Mutant/mutation

The mutant expresses a fusion of a drug resistance gene and a drug sensitivity gene, the so called postive-negative selectable marker (SM), constitutively expressed by the P. berghei eef1α promoter. Specifically, the mutant contains a fusion gene of hdhfr (human dihydrofolate reductase; positive SM) and yfcu (yeast cytosine deaminase and uridyl phosphoribosyl transferase; negative SM) stably integrated into the neutral S1 locus  through double cross-over recombination.

This line is named GIMO(S1) mother line (gene insertion/marker out): GIMO (S1)_PbANKA.

The GIMO mother line shows a normal development during the complete life cycle, including development in the mosquito and in the liver. This line can therefore be used as a reference line for introduction of transgenes.

The GIMO mother line is used for introduction of transgenes into the modified S1 locus through transfection with constructs that target the s1 locus. These constructs insert into the s1 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.

The use of this line greatly simplifies and speeds up the generation of mutants expressing heterologous proteins, free of drug-resistance genes, and requires far fewer laboratory animals.

Other mutants

Other GIMO mother lines have been generated in P. berghei and P. yoelii using different neutral loci (p230p, s1 etc) for introducing the hdhfr::yfcu selectable marker cassette