RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-4075
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_1228200; Gene model (P.falciparum): PF3D7_0802000; Gene product: glutamate dehydrogenase, putative (GDH3; GDHc)
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)
PhenotypeNo phenotype has been described
Last modified: 14 January 2017, 21:07
  *RMgm-4075
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) : 28027318
MR4 number
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).
The mutant parasite was generated by
Name PI/ResearcherSrivastava A, Waters AP
Name Group/DepartmentWellcome Centre for Molecular Parasitology, Institute of Infection, Immunity & Inflammation
Name InstituteCollege of Medical, Veterinary and Life Sciences, University of Glasgow
CityGlasgow
CountryUK
Name of the mutant parasite
RMgm numberRMgm-4075
Principal namegdh3-
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNot different from wild type
Fertilization and ookineteNot different from wild type
OocystNot different from wild type
SporozoiteNot different from wild type
Liver stageNot different from wild type
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of GDH3

Protein (function)
Glucose consumption by Plasmodium-infected RBC increases 10-fold and these stages rely primarily on glycolysis for energy generation. Notwithstanding their dependence on glycolysis, asexual blood stages maintain a single, poorly cristate mitochondrion and are dependent on electron transport chain (ETC) activity for the re-oxidation of inner membrane dehydrogenases and pyrimidine biosynthesis. The maintenance of the mitochondrial ETC is sustained in part, by the oxidation of pyruvate (diverted from glycolysis) and the uptake and catabolism of glutamine. Pyruvate can enter the TCA cycle via two pathways; through anaplerotic reactions involving the CO2-fixing enzyme, phosphoenolpyruvate carboxylase (PEPC), or through the activity of a repurposed branched chain α-keto acid dehydrogenase (BCKDH) complex, which substitutes for the activity of the missing mitochondrial pyruvate dehydrogenase in Plasmodium and other apicomplexan parasites. Despite the essentiality of the mitochondrion, operation of the TCA cycle is not required for intra-erythrocytic growth of P.falciparum.
Plasmodium spp. lack key enzymes involved in gluconeogenesis and all developmental stages are predicted to be dependent on the uptake of sugars. However, in contrast to the asexual blood stages, there is increasing evidence that the mosquito-infective stages of Plasmodium exhibit an increased dependence on the TCA cycle and mitochondrial metabolism.
Specifically, Plasmodium gametocytes develop more complex tubular mitochondrial cristae suggestive of increased mitochondrial function. Metabolomic analyses have confirmed increased TCA metabolism in P.falciparum gametocytes and demonstrated that this is essential for gametocyte maturation. Recent genetic studies have also shown that the TCA cycle is essential for the development of P.falciparum mosquito stages, consistent with earlier work in P.bergheid emonstrating that the TCA cycle and the electron transport chain are required for ookinete development and oocyst formation.

In this study  a combination of metabolomic and reverse genetic approaches was used to investigate the metabolic changes that occur in key mosquitostages of P.berghei and the potential impact of these changes on parasite infection in the mosquito. We find that these stages are highly sensitive to disruptions in multiple pathways in central carbon metabolism including the TCA cycle, the utilisation of glutamine as a carbon source, intermediary carbon metabolism and coenzyme A (CoA) synthesis.

Phenotype
Normal growth and development throughout the life cycle (slightly reduced growth rate of asexual blood stages; however not significant)

Additional information

Other mutants


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1228200
Gene Model P. falciparum ortholog PF3D7_0802000
Gene productglutamate dehydrogenase, putative
Gene product: Alternative nameGDH3; GDHc
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 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

  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameGFP (gfp-mu3)
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 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.
Other details transgene
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
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