Mutant/mutation
The mutant expresses both P. falciparum TRAP and LSAP2 under control of the sporozoite/liver stage promoter uis4 and expresses the reporter fusion protein GFP-Luciferase under control of the constitutive eef1a promoter

Both the TRAP and LSAP2 expression cassettes has been introduced into the genome by the GIMO transfection method ('gene insertion/marker out'). The trap gene has been introduced into the silent 230p locus and the lsap2 gene into the silent s1 locus.

The LSAP2 expression cassette has been introduced into the s1 locus of mutant 2353cl2; see RMgm-4067. This mutant expresses P. falciparum TRAP under control of the uis4 promoter and GFP-luciferase. Both expression cassettes are introduced into the silent 230p locus. In addition this line is a GIMO line since it contains the positive/negative selectable  marker cassette (yfcu/hdhfr) in the silent s1 locus (to be able to introduce additional transgenes in the s1 locus)

Protein (function)

Phenotype
Antigen expression by chimeric parasites was confirmed by immunofluorescence staining (IFA) of sporozoites with serum from mice vaccinated with single antigens PfTRAP and PfLSAP2.

From the abstract:

Cell mediated protection from liver-stage malaria is reliant on a sufficient number of antigen specific T cells reaching the liver during the time that parasites are present. A single vaccine expressing two antigens could potentially increase both the size and breadth of antigen specific response, while halving vaccine production cost. In this study we investigated combining two liver stage antigens PfLSA1 and PfLSAP2, and investigated induction of protective efficacy by co-administration of single-antigen vectors or vaccination with dual-antigen vectors, using simian Adenovirus and Modified Vaccinia Ankara Virus vectors. Efficacy of these vaccines was assessed in mouse malaria challenge models using chimeric P. berghei parasites expressing the relevant Pf antigens and challenging mice at the peak of T cell response. Vaccination with a combination of the single-antigen vectors expressing PfLSA1 or PfLSAP2 was shown to improve protective efficacy compared to vaccination with each single antigen vector alone. Vaccination with dual-antigen vectors expressing both PfLSA1 and PfLSAP2 resulted in responses to both antigens, particularly in outbred mice, and most importantly efficacy was equivalent to vaccination with a mixture of single-antigen vectors

Additional information
Double Additional Gene (DAGs) chimeric parasites were generated as previously described (38) by using a single additional gene (SAG) as the background parent line and stably inserting the additional P.falciparum gene into the neutral s1 gene locus in chromosome 12 through double-crossover recombination using a 2-step “gene insertion/marker out” (GIMO) transfection protocol.
In the first step, we deleted the Pbs1 CDS and replaced it with a positive-negative selectable marker to create a Pbs1 deletion GIMO line from the parent line. In order to do this, we generated the plasmid construct, based on the standard GIMO DNA construct pL0034, which contains a positive-negative (hdhfr::yfcu) SM cassette and was used to insert both the Pbs1 5′ and 3′ gene targeting regions (TRs). The linear pL1928 DNA construct was introduced into parent plasmid by using standard methods of transfection. Transfected parasites were selected in mice through addition of pyrimethamine in the drinking water. Transfected parasites were cloned by limiting dilution, resulting in the PbANKA-PfAg+PbΔs1 GIMO line.

Other mutant