above - temperatures in erythrocytic stages in the vertebrate host - and is maintained at 20°C –25°C, temperatures permissive for parasite development in the mosquito.

Other mutants
RMgm-201: A mutant expressing a mutated form of MSP-1 (Replacement of the P. berghei MSP-1 19 kD C-terminal with the P. falciparum MSP-1 19 kD C-terminal)
RMgm-326: Unsuccessful attempts to disrupt MSP-1