Summary

RMgm-13
Malaria parasiteP. berghei
Genotype
Transgene
Transgene not Plasmodium: GFP-aequorin (Ca2+ sensor protein).
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)
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; Sporozoite; Liver stage;
Last modified: 10 February 2009, 10:07
  *RMgm-13
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) : 15137943
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/ResearcherO. Billker
Name Group/DepartmentDepartment of Biological Sciences
Name InstituteImperial College London
CityLondon
CountryUnited Kingdom
Name of the mutant parasite
RMgm numberRMgm-13
Principal name1.7.6
Alternative nameCa2+ reporter line
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageAll stages express GFP-aequorin (Ca2+ sensor protein).
Gametocyte/GameteGFP-aequorin expression in female and male gametocytes. Strong GFP-aequorin-expression in female gametocytes. Higher expression in females, compared to males.
Fertilization and ookineteGFP-aequorin expression in ookinetes
OocystGFP-aequorin expression in oocysts
SporozoiteGFP-aequorin expression in sporozoites
Liver stageGFP-aequorin expression during liver stage development
Additional remarks phenotype

Mutant/mutation
The mutant expresses GFP-aequorin (Ca2+ sensor protein) under the control of the constitutive promoter of the elongation factor 1 alpha (eef1a) gene of P. berghei.

Protein (function)
The Ca2+-dependent bioluminescence of the aequorin protein offers a sensitive method to record cytosolic Ca2+ peaks from small numbers of cells. Sensitivity can be increased by exploiting the fluorescence energy transfer that occurs when aequorin is fused to GFP. This transgene mutant has been used to show the role of xanthurenic acid in activating signaling pathways in gametocytes that results in rapid increase in cytosolic free Ca2+ (Billker et al., Cell (2004) 117:503-14).


  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameGFP-aequorin (Ca2+ sensor protein).
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/constructPlasmid 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 gfp-aequorin gene has been integrated into the genome by single cross-over integration. Therefore the possibility exists of reversion to wild-type genotype by removal of the integrated DNA construct. The gfp-aequorin gene is integrated into the c/d-type small subunit ribosomal rna gene. No gene model is yet available for this integration locus.
Additional remarks selection procedure
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 / 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