Summary

RMgm-154
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_0408200; Gene model (P.falciparum): PF3D7_0310100; Gene product: calcium-dependent protein kinase 3 (CDPK3)
Phenotype Fertilization and ookinete; Oocyst;
Last modified: 21 December 2011, 14:35
  *RMgm-154
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) : 16796674
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/ResearcherI. Siden-Kiamos; O. Billker
Name Group/DepartmentDivision of Cell and Molecular Biology
Name InstituteImperial College London
CityLondon
CountryUnited Kingdom
Name of the mutant parasite
RMgm numberRMgm-154
Principal namecdpk3-
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNot different from wild type
Fertilization and ookineteNormal numbers of ookinetes are produced. These ookinetes have a (light microscope) morphology that is comparable to wild type ookinetes. Ookinetes show a strong reduction (98%) in the formation of oocysts in A. stephensi mosquitoes. Ookinetes fail to associate with the midgut epithelium. Ookinetes showed a strongly reduced ability to glide productively.
OocystOokinetes show a strong reduction (98%) in the formation of oocysts in A. stephensi mosquitoes.
Injection of in vitro cultured, mature ookinetes into the hemocoel and thereby by-passing the midgut wall resulted in normal development of oocysts and infectious sporozoites.
SporozoiteNot different from wild type
Liver stageNot different from wild type
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of CDPK3 (calcium-dependent protein kinase 3).

Protein (function)
CDPK3 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 analyses indicate a role of CDPK3 in the transformation of the mature ookinete into the oocyst stage. Specifically, ookinetes fail to bind and traverse the cell of the midgut wall and the motility of ookinetes is strongly affected.
Motility of  ookinetes was abnormal, showing frequent flexing, bending, twirling and pendular motions, but only rare bouts of translocation over short distances.
Injection of in vitro cultured, mature ookinetes into the hemocoel and thereby by-passing the midgut wall resulted in normal development of oocysts and infectious sporozoites, indicating that the role of CDPK3 is restricted to the ookinete stage.
These analyses suggest a role for CDPK3 in regulating productive gliding motility of ookinetes.

Additional information
An independent mutant lacking expression of CDPK3, RMgm-165, has been generated which show a comparable defect in ookinete to oocyst transition. However, instead of a general defect in  motility of the ookinetes it has been suggested on basis of the phenotype analysis that ookinetes are affected in a CDPK3-dependent mode of motility that is required to penetrate the layer surrounding the blood meal.
The cdpk3- parasite has been complemented by introducing an episomal plasmid in which the complete cdpk3 genomic sequence was fused to a double c-myc epitope tag (c-terminal tag) The plasmid was introduced by electroporation and maintained as an episome by selection for the hdhfr gene with WR99210. Complementation fully restored the infectivity of ookinetes.

Disruption of the P. falciparum ortholog has been attempted (Solyakov et al., 2011, Nat Commun, 2:565).
The gene is likely essential for asexual proliferation. After transfection with a KO vector a weak PCR signal diagnostic for integration was observed, indicating that integration does transiently occur but parasites with a disrupted locus do not persist. Cloning will be required to validate this interpretation for this gene.

Other mutants
RMgm-165: An independent 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.
RMgm-12: A mutant lacking expression CDPK4


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0408200
Gene Model P. falciparum ortholog PF3D7_0310100
Gene productcalcium-dependent protein kinase 3
Gene product: Alternative nameCDPK3
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 KpnI/BamHI
Partial or complete disruption of the genePartial
Additional remarks partial/complete disruption 5' targetting region: a 608 bp fragment comprising 5′ upstream sequence followed by the first 543 bp of exon1 of cdpk3

3' targetting region: A 686 bp fragment comprising the last two exons and 3′ flanking region of pbcdpk3

No additional information on primer sequences is available.
Selectable marker used to select the mutant parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationNo additional information on primer sequences used for the replacement construct is provided in the paper!
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