Summary

RMgm-3
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_0514900; Gene model (P.falciparum): PF3D7_1030900; Gene product: 28 kDa ookinete surface protein (Pbs21; P28)
DisruptedGene model (rodent): PBANKA_0515000; Gene model (P.falciparum): PF3D7_1031000; Gene product: 25 kDa ookinete surface antigen precursor (Pbs25; P25)
Phenotype Fertilization and ookinete; Oocyst;
Last modified: 31 July 2012, 17:52
  *RMgm-3
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption, Gene disruption
Reference (PubMed-PMID number) Reference 1 (PMID number) : 11483501
MR4 number MRA-443
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/ResearcherC.J. Janse, A.P. Waters, A.M. Thomas
Name Group/DepartmentLeiden Malaria Research Group
Name InstituteLeiden University Medical Center
CityLeiden
CountryThe Netherlands
Name of the mutant parasite
RMgm numberRMgm-3
Principal name73cl1; 73cl4; 108cl1
Alternative name25/28Dko
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 ookineteSlight inhibition of ookinete development in vitro (slight reduction in ookinete numbers in in vitro cultures); ookinetes do not form the typical clusters of multiple ookinetes in vitro; ookinetes are formed but with aberrant morphology.
A strong reduction in ookinete formation and oocyst production in vivo (in Anopheles stephensi).
OocystA strong reduction in ookinete formation and oocyst production (>95%) in vivo (in Anopheles stephensi).
SporozoiteNot different from wild type
Liver stageNot different from wild type
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of two ookinete surface proteins, P25 and P28.

Protein (function)
P25 and P28 are two major surface proteins of the plasma membrane of ookinetes. The proteins are conserved between different Plasmodium species. The proteins are characterized by a secretory N-terminal signal sequence followed by three or four epidermal growth factor (EGF) domains and a glycosylphosphatidylinositol (GPI) anchor. Synthesis of both proteins begins between 0.5 and 2 h after the formation of the female gametes, and the proteins are still present in young oocysts. The paralogous genes encoding P25 and P28 are located next to each other in the genome in a head to tail organization.

Phenotype
In the midgut of mosquitoes, the formation of ookinetes lacking both proteins is significantly inhibited due to decreased protection against lethal factors, including protease attack. In addition, ookinetes have a much reduced capacity to traverse the midgut epithelium and to transform into the oocyst stage.

Additional information
In this mutant the genes encoding P25 and P28 have been disrupted (double knock-out mutant) using a single DNA construct.
Disruption of either p25 or p28 ('single knock-out' mutants) has only a minor effect on ookinete and oocyst production, indicating redundancy in function between P25 and P28.
Motility and entry into insect cells in vitro of the mutant ookinetes are normal, but the number of ookinetes successfully transforming into oocysts in vitro is significantly reduced (Sidén-Kiamos et al., J. Cell Science (2000) 113, 3419-26).

Other mutants
A. P. berghei mutant has been generated lacking P28 (RMgm-1).
A. P. berghei mutant has been generated lacking P25 (RMgm-2).

 


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0514900
Gene Model P. falciparum ortholog PF3D7_1030900
Gene product28 kDa ookinete surface protein
Gene product: Alternative namePbs21; P28
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 parasitepbdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationIn this mutant line the genes encoding P25 (PB000266.01.0) and P28 (=Pbs21; PB000865.00.0) have been disrupted (double knock-out mutant) using a single DNA construct.
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

  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0515000
Gene Model P. falciparum ortholog PF3D7_1031000
Gene product25 kDa ookinete surface antigen precursor
Gene product: Alternative namePbs25; P25
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 modificationIn this mutant line the genes encoding P25 (PB000266.01.0) and P28 (=Pbs21; PB000865.00.0) have been disrupted (double knock-out mutant) using a single DNA construct
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