Summary

RMgm-12
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_0615200; Gene model (P.falciparum): PF3D7_0717500; Gene product: calcium-dependent protein kinase 4 (CDPK4)
Phenotype Gametocyte/Gamete; Fertilization and ookinete; Oocyst;
Last modified: 21 December 2011, 14:30
  *RMgm-12
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption
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-12
Principal name∆CDPK4
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNo male gametes are formed; Male gametocytes are not affected in the ability to 'round up'and lyse the host cell; in male gametocytes axoneme formation, formation of mitotic spindles and DNA synthesis is inhibited/blocked.
Female gametes are fertile; mutant female gametes fertilised by wild type male gametes produce ookinetes. However, these ookinetes are strongly affected in their ability to produce oocysts
Fertilization and ookineteSee the description of the phenotype of Gametocytes/Gametes and Additional remarks phenotype
OocystSee the description of the phenotype of Gametocytes/Gametes and 'Additional remarks phenotype'.
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of a (plant like) calcium-dependent protein kinase 4 (CDPK4).

Protein (function)
CDPK4 belongs to an expanded family of Ca2+ dependent protein kinases (CDPKs). CDPKs combine an amino-terminal serine/threonine kinase domain and a carboxy-terminal calmodulin-like domain, composed of four EF hands, in the same molecule. In plants, CDPKs translate Ca2+ signals generated by external stimuli into cellular responses, thereby regulating cell division and differentiation, the development of tolerance to stress stimuli and the specific defense responses to pathogens.

Phenotype
The phenotype of the male gametocytes/gametes indicate that CDPK4 controls S-phase entry in the male gametocyte. In the mutant DNA replication in the male gametocytes to form the 8 motile gametes is blocked.
The reduced infectivity of ookinetes, resulting from crossing mutant female gametes with wildtype male gametes suggests a second function of CDPK4 later during sporogonic development.

Additional information
Disruption of the P. falciparum ortholog has been succesful (Solyakov et al., 2011, Nat Commun, 2:565) indicating that this gene is not essential for asexual proliferation.
 

Other mutants
RMgm-154: A mutant lacking expression of CDPK3
RMgm-165: A mutant lacking expression of CDPK3
RMgm-168: A mutant lacking expression of CDPK3 and expressing GFP under control of the hsp70 promoter. This mutant has been generated by crossing mutant RMgm-165 lacking expression of CDPK3 with mutant RMgm-166 that expresses GFP.


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0615200
Gene Model P. falciparum ortholog PF3D7_0717500
Gene productcalcium-dependent protein kinase 4
Gene product: Alternative nameCDPK4
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct usedPlasmid double cross-over
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid
Partial or complete disruption of the geneComplete
Additional remarks partial/complete disruption
Selectable marker used to select the mutant parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe ∆CDPK4 mutant has been complemented with an intact copy of the cdpk4 gene, tagged with 2 copies of a carboxy-terminal c-myc epitope tag (insertion vector). Complementation resulted in the restoration of the wild type phenotype (wildtype male gamete formation, fertilisation and ookinete infectivity).
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