RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-4652
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_0515400; Gene model (P.falciparum): PF3D7_1031600; Gene product: conserved Plasmodium protein, unknown function (P. falciparum Gametocyte EXported Protein 15, PfGEXP15; PbGEXP15)
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; Oocyst; Sporozoite;
Last modified: 7 August 2019, 12:32
  *RMgm-4652
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) Reference 1 (PMID number) : 31348803
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 RMgm-1026
Other information parent lineA drug-selectable marker free reporter line expressing GFP under the constitutive hsp70 promoter
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The mutant parasite was generated by
Name PI/ResearcherHollin T, Khalife J
Name Group/DepartmentCenter for Infection and Immunity of Lille
Name InstituteUniv. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille
CityLille
CountryFrance
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Name of the mutant parasite
RMgm numberRMgm-4652
Principal nameΔgexp15cl1, cl2
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
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Phenotype
Asexual blood stageSlightly reduced growth rate of asexual blood stages (no ECM pathology in C57Bl6 mice; mice die from hyperparasitemia. BALB/c developed hyperparasitemia but were able to clear infection)
Gametocyte/GameteNot different from wild type
Fertilization and ookineteNot tested
Oocystno oocyst formation
SporozoiteNo oocyst formation; no sporozoite formation
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of PbGEXP15

Protein (function)
The P. falciparum ortholog has been identified as a ' gametocyte exported protein'  (PfGEXP15)

Phenotype
Slightly reduced growth rate of asexual blood stages (no ECM pathology in C57Bl6 mice; mice die from hyperparasitemia. BALB/c developed hyperparasitemia but were able to clear infection). No oocyst formation; no sporozoite formation

Additional information
Evidence is presented for:
- PbGEXP15 interacts with PP1 (serine/threonine protein phosphatase PP1; PBANKA_1028300). See also mutants RMgm-4653 and RMgm-4654 expressing mCherry-tagged PbGEX15 and mCherry-tagged PP1. Examination of PbGEXP15-mCherry by immunofluorescence assays showed a distribution in the cytoplasm of all parasite stages examined along with clear punctate localization, suggesting potential cytoplasmic organelle structures. Further, the PbGEXP15 signal clearly exhibited a pattern adjacent to and in the nucleus of trophozoite and gametocyte stages. With respect to PbPP1, the signal was observed throughout the cytoplasm with fluorescence partially overlapping DNA. 

Other mutants

  Disrupted: Mutant parasite with a disrupted gene
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Details of the target gene
Gene Model of Rodent Parasite PBANKA_0515400
Gene Model P. falciparum ortholog PF3D7_1031600
Gene productconserved Plasmodium protein, unknown function
Gene product: Alternative nameP. falciparum Gametocyte EXported Protein 15, PfGEXP15; PbGEXP15
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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 parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationFor the knock-out of pbgexp15, PCR amplifications were generated with the 5’ and 3’ UTR regions with primers p26-p27 (847 bp), p28-p29 (695 bp) and P. berghei gDNA as template. The inserts were subcloned into pBS-DHFR plasmid and the construct was linearized by XbaI-ApaI before transfection.
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 nameGFP
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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 parasiteNo selectable marker
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modificationThe 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
Additional remarks selection procedure
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Other details transgene
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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
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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 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
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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