Mutant/mutation
The mutant expresses mCherry under the control of the promoter of P. berghei b9 (PBANKA_080810). The mutant does not contain a drug-selectable marker.

The mutant has been generated and selected using the GIMO transfection method ('gene insertion/marker out') of transfection of GIMO mother lines that contain the hdhfr::yfcu selectable marker into the silent 230p locus.

The mutant has been generated in the reference line GIMO_PbANKA (RMgm-687). The GIMO mother line is used for introduction of transgenes into the modified 230p locus through transfection with constructs that target the 230p locus. These constructs insert into the 230p locus (‘gene insertion’), thereby removing the hdhfr::yfcu selectable marker (‘marker out’) from the genome of the mother lines. Transgenic parasites that are marker-free are subsequently selected by applying negative drug selection using 5-FC. This selection procedure is performed in vivo in mice

Protein (function)
The B9 protein contains a 4-cysteine domain with a structure that is highly similar to the structure of 4-cysteine domains in the known proteins of the 6-Cys family and is identified as a 6-Cys-related protein. B9 is predicted to be glycosylphophatidylinositol (GPI) anchored. B9 transcripts are found in sporozoites. Evidence is presented for translational repression of b9 transcripts in P. berghei sporozoites. The B9 protein is found in (young) liver stages. For P. falciparum B9 evidence is presented for a location at the plasmalemma membrane of liver stages. P. falciparum and P. berghei mutants lacking expression of B9 abort development during development of young liver stages (see RMgm-932, RMgm-933, RMgm-934 and RMgm-936 for P. berghei  and P. yoelli mutants lacking expression of B9)

Phenotype
No fluorescence signals were detected in blood-stages, oocysts and sporozoites, despite the presence of b9 transcripts in sporozoites. Strong fluorescence signals were detected in hepatocyte-culture 5 hours after the addition of mCherryb9 sporozoites. The non-fluorescent sporozoites indicate that b9 transcripts are translationally repressed and that the B9 protein is generated after a developmental switch to intrahepatic development. mCherry expression was observed both in intra-hepatic stages and in extracellular sporozoites that had been activated and  started to ‘round up’. Five hours post infection (hpi) fluorescence signals decreased; at 15hpi weak signals were detected in all parasites and no fluorescence was detected at 24hpi and 32hpi.

Additional information

Supporting Figure S2
Generation of transgenic mCherryb9 and mCherryhsp70 expressing mCherry under the control of the b9 and hsp70 promoter, respectively
A. Schematic representation of the introduction of two mCherry-expression cassettes (pL1694 and pL1695) into the GIMOpbANKA. Construct pL1694 contains the hsp70 promoter (5’hsp70)-mCherry-3’hsp70 cassette and construct pL1695 the b9 promoter-mCherry-3’b9 cassette. These constructs are integrated into the modified P. berghei 230p locus containing the hdhfr::yfcu selectable marker cassette (hatched box) by double cross-over homologous recombination at the target regions (grey boxes). Negative (Neg) selection with 5-FC selects for the transgenic parasite line (1804cl1 and 1867) that have the mCherry-expression cassettes introduced into the 230p locus and the hdhfr::yfcu marker removed. Location of primers used for PCR analysis (see B) and sizes of PCR products are shown (see Table S6 for primer sequences). B. Diagnostic PCRs analysis confirms the correct integration of construct pL1694 in line 1804cl1 and construct pL1695 in line 1867 parasites shown by the absence of the hdhfr::yfcu marker and the presence of the mCherry gene: 5' integration PCR (5’; primers 5510- 6836 or 7166), 3' integration PCR (3’; primers 6841 or 7167-5511), amplification of mCherry (mC; primers 6663-5514) and the selectable marker (M; primers 4698-4699). Primer locations and product sizes are shown in A (primer sequences in Table S1 primer).

Other mutants
See RMgm-932, RMgm-933, RMgm-934 and RMgm-936 for P. berghei  and P. yoelli mutants lacking expression of B9