RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-4140
Malaria parasiteP. chabaudi
Genotype
DisruptedGene model (rodent): PCHAS_0101300; Gene model (P.falciparum): Not available; Gene product: schizont membrane associated cytoadherence protein, putative (SMAC)
Transgene
 Transgene not Plasmodium: Luciferase (firefly)
Promoter: Gene model: PBANKA_1133300; Gene model (P.falciparum): PF3D7_1357100; Gene product: elongation factor 1-alpha (eef1a)
3'UTR: Gene model: Not available; Gene product: Not available
Replacement locus: Gene model: PCHAS_0101300; Gene product: schizont membrane associated cytoadherence protein, putative (SMAC)
Phenotype Asexual bloodstage;
Last modified: 9 May 2017, 17:55
  *RMgm-4140
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) : 28468674
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 Not applicable
Other information parent lineCloned lines of Plasmodium chabaudi chabaudi AS were originally obtained from David Walliker, University of Edinburgh, UK and subsequently passaged through mice by injection of infected red blood cells (iRBC) at the MRC National Institute for Medical Research, UK
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The mutant parasite was generated by
Name PI/ResearcherCunningham DA; Langhorne J
Name Group/DepartmentMalaria Immunology laboratory
Name InstituteFrancis Crick Institute
CityLondon
CountryUK
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Name of the mutant parasite
RMgm numberRMgm-4140
Principal namePcASΔsmacEFluc
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
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Phenotype
Asexual blood stageEvidence is presented that the absence of SMAC does not affect blood stage growth, course of parasitemia and tissue sequestration of Plasmodium chabaudi infected erythrocytes (see also RMgm-4139)
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of SMAC (Schizont Membrane Associated Cytoadherence protein) and expresses luciferase gene under control of the constitutive P. berghei elongation factor 1 alpha (eef1a) promoter integrated into the smac locus

Protein (function)
In P. berghei evidence has been presented that SMAC is a protein exported into the cytoplasm of infected erythrocytes and plays a role in CD36-mediated sequestration of schizonts in capillaries of inner organs.P. berghei SMAC  contains a signal sequence, a PEXEL motif (RFLVE) and a transmembrane domain. Orthologs are only found in the murine Plasmodium species. See P. berghei SMAC mutants

Phenotype
Evidence is presented that the absence of SMAC does not affect blood stage growth, course of parasitemia and tissue sequestration of Plasmodium chabaudi infected erythrocytes (see also RMgm-4139)

Additional information

Other mutants
See P. berghei SMAC mutants

  Disrupted: Mutant parasite with a disrupted gene
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Details of the target gene
Gene Model of Rodent Parasite PCHAS_0101300
Gene Model P. falciparum ortholog Not available
Gene productschizont membrane associated cytoadherence protein, putative
Gene product: Alternative nameSMAC
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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 modificationPlasmodium chabaudi AS parasites lacking expression of SMAC (PcASΔsmac; RMgm-4139) were generated by replacing the P. berghei smac (PBANKA_0100600) targeting region with the equivalent SMAC region of P. chabaudi (PCHAS_0101300; PCHAS_01_v3 52494-53200 and 53700-54284) using a published construct.
Δsmac parasites expressing luciferase constitutively were subsequently generated using a modified construct (PcASΔsmacEFluc; see RMgm-4140) whereby eef1a luciferase was inserted within the targeting region at EcoRV/EcoR1 sites. Both Δsmac and ΔsmacEFluc constructs were digested with Kpn1/SacII prior to 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 nameLuciferase (firefly)
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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 parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modification
Additional remarks selection procedure
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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 Not available
Gene productNot available
Gene product: Alternative name
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_0101300
Gene productschizont membrane associated cytoadherence protein, putative
Gene product: Alternative nameSMAC
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