RMgmDB - Rodent Malaria genetically modified Parasites


Malaria parasiteP. yoelii
TaggedGene model (rodent): PBANKA_0802000; Gene model (P.falciparum): PF3D7_0704300; Gene product: conserved Plasmodium membrane protein, unknown function (BLEB (basolateral expansion boundary))
Name tag: GFP
Phenotype Asexual bloodstage; Gametocyte/Gamete;
Last modified: 29 August 2022, 13:23
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) : 35972967
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. yoelii
Parent strain/lineP. y. yoelii 17XNL
Name parent line/clone Not applicable
Other information parent line
The mutant parasite was generated by
Name PI/ResearcherClements RL, Dvorin JD
Name Group/DepartmentDivision of Infectious Diseases
Name InstituteBoston Children's Hospital
Name of the mutant parasite
RMgm numberRMgm-5233
Principal namePyBLEB-GFP
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Asexual blood stageTo determine whether BLEB exhibits similar expression patterns in other Plasmodium species, we assessed the localization of PyBLEB-GFP in P. yoelii. We found that PyBLEB has a dynamic localization in P. yoelii schizonts, mirroring the basal complex localization of PfBLEB in P. falciparum schizonts. This provides further evidence that BLEB is a member of the basal complex found in multiple Plasmodium species.
Gametocyte/GameteExpression in the perihery of gametocytes
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

The mutant expresses a C-terminal GFP-tagged version of BLEB

Protein (function)
The inner membrane complex (IMC), a membranous scaffold together with associated proteins that lie below the parasite plasma membrane, strengthen the cytoskeleton, and  play a role in invasion, gametocyte formation, and cytokinesis. In gametocytes, the IMC serves as a critical structural support. The basal complex is believed to form a contractile ring at the edge of the emerging IMC and likely facilitates IMC expansion. PfBLEB, is a basal complex protein. Studies presented in this paper on PfBLEB shows that the protein is essential for gametocytogenesis. Parasites lacking PfBLEB harbor defects in IMC expansion and are unable to form mature gametocytes. 

To determine whether BLEB exhibits similar expression patterns in other Plasmodium species, we assessed the localization of PyBLEB-GFP in P. yoelii. We found that PyBLEB has a dynamic localization in P. yoelii schizonts, mirroring the basal complex localization of PfBLEB in P. falciparum schizonts. This provides further evidence that BLEB is a member of the basal complex found in multiple Plasmodium species.

Additional information 
In the paper evidence is presented that:
- PfBLEB Is Not Required for Asexual Parasite Replication In Vitro.
- PfBLEB Is Essential for the Formation of Mature Gametocytes
- PfBLEB Lines the Leading Edge of the Expanding IMC Throughout Gametocytogenesis
- Knockdown of PfBLEB Disrupts Expansion of the IMC and Microtubules in Gametocytes 
- PfBLEB Does Not Colocalize with Known Members of the Asexual Basal Complex in Gametocytes

From the paper: 'The major function of the IMC network in gametocytes is to generate and maintain cell shape. P. falciparum is unique within the Plasmodium genus in that it is the only species to undergo the dramatic transformation into a crescent shape. A handful of early electron microscopic studies suggest that the IMC in P. berghei and P. knowlesi does not completely surround these round gametocytes and instead lays discontinuously around the perimeter of the cell. This suggests that Plasmodium species with round gametocytes may not require a stable or continuous IMC, and therefore may not require BLEB in the development of transmission stages. Intriguingly, we have demonstrated that PyBLEB is expressed on the periphery of nonfalciform P. yoelii.gametocytes, but its function remains unclear. Future investigations into the IMC, BLEB, and its interacting partners in nonfalciform gametocytes may clarify how the development of transmission stages varies between species.'

Other mutants

  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0802000
Gene Model P. falciparum ortholog PF3D7_0704300
Gene productconserved Plasmodium membrane protein, unknown function
Gene product: Alternative nameBLEB (basolateral expansion boundary)
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 markerunknown
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationConventional gene editing approaches for P. yoelii were used to append a C-terminal GFP tag to PyBLEB.

pSL1528 (PyBLEB-GFP): 3’ HR and 3’ Open Reading Frame (ORF) were PCR amplified from Py17XNL genomic DNA using primers oSL28-59/oSL28-60 (3’ HR) and oSL28-63/28-64 (ORF). PCR SOE of ORF and 3’ HR was done with oSL28-59/oSL28-64. PCR SOE was ligated into the StuI site of pCR-Blunt (Thermo Fisher) to create pSL1526. PCR SOE sequence was verified in pSL1526 using Sanger Sequencing at the PSU Genomics Core. PCR SOE insert from pSL1526 was digested with
KpnI/SpeI and ligated into similarly digested vector pSL0442 to create pSL1528.
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