Summary

RMgm-311
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PBANKA_1008200; Gene model (P.falciparum): PF3D7_1436600; Gene product: cGMP-dependent protein kinase (PKG)
Name tag: GFP
Phenotype Asexual bloodstage; Gametocyte/Gamete; Fertilization and ookinete;
Last modified: 21 October 2010, 21:52
  *RMgm-311
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) : 19779564
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/ResearcherR.W. Moon; D.A Baker; O. Billker
Name Group/DepartmentDepartment of Cell and Molecular Biology
Name InstituteImperial College London
CityLondon
CountryUK
Name of the mutant parasite
RMgm numberRMgm-311
Principal namepkg-gfp
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageExpression of PKG-GFP in blood stages
Gametocyte/GameteExpression of PKG-GFP in gametocytes
Fertilization and ookineteExpression of PKG-GFP in ookinetes
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses a GFP-tagged (C-terminal) form of PKG (cGMP-dependent protein kinase).

Protein (function)
Cyclic GMP-dependent protein kinases (PKGs) are the major mediators of the cGMP signal transduction pathway and regulate a variety of physiological effects

Phenotype
Expression of PKG-GFP in blood stages, gametocytes and ookinetes. Attempts to disrupt the pkg gene were unsuccessful (RMgm-325, RMgm-544), suggesting an essential role of PKG in asexual blood stages.

Additional information
In the paper it is shown that a selective inhibitor of apicomplexan PKG, the ATP analog Compound 1, potently blocked ookinete motility.

In the paper evidence is presented that in ookinetes GCß is the main source for cGMP and that a cyclic GMP signalling module exists that regulates gliding motility of ookinetes. This evidence is partly based on the analysis of mutants lacking expression PDEδ (RMgm-308) and GCβ (RMgm-307) and a mutant  lacking expression of both GCβ and PDEδ (RMgm-309). These analyses indicate that PDEδ is the relevant cGMP degrading enzyme. Signalling via a cGMP-dependent protein kinase (PKG; PF14_0346; PB000726.02.0) may regulate ookinete differentiation and motility.

It the same paper it is reported that repeated attempts were made to disrupt the pkg gene (cGMP-dependent protein kinase: PB000726.02.0, PB300651.00.0; PF14_0346). These attempts were unsuccessful (see RMgm-325), suggesting its essential role in the blood stage parasites (no information is provided on the construct used to disrupt pkg and the sequence of primers used to amplify the target regions).

(Gene models for PKG PB000726.02.0; PB300651.00.0)

Other mutants
RMgm-357: A ‘conditional knock-out mutant’ lacking expression of PKG in liver stages. The Flp/FRT site-specific recombination (SSR) system of yeast has been used to silence PKG expression specifically in liver stages.
RMgm-544: Unsuccessful attempts to disrupt the pkg gene
RMgm-325: Unsuccessful attempts to disrupt the pkg gene





  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1008200
Gene Model P. falciparum ortholog PF3D7_1436600
Gene productcGMP-dependent protein kinase
Gene product: Alternative namePKG
Details of the genetic modification
Name of the tagGFP
Details of taggingC-terminal
Additional remarks: tagging
Commercial source of tag-antibodies
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 BlpI
Selectable marker used to select the mutant parasitehdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedureWR99210
Selection (negative) procedureNo
Additional remarks genetic modificationThe construct included 1.5 kb of the 3' end of the PKG coding region preceding the in frame GFP sequence.
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 1atatatGGTACCTCAGGGATCGTACTTCGGAGAGAGAG
Additional information primer 1Fwd (KpnI)
Sequence Primer 2aattatGGGCCCAAAATCAATATCCCAATTTTCATCATCAATTTC
Additional information primer 2Rev (ApaI)
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