Summary

RMgm-620
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PBANKA_0715500; Gene model (P.falciparum): PF3D7_0413500; Gene product: phosphoglucomutase-2 (PGM2)
Name tag: GFP
Phenotype Asexual bloodstage; Fertilization and ookinete;
Last modified: 24 July 2011, 12:11
  *RMgm-620
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene tagging
Reference (PubMed-PMID number) Reference 1 (PMID number) : 21689687
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 cl15cy1
Other information parent lineA reference wild type clone from the ANKA strain of P. berghei (PubMed: PMID: 17406255)
The mutant parasite was generated by
Name PI/ResearcherT. Hills; A. Srivastava; AP Waters; J.C. Pizarro
Name Group/DepartmentThe SGC (Structural Genomics Consortium)
Name InstituteUniversity of Toronto
CityToronto
CountryCanada
Name of the mutant parasite
RMgm numberRMgm-620
Principal namePbPGM2::GFP
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageAsexual blood stages express GFP-tagged PbPGM2
Gametocyte/GameteNot tested
Fertilization and ookineteZygotes and ookinetes express GFP-tagged PbPGM2
OocystNot different from wild type
SporozoiteNot different from wild type
Liver stageNot different from wild type
Additional remarks phenotype

Mutant/mutation
The mutant expresses a GFP-tagged (C-terminal) version of PbPGM2 (phosphoglucomutase-2)

Protein (function)
Two P. falciparum genes code for putative phosphoglycerate mutases (PGMases; PfPGM1 - PF11_0208 and PfPGM2 - PFD0660w), a widespread protein group characterized by the involvement of histidine residues in their catalytic mechanism. Phosphoglycerate mutases (EC 5.4.2.1; PGMase) catalyze the reversible conversion of 2-phosphoglycerate (2-PG) to 3-phosphoglycerate (3-PG).This is an essential component of the glycolysis pathway providing 2-PG to the enzyme enolase and also of the gluconeogenesis pathway where it supplies 3-PG to the phosphoglycerate kinase. Members of the PGM family function as phosphotransferases or phosphohydrolases acting upon a variety of substrates. Aside from metabolic functions, a proportion of PGM family members are involved in cell signaling and/or regulation. In this study the P. berghei homologue of PGM2 (PBANKA_071550) was studied. The P. berghei homolog of PGM1 is  PBANKA_092810

Phenotype
GFP-tagged PbPGM2 is expressed in asexual blood stages, zygotes and ookinetes as shown by Western and IFA asnalysis using anti-GFP antibodies. IFA-analysis shows a cytoplasmic location.

Additional information
Repeated failures to disrupt the gene (see 619) indicates that PbPGM2 is essential for P. berghei asexual blood stage development by the. No information is provided on the expression in gametocytes

Other mutants
RMgm-619: Unsuccessful attempt to disrupt the pbpgm2 gene


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0715500
Gene Model P. falciparum ortholog PF3D7_0413500
Gene productphosphoglucomutase-2
Gene product: Alternative namePGM2
Details of the genetic modification
Name of the tagGFP
Details of taggingC-terminal
Additional remarks: tagging
Commercial source of tag-antibodiesanti-GFP mouse monoclonal (Roche)
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 XbaI
Selectable marker used to select the mutant parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationC-terminal GFP-tagging of PGM2 was performed using a construct that integrates through single crossover homologous recombination into the PbPGM2-locus and contains the tgdhfr/ts selectable marker. Primers GU0626-SacII and GU0629-BamHI were used to amplify the 724bps targeting region of PbPGM2 (whilst incorporating an internal XbaI site using primers GU0627 and GU0628 by overlap extension PCR)
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 1GGccgcggGGGTTTAACTTATGCATATGAAAATTATG
Additional information primer 1GU0626 (SacII); targeting region forward
Sequence Primer 2GGggatccAAAATATGTAACACTATTGAAAGGAAGATG
Additional information primer 2GU0629 (BamHI); targeting region reverse
Sequence Primer 3TAATTTAtctagaGCATTTGGAAAATATTTACTTATGATTTC
Additional information primer 3GU0627 (XbaI); incorporation XbaI site
Sequence Primer 4CCAAATGCtctagaTAAATTAATAAATGATCCGAATTTAAATGAAG
Additional information primer 4GU0628 (XbaI); incorporation XbaI site
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6