Summary

RMgm-678
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PBANKA_1459300; Gene model (P.falciparum): PF3D7_1246200; Gene product: actin I (ACT1; actin1)
Name tag: GFP
Phenotype Asexual bloodstage; Fertilization and ookinete; Oocyst; Sporozoite; Liver stage;
Last modified: 1 July 2012, 14:57
  *RMgm-678
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) : 22138565
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone Not applicable
Other information parent line
The mutant parasite was generated by
Name PI/ResearcherF. Angrisano; J. Baum
Name Group/DepartmentWalter and Eliza Hall Institute of Medical Research
Name InstituteWalter and Eliza Hall Institute of Medical Research
CityParkville, Melbourne
CountryAustralia
Name of the mutant parasite
RMgm numberRMgm-678
Principal nameGFP-PbACTCON
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationNo
Phenotype
Asexual blood stageGFP-Actin I expression in all stages
Gametocyte/GameteNot tested
Fertilization and ookineteGFP-Actin I expression in ookinetes
OocystGFP-Actin I expression in oocysts
SporozoiteGFP-Actin I expression in sporozoites
Liver stageGFP-Actin I expression in liver stages
Additional remarks phenotype

Mutant/mutation
The mutant expresses an additional copy of a (N-terminal) GFP-tagged copy of Actin I. The endogenous actin I gene is not tagged. The (N-terminal) GFP tagged actin I gene is introduced into the genome into the (silent) c/d-ssuRNA gene locus (by single cross-over integration). The GFP-tagged copy is under control of the constitutive eef1α promoter.

Protein (function)
Actin, a cytoskeletal protein, has many diverse functions in eukaryotic cells ranging from roles in cell motility, cell division, vesicle trafficking to functions in cell signaling and regulation of transcription. A critical property of actin is its ability to form filamentous polymers (F-actin), and a plethora of proteins are involved in the highly dynamic regulation of F-actin formation . Actins are highly conserved proteins that often exist in multiple isoforms in the eukaryotic cell and their expression is regulated both spatially and temporally during development. The number of conventional actin genes varies among eukaryotic organisms. A few single cell eukaryotes, such as Saccharomyces cerevisiae, Toxoplasma gondii, and Trypanosoma brucei encode a single actin gene, which results in lethality when targeted with gene ablation approaches. Many organisms, however, have several conventional actin genes.Apicomplexan parasites all encode one major actin isoform, here termed Actin I. All apicomplexan parasites also contain a number of actin-related and actin-like proteins. Plasmodium species species stand out in that they all encode a second conventional actin, termed Actin II.

Phenotype
In the mutant the GFP-tagged actin I is introduced into the genome as an additional copy and is under the control of the constitutive eef1α promoter. All different life cycle stages express GFP-tagged Actin I and was found to be broadly cytoplasmic. Sporozoites suggested a pellicular concentration of actin whereas ookinetes and merozoites showed more uniform fluorescence. No obvious dynamics of parasite actin could be detected in gliding or turning ookinetes.

Additional information
See also the paper of Kiamos et al. (2011. Mol. Biochem Parasitol.). In this paper filamentous actin was analysed in ookinetes using peptide antibodies against P. berghei Actin I. In this paper they report  tagging of Actin I tagged with YFP. The YFP-tagged acrtin I gene is under the control of the promoter of the circumsporozoite and TRAP-related protein (CTRP). No information is provided on the mode of tagging. They write '... detected the presence of actin in a well-defined region in the apical tip of the parasite ...a similar localization was seen in transgenic parasites, expressing as a second copy YFP-tagged actin I fusion protein......Fluorescently tagged actin may interfere with the normal function of endogenous actin; although not well understood, this effect may be dependent on the ratio of non-modified to modified form....We, therefore, hypothesize that the observed accumulation of YFP-actin I might reflect the fusion protein in the globular (G−) form. Clearly, we did not detect YFP-actin in a pattern consistent with F− actin.'

Other mutants


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1459300
Gene Model P. falciparum ortholog PF3D7_1246200
Gene productactin I
Gene product: Alternative nameACT1; actin1
Details of the genetic modification
Name of the tagGFP
Details of taggingN-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 ApaI
Selectable marker used to select the mutant parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe endogenous actin I gene is not tagged. The (N-terminal) GFP tagged actin I gene is introduced into the genome into the (silent) c/d-ssuRNA gene locus (by single cross-over integration). The GFP-tagged copy is under control of the constitutive eef1α promoter.

Green fluorescent protein (GFP) was excised from the plasmid PbGFPCON by digesting plasmids with BamHI. In its place a stitched PCR product joining GFP to the N terminus of PbActin was created (using primers GFPfwd 5′-GATCggatccATGAGTAAAGGAGAAGAACTTTTCACTGGAG-3; GFPrev [PbAct overhang] 5-TGAACTTCTTCGTCACCCATactagtTTTGTATAGTTCATCCATGCCATGTGT-3; PbActfwd [GFP overhang] 5-GCATGGATGAACTATACAAAactagtATGGGTGACGAAGAAGTTCAAGCTTTAGTT-3; PbActrev 5-GATCggatcccctaggTTAGAAGCATTTTCTGTGGACAATACTTGG-3) that, when digested with BamHI, was re-ligated into the parent vector to create GFP-PbACTCON. This final vector was linearized at the unique ApaI site to integrate into the genome by single crossover recombination
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