Summary

RMgm-1026
Malaria parasiteP. berghei
Genotype
Transgene
Transgene not Plasmodium: GFP
Promoter: Gene model: PBANKA_0711900; Gene model (P.falciparum): PF3D7_0818900; Gene product: heat shock protein 70 (HSP70)
3'UTR: Gene model: PBANKA_0719300; Gene product: bifunctional dihydrofolate reductase-thymidylate synthase, putative (dhfr/ts)
Replacement locus: Gene model: PBANKA_0306000; Gene product: 6-cysteine protein (P230p)
Phenotype Asexual bloodstage;
Last modified: 28 April 2014, 12:57
  *RMgm-1026
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 24755823
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone P. berghei ANKA cl15cy1
Other information parent lineA reference wild type clone from the ANKA strain of P. berghei (PubMed: PMID: 17406255).
The mutant parasite was generated by
Name PI/ResearcherManzoni G; Silvie O
Name Group/DepartmentINSERM
Name InstituteINSERM
CityParis
CountryFrance
Name of the mutant parasite
RMgm numberRMgm-1026
Principal nameΔp230p-GFP
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageGFP expression in blood stages
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses GFP under the control of the HSP70 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. A below) used contains i) GFP 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 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+mCherry-, and can be easily distinguished from GFP+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+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+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 G, et. al. Sci Rep. 2014 Apr 23;4:4760.

(A). Construct for targeted gene deletion are assembled by cloning a 5′ and a 3′ homology sequences of the target gene on each side of a triple cassette, consisting of a GFP cassette (green box) under control of the PbHSP70 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 PbeEF1α promoter. Upon a double crossover recombination event, the target gene is replaced by the GFP/hDHFR-yFCU/mCherry triple cassette. A second recombination event between the two identical PbDHFR/TS 3′ 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


  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameGFP
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/constructPlasmid double cross-over
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid
Selectable marker used to select the mutant parasitehdhfr/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modification
Additional remarks selection procedureThe parasites are selected by a combination of positive selection (pyrimethamine), negative selection (5-FC) and FACS sorting.
1) Transfected parasites are first selected in a mouse by pyrimethamine treatment
2) GFP+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+mCherry- and marker free parasites are selected by FAC sorting and used to infect a mouse
Other details transgene
Promoter
Gene Model of Parasite PBANKA_0711900
Gene Model P. falciparum ortholog PF3D7_0818900
Gene productheat shock protein 70
Gene product: Alternative nameHSP70
Primer information details of the primers used for amplification of the promoter sequence  Click to view information
Primer information details of the primers used for amplification of the promoter sequence  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
3'-UTR
Gene Model of Parasite PBANKA_0719300
Gene productbifunctional dihydrofolate reductase-thymidylate synthase, putative
Gene product: Alternative namedhfr/ts
Primer information details of the primers used for amplification the 3'-UTR sequences  Click to view information
Primer information details of the primers used for amplification the 3'-UTR sequences  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
Insertion/Replacement locus
Replacement / InsertionReplacement locus
Gene Model of Parasite PBANKA_0306000
Gene product6-cysteine protein
Gene product: Alternative nameP230p
Primer information details of the primers used for amplification of the target sequences  Click to view information
Primer information details of the primers used for amplification of the target sequences  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
Sequence Primer 3
Additional information primer 3
Sequence Primer 4
Additional information primer 4