RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-4842
Malaria parasiteP. chabaudi
Genotype
DisruptedGene model (rodent): PCHAS_1304000; Gene model (P.falciparum): PF3D7_1475400; Gene product: cysteine repeat modular protein 4 (CRMP4)
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_1340000; Gene product: dihydrofolate synthase/folylpolyglutamate synthase, putative (PbDHFS-FPGS)
Replacement locus: Gene model: PCHAS_0308200; Gene product: 6-cysteine protein P230p, putative (P230p)
Phenotype Asexual bloodstage;
Last modified: 14 August 2020, 15:20
  *RMgm-4842
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) : 32500098
MR4 number
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Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. chabaudi
Parent strain/lineP. c.chabaudi AS
Name parent line/clone RMgm-4840
Other information parent lineRMgm-4840 is a reference P. c. chabaudi AS line (PcASGFP(ML)) that has a GFP expression cassette integrated into the neutral 230p locus and is drug-selectable marker free. GFP is under control of the constitutive P. berghei hsp70 promoter.
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The mutant parasite was generated by
Name PI/ResearcherMarr EJ, Thompson J
Name Group/DepartmentInstitute of Immunology and Infection Research, School of Biological Sciences
Name InstituteUniversity of Edinburgh
CityEdinburgh
CountryUK
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Name of the mutant parasite
RMgm numberRMgm-4842
Principal namePcAS.Δcrmp4
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationNo
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Phenotype
Asexual blood stageTransfected blood stages were selected in mice by pyrimethamine treatment
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of CRMP4 and and expresses GFP under control of the constitutive and strong P. berghei hsp70 promoter.

Protein (function)
The Cysteine Repeat Modular Proteins (CRMP1–4) of Plasmodium, are encoded by a small gene family that is conserved in malaria and other Apicomplexan parasites. They are very large, predicted surface proteins with multipass transmembrane domains containing motifs that are conserved within families of cysteine-rich, predicted surface proteins in a range of unicellular eukaryotes, and a unique combination of protein-binding motifs, including a > 100 kDa cysteine-rich modular region, an epidermal growth factor-like domain and a Kringle domain. CRMP4 is not essential for blood stage development and for the formation of oocysts. CRMP4 plays a role in egress of sporozoites from oocysts and also during development of the early liver stages (see mutant RMgm-585)..

Phenotype
Transfected blood stages were selected pyrimethamine treatment. The mutant has not been analysed in detail. This mutant has been generated to demonstrate the transfection efficiency of PlasmoGEM gene-deletion and gene-tagging vectors for P. chabaudi.

Additional information
This mutant has been generated to demonstrate the transfection efficiency of PlasmoGEM gene-deletion and gene-tagging vectors for P. chabaudi.

Other mutants

  Disrupted: Mutant parasite with a disrupted gene
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Details of the target gene
Gene Model of Rodent Parasite PCHAS_1304000
Gene Model P. falciparum ortholog PF3D7_1475400
Gene productcysteine repeat modular protein 4
Gene product: Alternative nameCRMP4
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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 usedYes
Name of PlasmoGEM construct/vectorPGEM-610764
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 modification
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 parasitehdhfr/yfcu
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modificationpCAT-230p-G6 was generated by replacing the sil6 target regions of pBAT-SIL6-G6 (Kooij et al., 2012; RMgm-757)) with bps 640-1680 and 3312-4255 of the P. chaubaudi 230p (PCHAS_0308200) locus. The 230p target regions were amplified from P.c. chabaudi AS genomic DNA using primers 5’230pF x 5’230p (5’ target region) and 3’230pF x 3’230pR (3’ target region) and cloned into the multiple cloning sites of pBAT-Sil6-G6 to generate pCAT-230p-G6
Additional remarks selection procedureThe selectable marker cassette has been removed by negative selection resulting in a drug-selectable marker free parasite
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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_1340000
Gene productdihydrofolate synthase/folylpolyglutamate synthase, putative
Gene product: Alternative namePbDHFS-FPGS
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 PCHAS_0308200
Gene product6-cysteine protein P230p, putative
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