Summary

RMgm-997
Malaria parasiteP. berghei
Genotype
Genetic modification not successful
DisruptedGene model (rodent): PBANKA_0203100; Gene model (P.falciparum): PF3D7_0110600; Gene product: phosphatidylinositol-4-phosphate 5-kinase (PIP5K)
PhenotypeNo phenotype has been described
Last modified: 10 March 2014, 13:03
  *RMgm-997
Successful modificationThe gene/parasite could not be changed/generated by the genetic modification.
The following genetic modifications were attempted Gene disruption
Number of attempts to introduce the genetic modification Unknown
Reference (PubMed-PMID number) Reference 1 (PMID number) : 24594931
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).
Attempts to generate the mutant parasite were performed by
Name PI/ResearcherBrochet, M; Billker, O.
Name Group/DepartmentWellcome Trust Sanger Institute, Hinxton
Name InstituteWellcome Trust Sanger Institute, Hinxton
CityHinxton, Cambridge
CountryUK

  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0203100
Gene Model P. falciparum ortholog PF3D7_0110600
Gene productphosphatidylinositol-4-phosphate 5-kinase
Gene product: Alternative namePIP5K
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct usedPlasmid double cross-over
PlasmoGEM (Sanger) construct/vector usedYes
Name of PlasmoGEM construct/vector-
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid
Partial or complete disruption of the geneUnknown
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 modificationNo details are provided for the PlasmoGEM vector used.

Enzymes in the inositol phospholipid biosynthetic pathway. Phosphorylated phosphatidylinositol lipids have important roles in vesicle trafficking and as a source of secondary messengers in signal transduction. Their biosynthesis from phosphatidyl-1D-myo-inositol (PI) is mediated by lipid kinases. The P. berghei genome encodes four putative lipid kinases to convert PI first to phosphatidylinositol 4-phosphate (PI4P) and then to phosphatidylinositol (4,5)- bisphosphate (PI(4,5)P2). Hydrolysis of the latter by a PI-specific phospholipase C (PI-PLC) gives rise to the secondary messenger inositol (1,4,5)-trisphosphate (IP3), which plays an important role in P. berghei gametocytes, where it is responsible for the mobilisation of Ca2+ from internal stores, leading to activation and gametogenesis.

To test whether the PKG-dependent phosphorylation of enzymes associated with phosphoinositide metabolism has a direct role in ookinete motility, an experimental genetics approach was used to infer the role of putative PI kinases. Both the putative PI4K (PBANKA_110940) and the putative PIP5K (PBANKA_020310) were unable to be genetically disrupted (unpublished data), suggesting these genes may be essential for asexual growth, although both loci could be modified.
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