Summary

RMgm-63
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_0933700; Gene model (P.falciparum): PF3D7_1113900; Gene product: mitogen-activated protein kinase 2 (MAP2; MAP-2; MAPK2)
Phenotype Fertilization and ookinete;
Last modified: 29 March 2013, 18:29
  *RMgm-63
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) : 16313614
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 line
The mutant parasite was generated by
Name PI/ResearcherR. Tewari, C. Doerig, O. Billker
Name Group/DepartmentDepartment of Biological Sciences
Name InstituteImperial College
CityLondon
CountryUK
Name of the mutant parasite
RMgm numberRMgm-63
Principal nameclone 8.2, clone 8.4
Alternative namePbmap-2 KO
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 ookineteMale and female gametocyte production is not affected. Genome replication occurs in the male but no nuclear division/ segmentation. No exflagellation of male gametocytes (no male gamete production).
No fertilization since male gametocytes do not produce gametes.
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of MAP2 protein.

Protein (function)
MAP kinases are serine/threonine protein kinases involved in a variety of functions including cell proliferation.

Phenotype
The mutant produces wild-type levels of gametocytes but complete male gamete formation was inhibited. Male gametogenesis progressed to genome replication but further development (cytokenisis) is arrested; the mutants are able to form mitotic spindles and assemble axonemes. Female gametes could develop to completion and were fertile, as was shown by the formation of ookinetes in cross-fertilization experiments with fertile males of the  mutant  (RMgm-60; which produces infertile females). This observation indicates that MAP2 is essential for male gamete formation and is only active after CDPK4 activation (RMgm-12; a mutant lacking expression of CDPK4).

See also the paper by Dorin-Semblat D. et al. (2007, Mol Microbiol 65, 1170-1180) for unsuccessful attempts to knock-out map2 in P. falciparum, indicating that in P. falciparum map2 has an essential function during asexual blood stage development.

Additional information
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 as integration of the KO vector was not achieved. Accesibility of the locus to recombination was verified by C-terminal tagging.

Other mutants
Independent P. berghei map2- mutants (RMgm-62;  RMgm-66) have been generated that also lacks MAP2 expression and have essentially the same phenotype.


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0933700
Gene Model P. falciparum ortholog PF3D7_1113900
Gene productmitogen-activated protein kinase 2
Gene product: Alternative nameMAP2; MAP-2; MAPK2
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, ApaI
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 modification
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 1ggtaccGTATTCTTGCGGTATAAAAATCATG
Additional information primer 1ol52; Primer F 5'
Sequence Primer 2gggcccTGTAATTATCTGGCACATGCAC
Additional information primer 2ol53; Primer R 5'
Sequence Primer 3ggatccTCGTCGAAAGGGGTGC
Additional information primer 3ol54; Primer F 3'
Sequence Primer 4gcggccGCTGTTATATATCCCAATTTCATTAG
Additional information primer 4ol55; Primer R 3'
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6