RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-766
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_1436600; Gene model (P.falciparum): PF3D7_1221400; Gene product: inner membrane complex protein 1h, putative (IMC1h)
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 Fertilization and ookinete; Oocyst; Sporozoite; Liver stage;
Last modified: 7 August 2012, 19:13
  *RMgm-766
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) : 22844474
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/ResearcherK. Volkmann, O. Billker, M. Brochet
Name Group/DepartmentWellcome Trust Sanger Institute
Name InstituteWellcome Trust Sanger Institute
CityCambridge Hinxton
CountryUK
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Name of the mutant parasite
RMgm numberRMgm-766
Principal nameIMC1h-KO
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
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Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNot different from wild type
Fertilization and ookineteIn the initial 16 hours, zygote/ookinete development appeared to be normal. However, advanced retort-forms showed abnormal development as the protruding area swelled up leaving a bottleneck between the latter and the remnant ‘‘zygote body’’. This bottleneck did not prevent the migration of the nucleus leading to ookinetes that were typically wider than WT ookinetes with a bulging area mostly in the anterior part of the cell. None of the mutant ookinetes were found to display the typical banana shape of WT ookinetes. Apart from the global shape abnormality, IMC1h-KO ookinetes appeared to possess a complete set of organelles and the assembly of the subpellicular microtubule and IMC appeared unaffected by the gene disruption.
Ookinetes showed abnormal gliding behaviour.
Ookinetes showed a 15-fold decrease in traversal of the midgut epithelium.
OocystOokinetes showed a 15-fold decrease in traversal of the midgut epithelium.
The number of IMC1h-KO oocysts in infected mosquitoes was 20-fold lower than in mosquitoes infected with WT parasites.
Mutant ookinetes that reached the basal epithelium developed into oocysts of normal size and morphology that gave rise to sporozoites.
SporozoiteMutant ookinetes that reached the basal epithelium developed into oocysts of normal size and morphology that gave rise to sporozoites.
Sporozoites invaded salivary glands but midgut IMC1h-KO sporozoites were four times less invasive than WT sporozoites. All IMC1h-KO sporozoites obtained from both oocysts and salivary glands had an abnormal shape, with a protruding bulge of variable size at the posterior end containing the nucleus.
IMC1h-KO sporozoites showed gliding motility defects.
IMC1h-KO sporozoites are able to invade hepatocytes but are not able to progress through the dermis in vivo.
IMC1h-KO sporozoites are able to transmigrate and invade hepatoma-cells in vitro but liver stages show developmental defects.
Liver stageIMC1h-KO sporozoites are able to invade hepatocytes but are not able to progress through the dermis in vivo.
IMC1h-KO sporozoites are able to transmigrate and invade hepatoma-cells in vitro but liver stages show developmental defects.
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of inner membrane complex protein 1h (IMC1h) and expresses GFP under the constitutive eef1α promoter

Protein (function)
The imc1h gene belongs to small 'family' of conserved genes that express putative membrane skeleton proteins which contain domains that share sequence homology to domains of articulins, proteins of membrane skeleton of free-living protists and show homology to the inner membrane complex protein 1 (TgIMC1) of the subpellicular network of Toxoplasma gondii tachyzoites (Khater, E.I., et al. 2004, J. Cell. Biol. 167, 425-32) . In Plasmodium eight conserved IMC1 protein family members have been identified, named IMC1a-IMC1h. Two of these, IMC1a and IMC1b, were shown to be differentially expressed in sporozoites and ookinetes, respectively, and to form part of their pellicle structures in P. berghei. IMC1a and IMC1b are structurally and functionally homologous and involved in parasite morphology, mechanical strength, gliding motility and infectivity, in accordance with their roles as membrane skeleton proteins (see also mutants  RMgm-147 and RMgm-148 lacking expression of IMC1b and IMC1a). IMC1h, is found in the pellicle of both ookinetes and sporozoites (see mutant RMgm-600) . Phenotype analyses of the mutant lacking IMC1h (RMgm-601) indicates a role of this protein in morphology/shape of ookinetes and sporozoites and in motility of both stages (see also below)

Phenotype
Phenotype analyses indicate that IMC1h is involved in maintaining the cellular architecture supporting typical movement behaviour of both ookinetes and sporozoites and plays a role during development of liver stages

Additional information
See also phenotype analyses of an independent mutant lacking IMC1h (RMgm-601). The phenotype of ookinetes and sporozoites of this mutant are highly similar to the phenotype of the mutant described here, but no salivary gland sporozoites were detected.

Immunofluorescence analysis of a mutant expressing a HA-tagged form of IMC1h (RMgm-767) showed expression in ookinetes, sporozoites and liver stages

Other mutants
RMgm-147: A mutant lacking expression of IMC1b
RMgm-148: A mutant lacking expression of IMC1a
RMgm-163: A mutant expressing a GFP-tagged form of IMC1b
RMgm-600: A mutant expressing a GFP-tagged form of IMC1h
RMgm-601: A mutant lacking expression of IMC1h
RMgm-602: A mutant lacking expression of both IMC1h and IMC1b
RMgm-767: A mutant expressing a HA-tagged form of IMC1h

  Disrupted: Mutant parasite with a disrupted gene
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Details of the target gene
Gene Model of Rodent Parasite PBANKA_1436600
Gene Model P. falciparum ortholog PF3D7_1221400
Gene productinner membrane complex protein 1h, putative
Gene product: Alternative nameIMC1h
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Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct usedPlasmid double cross-over
PlasmoGEM (Sanger) construct/vector usedYes
Name of PlasmoGEM construct/vectorpBGEM-72290.5620
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 modificationThe mutant has been generated using a PlasmoGEM construct as described in Pfander, C. et al. (2011) Nature Methods 8, 1078-1082. However, in the paper no details or name of the construct are provided.
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/constructPlasmid 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.
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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