Mutant/mutation
The mutant expresses the yeast Flp recombinase under the control of the promoter of uis4. The flp gene is integrated into the silent 230p locus by double cross-over integration.
The mutant does not contain a selectable marker. This marker (the hdhfr gene) has been removed from the genome by using the Flp/FRT site-specific recombination (SSR) system (see below 'Protein-function'). Removal of the hdhfr gene has been achieved by  transmission of the mutant through mosquitoes, thereby activating expression of the Flp recombinase that resulted in the excision of the hdhfr gene which was flanked by FRT sequences.

Protein (function)
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 FlpL (Low activity) recombinase is greatly 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.

Phenotype
Expression of Flp is specific for salivary gland sporozoites. Expression of Flp does not affect viability of infectivity of the different life cycle stages.

Additional information
This mutant expressing Flp can be used for introducing additional DNA sequences (genes) that are flanked by FRT sequences. Subsequently these sequences can be removed by mosquito transmission. This 'conditional mutagenesis' approach allows for the analysis of the effect of removal of genes essential for blood stage development.

Different Flp-expressing P. berghei lines have been generated that can be used as parasite receiver lines for conditional mutation/disruption of genes of interest. Several of these lines express also GFP (under the control of a constitutive promoter). Several lines have been generated in the P. berghei NK65 background. A disadvantage of several lines is that the flp gene is integrated by single cross-over recombination (insertion construct). The disadvantage of using an insertion construct is that the construct can be removed from the genome, thereby restoring the wild type genotype. in addition, some of these mutante contain the pbdhfr selectable marker. Therefore, introduction of additional mutations is dependent on a second drug selectable marker (hdhfr).

The line described here is made in P. berghei ANKA and the flp gene is integrated into the silent 230p locus by double cross-over integration.

See also mutant PbA FlpL@TRAP (RMgm-747) for a mutant expressing FlpL under the control of the trap promoter that is switched on in midgut and salivary gland sporozoites. The line is also made in P. berghei ANKA and the flpl gene is integrated into the silent 230p locus by double cross-over integration.

Other mutants
RMgm-268: A A P. berghei NK65 mutant expressing FlpL under the control of the promoter of trap. It does not contain a drug-selectable marker.
RMgm-269: A P. berghei NK65 mutant expressing Flp under the control of the uis4 promoter that is specifically switched on in sporozoites after salivary gland invasion. In addition it expresses GFP under the control of the constitutive hsp70 promoter.
RMgm-278: A P. berghei NK65 mutant expressing Flp under the control of the uis4 promoter that is specifically switched on in sporozoites after salivary gland invasion. It does not contain a drug-selectable marker and does not express GFP.
RMgm-683: A P. berghei NK65 mutant expressing FlpL under the control of the trap promoter. It does not contain a drug-selectable marker. In addition it expresses GFP under the control of the hsp70 promoter. The gfp gene is stably integrated into the genome.
RMgm-747: A P. berghei ANKA mutant expressing FlpL under the control of the trap promoter that is switched on in midgut and salivary gland sporozoites. The line is  made in P. berghei ANKA and the flpl gene is integrated into the silent 230p locus by double cross-over integration. The mutant does not express GFP.