RMgmDB - Rodent Malaria genetically modified Parasites

Back to search results

Summary

RMgm-602
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_0907100; Gene model (P.falciparum): PF3D7_1141900; Gene product: inner membrane complex protein 1b, putative (IMC1b, inner membrane complex protein 1b)
DisruptedGene model (rodent): PBANKA_1436600; Gene model (P.falciparum): PF3D7_1221400; Gene product: inner membrane complex protein 1h, putative (IMC1h, inner membrane complex protein 1h)
Transgene
Transgene not Plasmodium: EGFP
Promoter: Gene model: PBANKA_0907100; Gene model (P.falciparum): PF3D7_1141900; Gene product: inner membrane complex protein 1b, putative (IMC1b, inner membrane complex protein 1b)
3'UTR: Gene model: PBANKA_0719300; Gene product: bifunctional dihydrofolate reductase-thymidylate synthase, putative (dhfr/ts)
Replacement locus: Gene model: PBANKA_0907100; Gene product: inner membrane complex protein 1b (IMC1b)
Transgene
Transgene not Plasmodium: EGFP
Promoter: Gene model: PBANKA_1436600; Gene model (P.falciparum): PF3D7_1221400; Gene product: inner membrane complex protein 1h, putative (Inner membrane complex protein 1h; IMC1h)
3'UTR: Gene model: PBANKA_0719300; Gene product: bifunctional dihydrofolate reductase-thymidylate synthase, putative (dhfr/ts)
Replacement locus: Gene model: PBANKA_1436600; Gene product: inner membrane complex protein 1h (IMC1h)
Phenotype Fertilization and ookinete; Oocyst; Sporozoite;
Last modified: 15 January 2011, 22:07
  *RMgm-602
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption, Gene disruption, Introduction of a transgene, Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 21098480
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 2.34
Other information parent lineP. berghei ANKA 2.34 is a cloned, gametocyte producer line of the ANKA strain (PubMed: PMID: 15137943).
The mutant parasite was generated by
Name PI/ResearcherA.Z. Tremp; J.T. Dessens
Name Group/DepartmentDepartment of Pathogen Molecular Biology, Faculty of Infectious and Tropical Diseases
Name InstituteLondon School of Hygiene & Tropical Medicine
CityLondon
CountryUK
Name of the mutant parasite
RMgm numberRMgm-602
Principal nameIMC1h/b-dKO
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 ookineteNormal numbers of ookinetes are produced. Mature ookinetes showed abnormal morphology. Ookinetes were wider, shorter and possessed a bulging area typically in the central part of the cell. Gliding motility of ookinetes was reduced. Infectivity of ookinetes was reduced (ookinetes produced ~98-fold less oocysts in A. stephensi).
OocystInfectivity of ookinetes was reduced (ookinetes produced ~98-fold less oocysts in A. stephensi). Oocysts appeared to develop normally, forming large numbers of sporozoites. However, all sporozoites were of abnormal shape, typically possessing a bulging area near the middle of the sporozoite. No sporozoites were detected in salivary glands.
SporozoiteInfectivity of ookinetes was reduced (ookinetes produced ~98-fold less oocysts in A. stephensi). Oocysts appeared to develop normally, forming large numbers of sporozoites. However, all sporozoites were of abnormal shape, typically possessing a bulging area near the middle of the sporozoite. No sporozoites were detected in salivary glands.
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of Inner membrane complex protein 1h (IMC1h) and  inner membrane complex protein 1b (IMC1b). This 'double knock-out' mutant has been obtained by genetic crossing the single imc1h knock-out mutant (RMgm-601) with the single imc1b knock-out mutant (RMgm-147).

Protein (function)
The imcb1h and imcb1b genes 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 mutanst 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) and acts in a very similar way to IMC1b and IMC1a. It plays a role in in morphology/shape of ookinetes and sporozoites and in motility of both stages. See mutant RMgm-601 which lacks expression of IMC1h and see below

Phenotype
Phenotype analyses of a mutant lacking IMC1h (RMgm-601, IMC1h-sKO) indicate a role of this protein in morphology/shape of ookinetes and sporozoites and in motility of both stages.
Phenotype analyses of a mutant lacking IMC1b (RMgm-147) indicate a role of this protein in morphology/shape and motility of ookinetes. 
Analysis of a mutant lacking expression of both IMC1h and IMC1b (IMC1h/b-dKO) provides evidence that for a direct involvement of IMC1 proteins in gliding motility uncoupled from cell shape. The shape IMC1h/b-dKO ookinetes was not significantly different to that of IMC1h-sKO ookinetes (RMgm-601). Thus, IMC1h-KO ookinete cell shape appears not to be further affected by the additional imc1b gene disruption. Both  gliding motility and infectivity of IMC1b/h-dKO were more strongly reduced compared to IMC1h-sKO ookinetes. The morphology of IMC1b/h-dKO sporozoites was similar to that of IMC1h-sKO sporozoites. These phenotype analyses of a mutant lacking expression of both IMC1h and IMC1b provides evidence that for a direct involvement of IMC1 proteins in gliding motility uncoupled from cell shape.

Additional information
Cell death in response to osmotic shock was increased  in the IMC1b/h-dKO ookinetes compared to the IMC1h-sKO. These results indicate that IMC1h, like IMC1b, is involved in the mechanical stability of ookinetes, which is consistent with it being an ookinete membrane skeleton component. It also indicates that there is a cumulative effect of multiple ookinete-specific imc1 gene disruptions on the ookinetes’ mechanical strength. Both gliding motility and infectivity of IMC1b/h-dKO were more strongly reduced compared to IMC1h-sKO ookinetes.

