RMgmDB - Rodent Malaria genetically modified Parasites


Malaria parasiteP. berghei
TaggedGene model (rodent): PBANKA_1446100; Gene model (P.falciparum): Pf3D7_ 1231400; Gene product: membrane protein ICM1 (ICM1)
Name tag: triple-HA
Phenotype Asexual bloodstage; Gametocyte/Gamete;
Last modified: 18 May 2021, 17:54
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) : 33762339
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/ResearcherBalestra AC, Brochet M
Name Group/DepartmentDepartment of Microbiology and Molecular Medicine, Faculty of Medicine
Name InstituteUniversity of Geneva
Name of the mutant parasite
RMgm numberRMgm-4993
Principal namePbICM1-HA3
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Asexual blood stageSee phenotype and additional information below
Gametocyte/GameteSee phenotype and additional information below
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

The mutant expresses a 3xHA-tagged version of ICM1

Protein (function)
See additional information below.

No signal was observed for PbICM1-HA3 in Western blot analysis of schizont or gametocyte extracts from this line, possibly due to low abundance of PbICM1. Consistent with this observation, no specific immunofluorescence signal could be observed in both schizonts and gametocytes. Nevertheless, immunoprecipitation (IP) of PbICM1-HA3 from extracts of both schizonts and gametocytes recovered multiple peptides, identified by MS to encompass up to 28% coverage of PbICM1. As expected, based on the PbPKG IPs, PbPKG was the most enriched protein coimmunoprecipitated with PbICM1-HA3 in both stages. Notably, no peptides from PbCDPK1 or PbCDPK4 were recovered from either immunoprecipitate, confirming that these kinases do not interact with PbPKG or PbICM1 as previously suggested.

Additional information
From the paper:
'We identify a multipass membrane protein, termed important for calcium mobilization-1 (ICM1; Pf3D7_1231400) that interacts with and is phosphorylated by PKG in two different Plasmodium developmental stages and species. Through stage-specific knockdown and reverse genetic approaches, we show that ICM1 is essential for calcium mobilization in both the clinically relevant asexual blood stages and in gametocytes that mediate transmission to mosquitoes. Our findings highlight this putative transporter or channel as a crucial link between PKG function and calcium signaling'.
PKG is involved in release of merozoites from red blood cells and hepatocytes, induction of gametogenesis and parasite transmission upon ingestion of gametocytes by a blood-feeding mosquito, and sustaining cellular motility necessary for parasite dissemination. A major function of PKG in all these processes is the tightly regulated mobilization of calcium from intracellular stores within seconds of activation

In the paper evidence is presented that:
- ICM1 may represent a structurally divergent polytopic membrane protein with architectural similarities to mammalian channels, transporters, and IP3 receptors. 
- ICM1 is phosphorylated in a PKG-dependent manner 
- Stage-specific knockdown of PbICM1 reveals a crucial role in early calcium mobilization required to initiate gametogenesis

Other mutants

  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1446100
Gene Model P. falciparum ortholog Pf3D7_ 1231400
Gene productmembrane protein ICM1
Gene product: Alternative nameICM1
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) PCR construct double cross-over
PlasmoGEM (Sanger) construct/vector usedYes
Name of PlasmoGEM construct/vectorPbGEM-121338
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 modificationICM1-HA3 and ICM1-AID/HA tagging constructs were generated using phage recombineering in Escherichia coli TSA strain with PlasmoGEM vectors (http://plasmogem.sanger.ac.uk/). The modified library inserts were then released from the plasmid backbone using Not I. The ICM1-AID/HA (PbGEM-645751) targeting vector was transfected into the 615 line. The ICM1-HA3 (PbGEM-121338) vector was 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