RMgmDB - Rodent Malaria genetically modified Parasites


Malaria parasiteP. berghei
TaggedGene model (rodent): PBANKA_1128600; Gene model (P.falciparum): PF3D7_0629800; Gene product: cullin-2, putative (CUL4)
Name tag: triple-HA
Phenotype Asexual bloodstage; Gametocyte/Gamete;
Last modified: 21 February 2024, 21:50
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/ResearcherRashpa R, Brochet, M
Name Group/DepartmentFaculty of Medicine, Department of Microbiology and Molecular Medicine
Name InstituteUniversity of Geneva
Name of the mutant parasite
RMgm numberRMgm-5393
Principal nameCUL4::3xHA
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Asexual blood stageImmunofluorescence assays showed a cytoplasmic distribution of CUL4-HA both in P. berghei schizonts and gametocytes
Gametocyte/GameteImmunofluorescence assays showed a cytoplasmic distribution of CUL4-HA both in P. berghei schizonts and gametocytes. Immunoblotting confirmed expression of the fusion protein in mature gametocytes with the expected mobility
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

The mutant expresses a 3xHA-tagged version of CUL4.
The mutant was published in bioRxiv preprint doi: https://doi.org/10.1101/2023.07.19.549332

Protein (function)
From the paper: 'Plasmodium encodes a second cullin annotated as cullin-2 (CUL4 - PF3D7_0629800) that, compared to human cullins, shares maximum sequence identity with Cullin-4B. To keep in line with the nomenclature, we here propose to rename this protein CUL4.
Here we confirm that CUL4 forms a CRL4-related complex in P. falciparum asexual blood stages that consists of RBX1, the adaptor protein DDB1 and a distinct set of putative receptor proteins containing WD40 repeat domains.
We further show that this CRL4-related complex is expressed in P. berghei gametocytes with at least one putative receptor WD protein expressed at both stages. While this WD protein is not required for the proliferation of asexual blood stages, its disruption leads to a complete block in microgamete formation (see RMgm-5394). We thus decided to name this protein WD repeat protein Important for Gametogenesis 1 (WIG1). Proteomic analyses indicate that WIG1 disruption alters proteostasis of ciliary proteins and components of the DNA replication machinery during gametocytogenesis. Further analysis by ultrastructure expansion microscopy (U-ExM) indicates that WIG1-dependent depletion of ciliary proteins is associated with altered formation of the microgametocyte MTOCs and defects in DNA replication

Immunofluorescence assays showed a cytoplasmic distribution of CUL4-HA both in P. berghei schizonts and gametocytes. Immunoblotting confirmed expression of the fusion protein in mature gametocytes with the expected mobility.

Additional information
Affinity purification of PbCUL4-HA from 4 min activated P. berghei gametocytes also identified  RBX1 (PBANKA_0806200), DDB1 (PBANKA_0807500), Nedd8 (PBANKA_1411400), and polyubiquitin 140 (PolyUb – PBANKA_0610300) highlighting the conservation of a CRL4-based complex in multiple stages of  the Plasmodium lifecycle. The orthologues of the WD containing protein WIG1 (PBANKA_1216700) and the ubiquitin carboxyl-terminal hydrolase (PbUCH7 -143 PBANKA_0210600) were also enriched. To confirm these interactions, we generated P. berghei lines expressing HA-tagged alleles of PbDDB1 (RMgm-5395) and PbWIG1(RMgm-5396).
We took advantage of the highly efficient PlasmoGEM vectors to attempt to delete the last 217 codons of the WIG1 gene (PBANKA_1216700) in P. berghei. A WIG1-GD line was readily obtained (RMgm-5394) and cloned suggesting no defect in the proliferation of asexual blood stages. Similarly, the WIG1-GD line produced microscopically normal female and male gametocytes, as assessed by Giemsa staining. However, upon activation by XA, no exflagellation events could be observed. 

Other mutants

  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1128600
Gene Model P. falciparum ortholog PF3D7_0629800
Gene productcullin-2, putative
Gene product: Alternative nameCUL4
Details of the genetic modification
Name of the tagtriple-HA
Details of taggingC-terminal
Additional remarks: tagging
Commercial source of tag-antibodies
Type of plasmid/construct(Linear) plasmid double 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/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationTriple HA or KO targeting vectors were generated using phage recombineering in Escherichia coli TSA strain with PlasmoGEM vectors (https://plasmogem.umu.se/pbgem/). For final targeting vectors not available in the PlasmoGEM repository, generation of knock-out and tagging constructs were performed using sequential recombineering and gateway steps as previously described. For each gene of interest (goi), the Zeocin-resistance/Phe-sensitivity cassette was introduced using oligonucleotides goi HA-F x goi HA-R and goi KO-F x goi KO-R for 3xHA, AID/HA tagging and KO targeting vectors, respectively. Insertion of the GW cassette following gateway reaction was confirmed using primer pairs GW1 x goi QCR1 and GW2 x goi QCR2. The modified library inserts were then released from the plasmid backbone using NotI. The GD and triple HA targeting vectors were transfected into the 2.34 line.
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