RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-4958
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PbANKA_0800600; Gene model (P.falciparum): PF3D7_0702900; Gene product: centrin, putative
Transgene
Transgene not Plasmodium: A fusion of GFP (gfp-mu3) and Luciferase Firefly (LucIAV)
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: 15 February 2021, 16:55
  *RMgm-4958
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) : 33515807
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 676m1cl1 (RMgm-29)
Other information parent line676m1cl1 (RMgm-29) is a reference ANKA mutant line which expresses GFP-luciferase 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/ResearcherKnöckel J, Billker O, Wright GJ
Name Group/DepartmentCell Surface Signalling Laboratory; Malaria Programme
Name InstituteWellcome Sanger Institute
CityCambridge
CountryUK
Name of the mutant parasite
RMgm numberRMgm-4958
Principal nameΔPbANKA_0800600
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 PbANKA_0800600 and expresses the fusion protein GFP-luciferase under control of the constitutive eef1a promoter

Protein (function)
A library of recombinant proteins representing the repertoire of cell surface and secreted proteins from the P. falciparum sporozoite was generated and an assay was designed to detect extracellular interactions to systematically identify complexes. We identify three protein complexes including an interaction between two components of the p24 complex that is involved in the trafficking of glycosylphosphatidylinositol (GPI)-anchored proteins through the secretory pathway. The (3 complexes between PIESP15 (PF3D7_0103900; PBANKA_0209200) and a secreted protein: PF3D7_0702900 (PbANKA_0800600); a second between two members of the p24 family of proteins PF3D7_0422100 (PBANKA_0522500) and PF3D7_0526900 (PBANKA_1241700), and the third between CelTOS and a predicted secreted protein PF3D7_0721100 (PBANKA_0618600).

Phenotype
No phenotype different from wild type parasites was detected throughout the life cycle

Additional information
From the paper: 
'We observed that both the ΔPbPIESP15 and ΔPbANKA_0800600 parasite lines had no overt infection phenotype in either the mosquito or in mice compared to the wild-type parental line. This lack of infection phenotype was consistent with the inability of polyclonal antibodies to both PIESP15 and PF3D7_0702900 to prevent invasion of P. falciparum sporozoites in human cells'

Other mutants


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PbANKA_0800600
Gene Model P. falciparum ortholog PF3D7_0702900
Gene productcentrin, putative
Gene product: Alternative name
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/vectorPbGEM-331155
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 modificationP. berghei targeting plasmids for PIESP15 (PBANKA_0209200, PbGEM-268595) and PBANKA_0800600, PbGEM-331155 were obtained from the PlasmoGEM vector resource (plasmogem.sanger.ac.uk). To make targeting plasmids that disrupt the PBANKA_0522500 and PBANKA_1241700 loci, oligonucleotides were designed to amplify 5’- and 3’ homology regions of each gene. Each homology arm was approximately 1 kbp long and spans the 5’ UTR into the open reading frame, and from inside the open reading frame into the 3’UTR of each gene respectively. Restriction sites were added to the primer sequence to enable cloning of the PCR products.
PCR products were sequentially cloned into the KpnI and XmaI or the XhoI and SacII restriction sites of the pR6K attL1-3xHA-hDHFR-yfcu-attL2 plasmid backbone and used to transfect P. berghei parasites.
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 nameA fusion of GFP (gfp-mu3) and Luciferase Firefly (LucIAV)
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-Luciferase gene (1 copy) has been inserted into the 230p locus (PB000214.00.0) by double cross-over integration.
Additional remarks selection procedureThis reporter mutant expressing GFP-Luciferase 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