RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-4437
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_1315100; Gene model (P.falciparum): PF3D7_1451400; Gene product: transcriptional regulatory protein sir2b (SIR2B)
DisruptedGene model (rodent): PBANKA_1343800; Gene model (P.falciparum): PF3D7_1328800; Gene product: transcriptional regulatory protein sir2a (SIR2A)
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;
Last modified: 26 April 2018, 10:03
  *RMgm-4437
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
Reference (PubMed-PMID number) Not published (yet)
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 RMgm-4436
Other information parent lineThis mutant lacks sir2b. The negative selectable marker (SM) hdfr/yfcu has been removed by negative selection of parasites of 1079cl1. Cloning after negative selection resulted in line 1079cl1m4cl1 that is SM free. These knock out parasites lacking sir2b have been used to make a double gene knockout lacking both sir2b and sir2a
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The mutant parasite was generated by
Name PI/ResearcherReliga AA, Janse CJ, Waters AP
Name Group/DepartmentLeiden Malaria Research Group
Name InstituteLeiden University Medical Center
CityLeiden
CountryThe Netherlands
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Name of the mutant parasite
RMgm numberRMgm-4437
Principal name1184m1cl1
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 ookineteNormal gamete production, fertilisation and ookinete production. Normal formation of mature ookinetes at the light microscope level. Evidence is found that ookinetes do not traverse the midgut epithelium.
OocystNo oocyst formation
SporozoiteNo oocyst formation. No sporozoite formation
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of SIR2b and SIR2a

Protein (function)

Phenotype
The phenotype is similar to the phenotype of a mutant lacking only SIR2a (RMgm-4435).

Normal blood stage development and gametocyte production. Normal gamete production, fertilisation and ookinete production. Normal formation of mature ookinetes at the light microscope level. Evidence is found that ookinetes do not traverse the midgut epithelium.  No oocyst formation. No sporozoite formation.

Additional information
The negative selectable marker (SM) hdfr/yfcu has been removed by negative selection of parasites of 1079cl1 (RMgm-4436). Cloning after negative selection resulted in line 1079cl1m4cl1 that is SM free. These knock out parasites lacking sir2b have been used to make the double gene knockout mutant lacking both sir2b and sir2a

Other mutants
- a mutant lacking sir 2a - RMgm-4435
- a mutant lacking sir2b - RMgm-4436

  Disrupted: Mutant parasite with a disrupted gene
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Details of the target gene
Gene Model of Rodent Parasite PBANKA_1315100
Gene Model P. falciparum ortholog PF3D7_1451400
Gene producttranscriptional regulatory protein sir2b
Gene product: Alternative nameSIR2B
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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 parasitehdhfr/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe negative selectable marker (SM) hdfr/yfcu has been removed by negative selection of parasites of 1079cl1. Cloning after negative selection resulted in line 1079cl1m4cl1 that is SM free. These parasites lacking sir2b have been used to make a double gene knockout lacking both sir2b and sir2a (see mutant RMgm-4437).
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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  Disrupted: Mutant parasite with a disrupted gene
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Details of the target gene
Gene Model of Rodent Parasite PBANKA_1343800
Gene Model P. falciparum ortholog PF3D7_1328800
Gene producttranscriptional regulatory protein sir2a
Gene product: Alternative nameSIR2A
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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 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
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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/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 (PBANKA_030600) by double cross-over integration.
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 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