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 both IMC1h


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0907100
Gene Model P. falciparum ortholog PF3D7_1141900
Gene productinner membrane complex protein 1b, putative
Gene product: Alternative nameIMC1b, inner membrane complex protein 1b
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct usedGenetic cross
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 genePartial
Additional remarks partial/complete disruption A genetically modified P. berghei parasite was constructed in which all of the imc1b coding sequence was removed except for the first 30 residues
Selectable marker used to select the mutant parasitetgdhfr/hdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedureWR99210
Selection (negative) procedureNo
Additional remarks genetic modificationThe imc1b gene has been disrupted using a construct that integrates by double cross-over integration that contains the tgdhfr selectable marker and a copy of gfp under control of the native imc1b promoter. This mutant has been described as mutant RMgm-147.
The imc1h gene has been disrupted using a construct that contains the hgdhfr selectable marker and a copy of gfp under control of the native imc1h promoter that integrates by double cross-over recombination. This mutant has been described as mutant RMgm-601.
The imc1b/imc1h double KO mutant was produced by crossing (in the mosquito) the imc1b KO mutant (RMgm-147) with the imc1h KO mutant (RMgm-601). The resulting sporozoite population was transmitted to a naïve mouse by mosquito bites. Clones were obtained by limiting dilution from the ensuing blood stage infection after WR99210 selection.
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

  Disrupted: Mutant parasite with a disrupted gene
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, inner membrane complex protein 1h
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct usedGenetic cross
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 genePartial
Additional remarks partial/complete disruption All but the first 25 amino acids of the imc1h ORF have been removed.
Selectable marker used to select the mutant parasitetgdhfr/hdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedureWR99210
Selection (negative) procedureNo
Additional remarks genetic modificationThe imc1b gene has been disrupted using a construct that integrates by double cross-over integration that contains the tgdhfr selectable marker and a copy of gfp under control of the native imc1h promoter. This mutant has been described as mutant RMgm-147.
The imc1h gene has been disrupted using a construct that contains the hgdhfr selectable marker and a copy of gfp under control of the native imc1h promoter that integrates by double cross-over recombination. This mutant has been described as mutant RMgm-601.
The imc1b/imc1h double KO mutant was produced by crossing (in the mosquito) the imc1b KO mutant (RMgm-147) with the imc1h KO mutant (RMgm-601). The resulting sporozoite population was transmitted to a naïve mouse by mosquito bites. Clones were obtained by limiting dilution from the ensuing blood stage infection after WR99210 selection.
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 nameEGFP
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/constructGenetic cross
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/hdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedureWR99210
Selection (negative) procedureNo
Additional remarks genetic modificationThe imc1b gene has been disrupted using a construct that integrates by double cross-over integration that contains the tgdhfr selectable marker and a copy of gfp under control of the native imc1b promoter. This mutant has been described as mutant RMgm-147.
The imc1h gene has been disrupted using a construct that contains the hgdhfr selectable marker and a copy of gfp under control of the native imc1h promoter that integrates by double cross-over recombination. This mutant has been described as mutant RMgm-601.
The imc1b/imc1h double KO mutant was produced by crossing (in the mosquito) the imc1b KO mutant (RMgm-147) with the imc1h KO mutant (RMgm-601). The resulting sporozoite population was transmitted to a naïve mouse by mosquito bites. Clones were obtained by limiting dilution from the ensuing blood stage infection after WR99210 selection.
Additional remarks selection procedure
Other details transgene
Promoter
Gene Model of Parasite PBANKA_0907100
Gene Model P. falciparum ortholog PF3D7_1141900
Gene productinner membrane complex protein 1b, putative
Gene product: Alternative nameIMC1b, inner membrane complex protein 1b
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_0907100
Gene productinner membrane complex protein 1b
Gene product: Alternative nameIMC1b
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

  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameEGFP
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/constructGenetic cross
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/hdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedureWR99210
Selection (negative) procedureNo
Additional remarks genetic modificationThe imc1b gene has been disrupted using a construct that integrates by double cross-over integration that contains the tgdhfr selectable marker and a copy of gfp under control of the native imc1b promoter. This mutant has been described as mutant RMgm-147.
The imc1h gene has been disrupted using a construct that contains the hgdhfr selectable marker and a copy of gfp under control of the native imc1h promoter that integrates by double cross-over recombination. This mutant has been described as mutant RMgm-601.
The imc1b/imc1h double KO mutant was produced by crossing (in the mosquito) the imc1b KO mutant (RMgm-147) with the imc1h KO mutant (RMgm-601). The resulting sporozoite population was transmitted to a naïve mouse by mosquito bites. Clones were obtained by limiting dilution from the ensuing blood stage infection after WR99210 selection.
Additional remarks selection procedure
Other details transgene
Promoter
Gene Model of Parasite PBANKA_1436600
Gene Model P. falciparum ortholog PF3D7_1221400
Gene productinner membrane complex protein 1h, putative
Gene product: Alternative nameInner membrane complex protein 1h; IMC1h
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_1436600
Gene productinner membrane complex protein 1h
Gene product: Alternative nameIMC1h
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