Mutant/mutation
The mutant expresses the 'P. vivax like' CSP (G10; locus PVU09738; Accession U09738). The gene is introduced as an additional copy into the silent p230p locus under control of the sporozoite/liver stage promoter uis4 and expresses the reporter fusion protein GFP-Luciferase under control of the constitutive eef1a promoter

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

The mutant has been generated in the reference line GIMO_PbANKA (RMgm-687). This 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 CS protein is the major protein on the surface of sporozoites and is critical for development of sporozoites within the oocysts and is involved in motility and invasion of both the salivary gland of the mosquito and the liver cells. The protein is also found on the oocyst plasma membrane and on the inner surface of the oocyst capsule. Specific motifs in CS are involved in sporozoite binding to mosquito salivary glands and in sporozoite attachment to heparan sulfate proteoglycans in the liver of the mammalian host. During substrate-dependent locomotion of sporozoites, CS is secreted at the sporozoite anterior pole, translocated along the sporozoite axis and released on the substrate at the sporozoite posterior pole. Following sporozoite invasion of hepatocytes, the CS is released in the host cell cytoplasm.

Phenotype
Both oocyst and sporozoite production in A. stephensi mosquitoes was comparable to wild-type P. berghei parasites. The infectivity of chimeric spz, as determined by the length of the prepatent period after intravenous injection of 1000 spz in BALB/c mice, was similar to that in mice infected with wild-type P. berghei spz.

The expression of the PvCSP-P. vivax-like protein in Pb-PvCSP-like G10 spz was determined by immunofluorescence analysis using sera from mice immunized with the recombinant proteins. The 3D11 antibody recognizing P. berghei CSP was used as a control. Pb-PvCSP-like G10 spz stained both with the antiserum and the 3D11 antibody, demonstrating the expression of both P. berghei CSP and PvCSP-P. vivax-like protein in spz of the Pb-PvCSP-like G10 line.

Additional information

Other mutant