RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-4450
Malaria parasiteP. berghei
Genotype
Transgene
 Transgene not Plasmodium: GFP (gfp-mut3)
Promoter: Gene model: PBANKA_0522700; Gene model (P.falciparum): PF3D7_0422300; Gene product: alpha tubulin 2
3'UTR: Gene model: PBANKA_0719300; Gene product: bifunctional dihydrofolate reductase-thymidylate synthase, putative (dhfr/ts)
Insertion locus: Gene model: Not available; Gene product: Not available (small subunit ribosomal rna gene (c- or d-type unit))
Phenotype Asexual bloodstage; Gametocyte/Gamete; Fertilization and ookinete; Oocyst;
Last modified: 18 May 2018, 14:04
  *RMgm-4450
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 15935755
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 cl15cy1
Other information parent lineA reference wild type clone from the ANKA strain of P. berghei (PubMed: PMID: 17406255).
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The mutant parasite was generated by
Name PI/ResearcherS.M. Khan, G.R. Mair, C.J. Janse, A.P. Waters
Name Group/DepartmentLeiden Malaria Research Group
Name InstituteLeiden University Medical Center (LUMC)
CityLeiden
CountryThe Netherlands
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Name of the mutant parasite
RMgm numberRMgm-4450
Principal name357cl1
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationNo
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Phenotype
Asexual blood stageWeak GFP expression in trophozoites
Gametocyte/GameteWeak GFP expression in female gametocytes.
Strong GFP expression in male gametocytes.
Fertilization and ookineteGFP expression in ookinetes
OocystGFP expression in oocysts
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses GFP under the control of the promoter of the gene encoding alpha tubulin 2

Protein (function)
Alpha-tubulin II is one of the two alpha-tubulin genes (I and II) in Plasmodium. This gene is highly expressed in male gametocytes. Expression was also observed in the asexual blood stages, female gametocytes, ookinetes and oocysts

Phenotype
Weak GFP expression in trophozoites. Weak GFP expression in female gametocytes. Strong GFP expression in male gametocytes.

Additional information
The construct GFPcon (RMgm-5) containing GFP under the control of the eef1a promoter formed the basis for the construct driving expression of GFP under control of the alpha tubulin 2 promoter. This constructs contains a c/d-ssurrna target fragment for integration into the c/d-rrna genomic locus. GFP is flanked by the 3'-UTR sequence of the dhfr gene of P. berghei. The eef1a promoter region is replaced by the promoter region of alpha tubulin 2.

Other mutants

  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-mut3)
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Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct(Linear) plasmid single 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 parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe construct GFPcon (RMgm-5) containing GFP under the control of the eef1a promoter formed the basis for the construct driving expression of GFP under control of the nek4 promoter.
This constructs contains a c/d-ssurrna target fragment for integration into the c/d-rrna genomic locus. GFP is flanked by the 3'-UTR sequence of the dhfr gene of P. berghei. The eef1a promoter region is replaced by the promoter region of alpha tubulin 2.

The gfp gene has been integrated into the genome by single cross-over integration. Therefore the possibility exists of reversion to the wild-type genotype by removal of the integrated DNA construct.
Additional remarks selection procedure
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Other details transgene
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Promoter
Gene Model of Parasite PBANKA_0522700
Gene Model P. falciparum ortholog PF3D7_0422300
Gene productalpha tubulin 2
Gene product: Alternative name
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 / InsertionInsertion locus
Gene Model of Parasite Not available
Gene productNot available
Gene product: Alternative namesmall subunit ribosomal rna gene (c- or d-type unit)
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