RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5188
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_0701900; Gene model (P.falciparum): PF3D7_0828800; Gene product: GPI-anchored micronemal antigen (GAMA, Putative Secreted Ookinete Protein 9, PSOP9)
Transgene
 Transgene not Plasmodium: A fusion of GFP (gfp-mu3) and Luciferase Firefly (LucIAV)
Promoter: Gene model: PBANKA_1133300; Gene model (P.falciparum): PF3D7_1357100; Gene product: elongation factor 1-alpha (eef1a)
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 (230p)
Phenotype Fertilization and ookinete; Oocyst; Sporozoite; Liver stage;
Last modified: 29 March 2022, 17:35
  *RMgm-5188
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption, Introduction of a transgene
Reference (PubMed-PMID number) Not published (yet)
MR4 number
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Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone P. berghei ANKA 676m1cl1 (RMgm-29)
Other information parent line676m1cl1 (RMgm-29) is a reference ANKA mutant line which expresses GFP-luciferase under control of a constitutive promoter. This reference line does not contain a drug-selectable marker (PubMed: PMID: 16242190).
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The mutant parasite was generated by
Name PI/ResearcherJennison C, Vaughan AM
Name Group/DepartmentCenter for Global Infectious Disease Research
Name InstituteSeattle Children’s Research Institute
CitySeattle
CountryUSA
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Name of the mutant parasite
RMgm numberRMgm-5188
Principal name∆GAMA
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
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Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNot different from wild type
Fertilization and ookineteWild type numbers of ookinetes are produced
Oocyst(Strongly) reduced oocyst production. Evidence is provided for a defect in the egress of sporozoites from oocysts.
Sporozoite(Strongly) reduced oocyst production and strongly reduced numbers of midgut- and salivary gland sporozoites. Evidence is provided for a defect in the egress of sporozoites from oocysts. Sporozoites show strongly reduced motility
Liver stage(Midgut) sporozoites are unable to establish an infection in mice (after intravenous injection).
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of GAMA and expresses the reporter GFP-Luciferase under the constitutive eefia promoter.

Published in: bioRxiv preprint doi: https://doi.org/10.1101/2022.02.24.481744

Protein (function)
GAMA is a micronemal protein with a C-terminal GPI-anchor. P. berghei GAMA consists of 625 aa with a predicted molecular mass of ~71 kDa, (signal peptide cleavage results in a 69 kDa mass). In P. falciparum, two additional asparagine rich repeat regions result in a larger 738 aa protein (predicted molecular weight ~85 kDa, 83 kDa after cleavage of the signal peptide). For P. berghei, SignalP-5.0 predicts a signal peptide with a cleavage site at S21/L22. For P.falciparum, cleavage is predicted at the same locus, A21/L22.
P. berghei and P. falciparum share 44.8% identity across the full-length sequence, with areas of higher conservation found towards the N-terminus (residues 64-347 = 60% identity) and C-terminus (residues 501-700 = 60% identity) split by a central repeat region of low shared identity (residues 348-500, with predicted disorderly structure. The C-terminal transmembrane domain is predicted to be cleaved and appended with a GPI anchor. Downstream of signal peptide cleavage, P. falciparum contains seven cysteine residues prior to the central repeat region and one downstream which are conserved across all Plasmodium species investigated. P. berghei and other rodent infective species of Plasmodium bear an additional cysteine at position 152 in the alignment. GAMA is present in all Plasmodium species and indeed all other intracellular apicomplexans.

Phenotype
Wild type-like blood stage development. Wild type numbers of ookinetes are produced. (Strongly) reduced oocyst production and strongly reduced numbers of midgut- and salivary gland sporozoites. Evidence is provided for a defect in the egress of sporozoites from oocysts. Sporozoites show strongly reduced motility. (Midgut) sporozoites are unable to establish an infection in mice (after intravenous injection). 

Additional information

Other mutants

  Disrupted: Mutant parasite with a disrupted gene
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Details of the target gene
Gene Model of Rodent Parasite PBANKA_0701900
Gene Model P. falciparum ortholog PF3D7_0828800
Gene productGPI-anchored micronemal antigen
Gene product: Alternative nameGAMA, Putative Secreted Ookinete Protein 9, PSOP9
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Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct used(Linear) plasmid double cross-over
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid
Partial or complete disruption of the geneComplete
Additional remarks partial/complete disruption
Selectable marker used to select the mutant parasitehdhfr/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationTo generate the knock-out and P. falciparum GAMA knock-in parasite lines the Gene In Marker Out (GIMO) methodology was employed. In brief, the GIMO plasmid was modified with the addition of 5’ and 3’ homology arms corresponding with the 5’ and 3’ UTR of GAMA, flush to the ORF of GAMA, so that a KO parasite would be generated expressing the hdhfr/yfcu cassette in place of the GAMA gene. Transfections were performed in the marker-free, genome integrated GFPLuc expressing P. berghei parasite line Pb676, which was used as wild type throughout this study. Transfectant parasites obtained after negative selection with pyrimethamine were cloned by limiting dilution into naïve recipient mice to obtain clonal parasites. To generate the Pb PfGAMA parasite line, the full-length P. falciparum GAMA gene was amplified from P. falciparum genomic DNA and inserted via the KpnI and SacII restriction sites into the PbGAMA knockout GIMO plasmid. Transfections were performed on ∆GAMA parasites and integration of PfGAMA was achieved by negative selection with 5-fluorocytosine. Clonal parasites were obtained.
Additional remarks selection procedure
Primer information: Primers used for amplification of the target sequences  Click to view information
Primer information: 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
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6
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  Transgene: Mutant parasite expressing a transgene
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Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameA fusion of GFP (gfp-mu3) and Luciferase Firefly (LucIAV)
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Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct(Linear) plasmid 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 parasitegfp (FACS)
Promoter of the selectable markereef1a
Selection (positive) procedureFACS (flowsorting)
Selection (negative) procedureNo
Additional remarks genetic modificationThe GFP-Luciferase gene (1 copy) has been inserted into the 230p locus (PB000214.00.0) by double cross-over integration.
Additional remarks selection procedureThis reporter mutant expressing GFP-Luciferase does not contain a drug-selectable marker. This mutant has been selected by FACS sorting after transfection based on GFP fluorescence.
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Other details transgene
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Promoter
Gene Model of Parasite PBANKA_1133300
Gene Model P. falciparum ortholog PF3D7_1357100
Gene productelongation factor 1-alpha
Gene product: Alternative nameeef1a
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
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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 name230p
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
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Conceptual design of the database: Dr. C.J. Janse (Leiden Malaria Research Group, Leiden, The Netherlands).
Technical design and realization: S. Mededovic and A. van Wigcheren