RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-1334
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PBANKA_1365500; Gene model (P.falciparum): Not available; Gene product: intra-erythrocytic P. berghei-induced structures protein 1 exported protein (IBIS1)
Name tag: mCherry
Transgene
 Transgene not Plasmodium: GFP (gfp-mu3)
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 Asexual bloodstage;
Last modified: 3 October 2015, 13:30
  *RMgm-1334
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene tagging, Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 26219962
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 507cl1 (RMgm-7)
Other information parent lineP.berghei ANKA 507cl1 (RMgm-7) is a reference ANKA mutant line which expresses GFP 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/ResearcherMatz, JM; Kooij, TW
Name Group/DepartmentMedical Microbiology
Name InstituteRadboudumc
CityNijmegen
CountryThe Netherlands
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Name of the mutant parasite
RMgm numberRMgm-1334
Principal namemCherry(pv)
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
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Phenotype
Asexual blood stageStrong staining of the parasitophorous vacuole (PV) including motile tubular extensions and vesicular structures
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses part of ibis1 fused to mCherry. This reporter transgene localizes to the parasitophorous vacuole (PV) in blood stages. The lack of a spacer between the IBIS1 PEXEL/VTS motif and mCherry prevents export of the fusion protein into the cytoplasm of the host red blood cell.

Protein (function)
IBIS1 is a PEXEL/HT-containing protein, expressed in both blood- and liver stages. In blood stages the protein is exported into the cytoplasm of the host erythrocyte.  IBIS is localized to discrete punctate structures and evidence is presented that IBIS1 localizes in membranous structures inside the cytoplasm of the host red blood cell, resembling Maurer's clefts-like structures (RMgm-735).

Phenotype
Strong staining of the parasitophorous vacuole (PV) including motile tubular extensions and vesicular structures. The lack of a spacer between the IBIS1 PEXEL/VTS motif and mCherry prevents export of the fusion protein into the cytoplasm of the host red blood cell.

Additional information

Other mutants
See link for other IBIS1 mutants

  Tagged: Mutant parasite with a tagged gene
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Details of the target gene
Gene Model of Rodent Parasite PBANKA_1365500
Gene Model P. falciparum ortholog Not available
Gene productintra-erythrocytic P. berghei-induced structures protein 1 exported protein
Gene product: Alternative nameIBIS1
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Details of the genetic modification
Name of the tagmCherry
Details of taggingC-terminal
Additional remarks: taggingThe first 484 bp of the coding sequence of IBIS1 and 1,282 bp of the 5′flanking region were cloned into the b3D+ mCherry vector, using the SacII and SpeI restriction sites. Following linearization, the mCherryPV transfection vector was integrated into the genome of P. berghei GFPcon42 through single crossover homologous recombination. The lack of a spacer between the IBIS1 PEXEL/VTS motif and mCherry prevents export of the fusion protein into the cytoplasm of the host red blood cell.
Commercial source of tag-antibodies
Type of plasmid/construct(Linear) plasmid single 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 markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe first 484 bp of the coding sequence of IBIS1 and 1,282 bp of the 5′flanking region were cloned into the b3D+ mCherry vector, using the SacII and SpeI restriction sites. Following linearization, the mCherryPV transfection vector was integrated into the genome of P. berghei GFPcon42 through single crossover homologous recombination. The lack of a spacer between the IBIS1 PEXEL/VTS motif and mCherry prevents export of the fusion protein into the cytoplasm of the host red blood cell.
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 (gfp-mu3)
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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 gene (1 copy) has been inserted into the 230p locus (PBANKA_030600) by double cross-over integration.
Additional remarks selection procedureThis reporter mutant expressing GFP 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