RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5132
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PBANKA_1322200; Gene model (P.falciparum): PF3D7_1458500; Gene product: spindle assembly abnormal protein 4, putative (SAS4)
Name tag: GFP
Phenotype Gametocyte/Gamete;
Last modified: 14 December 2021, 16:09
  *RMgm-5132
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene tagging
Reference (PubMed-PMID number) Not published (yet)
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/ResearcherZeeshan M, Tewari R
Name Group/DepartmentSchool of Life Sciences
Name InstituteUniversity of Nottingham
CityNottingham
CountryUK
Name of the mutant parasite
RMgm numberRMgm-5132
Principal nameSAS4-GFP
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteSAS4-GFP was not detectable in asexual blood stages but was located in the cytoplasm of male gametocytes.
Multiple discrete SAS4-GFP foci in the cytoplasm of
activated male gametocytes, with the number of foci depending on the length of time after activation (during formation of male gametes during exflagellation).
Evidence is presented for:
- SAS4 associating with Kinesin-8B, a molecular motor that regulates basal body formation and axoneme assembly
- spatiotemporal locations of SAS4 and the kinetochore protein NDC80 reveal basal body formation and mitotic spindle dynamics are coupled processes
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses a C-terminal GFP tagged version of SAS4 

Published in bioRxiv preprint doi: https://doi.org/10.1101/2021.12.03.471138

Protein (function)
The centriole/basal body (CBB) is an evolutionarily conserved organelle acting as a microtubule organising centre (MTOC) to nucleate cilia, flagella and the centrosome. SAS4/CPAP is a conserved component associated with BB biogenesis in many model flagellated cells. SAS4 and SAS6 are both ancestral components of BB formation. Plasmodium, a divergent unicellular eukaryote, displays an atypical closed mitosis with an MTOC, reminiscent of the acentriolar MTOC, embedded in the nuclear membrane at most proliferative stages. Mitosis during male gamete formation is accompanied by flagellum formation.

Phenotype
SAS4-GFP was not detectable in asexual blood stages but was located in the cytoplasm of male gametocytes. Multiple discrete SAS4-GFP foci in the cytoplasm of activated male gametocytes, with the number of foci depending on the length of time after activation (during formation of male gametes during exflagellation). 
Evidence is presented for: 
- SAS4 associating with Kinesin-8B, a molecular motor that regulates basal body formation and axoneme assembly
- spatiotemporal locations of SAS4 and the kinetochore protein NDC80 reveal basal body formation and mitotic spindle dynamics are coupled processes

Additional information
Electron microscopy analysis suggests that SAS4 is part of an outer centriolar BB MTOC in male gametocytes. 

Other mutants


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1322200
Gene Model P. falciparum ortholog PF3D7_1458500
Gene productspindle assembly abnormal protein 4, putative
Gene product: Alternative nameSAS4
Details of the genetic modification
Name of the tagGFP
Details of taggingC-terminal
Additional remarks: tagging
Commercial source of tag-antibodies
Type of plasmid/construct(Linear) plasmid 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 markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe C-terminus of SAS4 was tagged with GFP by single crossover homologous recombination in the parasite. To generate the SAS4-GFP line, a region of the sas4 gene downstream of the ATG start codon was amplified using primers T2011 and T2012, ligated to p277 vector.
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