RMgmDB - Rodent Malaria genetically modified Parasites


Malaria parasiteP. berghei
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; Fertilization and ookinete;
Last modified: 8 June 2022, 16:53
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) : 35550346
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
Name of the mutant parasite
RMgm numberRMgm-5212
Principal nameSAS4-GFP
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Asexual blood stageNot different from wild type
Gametocyte/GameteSAS4 was not detectable in asexual blood stages and female gametocytes but was located in the cytoplasm of male gametocytes.
Fertilization and ookineteSAS4-GFP shows discrete foci at apical end during zygote to ookinete development and has a nuclear location during meiosis.
OocystNot different from wild type
SporozoiteNot different from wild type
Liver stageNot different from wild type
Additional remarks phenotype

The mutant  expression a C-terminal GFP-tagged version of SAS4.

Protein (function)
Centriole/basal bodies (CBBs) are associated with the microtubule organising centre (MTOC) that nucleates cilia, flagella, and centrosomes and are conserved ancestral organelles in eukaryotes. Centrioles and basal bodies (BBs) share structural features and BBs are mainly associated with flagella or cilia organisation, and extend to produce an axoneme. The canonical view of CBB biogenesis includes centriole duplication and segregation to a daughter cell during mitosis. However, in some organisms, BB biology is more diverse, for example, where the centrioles or BBs form de novo or exhibit non- canonical biogenesis, as observed in Naegleria and in some parthenogenic insect eggs.SAS4/SAS6 are ancestral core proteins involved in BB biogenesis, as predicted by phylogenetic analysis. Clear centrioles with 9 + 1 or 9 + 2 microtubules can only be seen in flagella biogenesis during male gamete formation in Plasmodium.

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. Plasmodium, a divergent unicellular eukaryote and causative agent of malaria, displays an atypical, closed mitosis with an MTOC (or centriolar plaque), reminiscent of an acentriolar MTOC, embedded in the nuclear membrane. Mitosis during male gamete formation is accompanied by flagella formation. There are two MTOCs in male gametocytes: the acentriolar nuclear envelope MTOC for the mitotic spindle and an outer centriolar MTOC (the basal body) that organises flagella assembly in the cytoplasm. 

SAS4 was not detectable in asexual blood stages and female gametocytes but was located in the cytoplasm of male gametocytes.
SAS4-GFP shows discrete foci at apical end during zygote to ookinete development and has a nuclear location during meiosis. 

Evidence is presented that: 
- Live cell imaging of SAS4-GFP reveals de novo BB formation and its rapid dynamics during male gametogenesis 
- SAS4 associates with kinesin-8B, a molecular motor that regulates BB formation and axoneme assembly
- BB formation and mitotic spindle dynamics are coupled processes (based on spatiotemporal locations of SAS4 and the kinetochore protein NDC80)

Additional information
The SAS4-GFP parasites were mixed with kinesin-8B-mCherry or NDC80-mCherry parasites in equal numbers and injected into a mouse. Mosquitoes were fed on this mouse 4–5 d after infection when gametocyte parasitaemia was high. These mosquitoes were checked for oocyst development and sporozoite formation at day 14 and day 21 after feeding. Infected mosquitoes were then allowed to feed on naive mice and after 4–5 d these mice were examined for blood stage parasitaemia by microscopy with Giemsa-stained blood smears. In this way, some parasites expressed both SAS4- GFP and kinesin-8B-mCherry or SAS4-GFP and NDC80-mCherry in the resultant gametocytes. These gametocytes were purified, and fluorescence microscopy images were collected.

From the Abstract: 
'We show the coordinated location, association and assembly of SAS4 with the BB component, kinesin- 8B, but no association with the kinetochore protein, NDC80, indicating that SAS4 is part of the BB and outer centriolar MTOC in the cytoplasm. Deletion of the SAS4 gene produced no phenotype, indicating that it is not essential for either male gamete formation or parasite transmission'.

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 parasitehdhfr
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