Mutant/mutation
The mutant expresses a fusion protein of GFP and luciferase under the control of the eef1a promoter. The reporter gene is introduced into the silent 230p locus using the GOMO method of transfection (see 'Additional information')

Protein (function)

Phenotype

Additional information
The parasites are selected by a combination of positive selection (pyrimethamine), negative selection (5-FC) and FACS sorting (GOMO transfection; 'Gene Out Marker Out').

The construct (see Fig. B below) used contains i) GFP-Luciferase under control of the constitutive promoter of PbHSP70 and ii) a hDHFR-yFCU fusion gene, for positive negative selection, coupled to a second fluorescent cassette, encoding the red fluorescent protein mCherry. Both hDHFR-yFCU and mCherry are placed under control of a single bidirectional promoter of PbeEF1a (PBANKA_113330 and PBANKA_113340). The GFP-Luciferase and mCherry reporter genes are followed by an identical 1 kb fragment corresponding to the 3' untranslated region (UTR) of P. berghei DHFR-TS, placed in the same orientation, which serves both as a transcription terminator, for proper expression of the reporters, and for recycling of the drug resistance cassette. Spontaneous recombination between the two homologous PbDHFR-TS 3' UTR fragments results in excision of both the hDHFR-yFCU and the mCherry expression cassettes Therefore, with this strategy, parasites that have excised the drug-selectable marker become GFP-LUC+mCherry-, and can be easily distinguished from GFP-LUC+ mCherry+ parasites still harbouring the hDHFR-yFCU marker.

Selection procedure (see Fig. C below):
1) Transfected parasites are first selected in a mouse by pyrimethamine treatment
2) GFP-LUC+mCherry+ parasites are selected by FACS sorting and used to infect a mice
3) This mouse is treated with 5-FC to select for parasites that have the selectable marker removed
4) GFP-LUC+mCherry- and marker free parasites are selected by FAC sorting and used to infect a mouse.

This new selection method eliminates the need for in vivo cloning of the parasites, and allows the rapid generation of drug-selectable marker-free P. berghei and P. yoelii mutants expressing a GFP or GFP-LUC cassette, using as few as three mice.

From: A rapid and robust selection procedure for generating drug-selectable marker-free recombinant malaria parasites. Manzoni, et al. Sci Rep. 2014 Apr 23;4:4760.

(B) Construct for targeted gene deletion are assembled by cloning a 59 and a 39 homology sequences of the target gene on each side of a triple cassette, consisting of a  GFP-LUC cassette (green box) under control of the  PbeEF1a promoter, a hDHFR-yFCU cassette (blue box) and a mCherry cassette (red box). The hDHFR-yFCU and mCherry genes are both under control of a single bidirectional PbeEF1a promoter. Upon a double crossover recombination event, the target gene is replaced by the GFP-LUC/hDHFR-yFCU/mCherry triple cassette. A second recombination event between the two identical PbDHFR/TS 39 UTR sequences (pink lollipops) results in excision of the hDHFR-yFCU and mCherry cassettes.
(C) Overview of the selection procedure. After transfection, parasites are injected into a first mouse, followed by positive selection of recombinant parasites with pyrimethamine. GFP+ mCherry+ pyrimethamine-resistant parasites are then recovered and transferred into a second mouse, and exposed to 5-FC for negative selection of parasites that have not excised the hDHFR-yFCU marker. GFP+ mCherry− 5-FC-resistant parasites, which harbour the intended recombined locus after marker excision, are then sorted by FACS and injected into a third mouse, allowing the isolation of a pure population of drug-selectable marker-free recombinant parasites.

Other mutants