RMgmDB - Rodent Malaria genetically modified Parasites


Malaria parasiteP. berghei
TaggedGene model (rodent): PBANKA_1301300; Gene model (P.falciparum): PF3D7_1474900; Gene product: trailer hitch homolog, putative (CITH; CAR-I/Trailer Hitch Homolog)
Name tag: GFP
Phenotype Gametocyte/Gamete; Fertilization and ookinete;
Last modified: 21 February 2010, 13:35
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) : 20169188
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 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/ResearcherG.R. Mair; C.J. Janse; A.P. Waters
Name Group/DepartmentLeiden Malaria Research Group
Name InstituteLeiden University Medical Center
CountryThe Netherlands
Name of the mutant parasite
RMgm numberRMgm-358
Principal name909cl1
Alternative nameCITH::GFP
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Asexual blood stageNot different from wild type
Gametocyte/GameteNormal production of gametocytes and gametes. A punctate GFP-fluorescence pattern that appeared to be restricted to the cytoplasm of female gametocytes was observed. The mutant showed wild-type fertilization rates and zygote/ookinete production.
Fertilization and ookineteThe mutant showed wild-type fertilization rates and zygote/ookinete production.
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

The mutant expresses a c-terminal GFP-tagged  version of of CITH (CAR-I/Trailer Hitch Homolog).

Protein (function)
The Plasmodium protein CITH shows homology with proteins that co-locolaize with germ cell and P granules in other organisms such as worm CAR-I and fly Trailer Hitch but also Xenopus Rap55. The Plasmodium protein contains both the conserved LSM14 domain and the extended FDF motif, known to compete with the enhancer of mRNA decapping EDC3 for binding to DDX6 helicases.
The phenotype analysis of mutants lacking expression of CITH (RMgm-359, RMgm-360)  indicate that CITH plays an important role in translational repression of mRNAs in female gametocytes.

The phenotype analyses demonstrate normal fertilisation and zygote/ookinete development indicating that the function of CITH is not affected by the GFP-tag (see RMgm-359, RMgm-360 for the phenotype of mutants lacking expression of CITHI).
A punctate GFP-fluorescence pattern that appeared to be restricted to the cytoplasm of female gametocytes was observed. Fluorescence in situ hybridization (FISH) analysis of the localization of translationally repressed transcripts of genes encoding ookinete surface proteins P25 and P28, showed co-localisation of the repressed transcripts and CITH::GFP. Immunoprecipitations of the CITH::GFP fusion protein from gametocyte lysates have been made with monoclonal anti-GFP antibodies. The eluates have been analyzed for DOZI protein and transcript content, showing the presence of (known) translational repressed transcripts.

Additional information
This mutant has been used for analysis of the protein content by LC-MS/MS mass-spectrometry of imuunoprecipitations of the CITH::GFP fusion protein from gametocyte lysates. This analysis identified a number of proteins that show homology to proteins found in metazoan mRNP granules involved in translational repression of mRNAs indication the presence of messenger ribonucleoprotein (mRNP) bodies in gametocytes with homology to metazoan mRNP granules/P-bodies. See also mutant RMgm-131 for characterization of another protein, DOZI (protein development of zygote inhibited; ATP-dependent RNA helicase, putative)  that is present in the mRNPs of gametocytes.

Other mutants
RMgm-359: A mutant lacks expression of CITH whch expresses GFP under control of the constitutive eef1a promoter
RMgm-360: A mutant lacking expression of CITH which expresses GFP under control of a male and RFP under control of a female gametocyte specific promoter.

  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1301300
Gene Model P. falciparum ortholog PF3D7_1474900
Gene producttrailer hitch homolog, putative
Gene product: Alternative nameCITH; CAR-I/Trailer Hitch Homolog
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
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 1aaagaattcAGATATGAAGGAATTTTG
Additional information primer 12831 (EcoRI); 950bp CITH c-terminus forward
Sequence Primer 2aaagcggccgctATAGGCTGGGTATCTGTTAAATG
Additional information primer 22832 (NotI); 950bp CITH c-terminus reverse
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