RMgmDB - Rodent Malaria genetically modified Parasites


Malaria parasiteP. berghei
TaggedGene model (rodent): PBANKA_1422900; Gene model (P.falciparum): PF3D7_0814600; Gene product: PIMMS22 protein, putative (PIMMS22)
Name tag: GFP
Phenotype Fertilization and ookinete; Sporozoite;
Last modified: 24 May 2021, 15:50
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) : 33791240
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/ResearcherUkegbu CV, Vlachou D
Name Group/DepartmentDepartment of Life Sciences
Name InstituteImperial College London
Name of the mutant parasite
RMgm numberRMgm-5002
Principal namec22::gfp
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Asexual blood stageNot different from wild type
Gametocyte/GameteNot different from wild type
Fertilization and ookineteSee additional information below
OocystNot tested
SporozoiteNormal sporozoite production. See additional information below for GFP expression
Liver stageNot tested
Additional remarks phenotype

The mutant c22::gfp lacks expresses a C-terminal GFP-tagged version of PIMMS22

Protein (function)
PfPIMMS22 encodes a 393 amino acid (45 kDa) protein and PbPIMMS22 encodes a 393 aa (44 kDa) protein, both with no predicted signal peptide or transmembrane domain. The protein is highly conserved amongst Plasmodium orthologues. PyPIMMS22 (PY17X_1424900) was previously identified in salivary gland sporozoites through a  subtractive hybridization (SSH) profiling and termed sporozoite protein S15. The same protein was also identified in midgut oocyst sporozoites as an interacting partner to the apicomplexan specific RNA-binding protein, ALBA4, which is involved in mRNA regulation in gametocyte and midgut oocyst sporozoite development. PIMMS22 homologues are found in other apicomplexan parasites including Toxoplasma gondii, Neospora caninum and Eimeria with sequence identities to PIMMS22 ranging from 36% to 42%. InterPro domain analysis revealed no recognizable domain in PIMMS22.

See below for details of GFP-expression
See RMgm-4999 for a mutant lacking expression of PIMMS22, showing reduced oocyst and sporozoite formation

Additional information
Three genes were selected for analysis that exhibit highly abundant transcripts 24 h post blood feeding in the midguts of Anopheles coluzzii mosquitoes.
PIMMS01: PF3D7_0112100; PBANKA_0201700 
PIMMS57: PF3D7_1244500; PBANKA_1457700
PIMMS22: PF3D7_0814600; PBANKA_1422900 
((PIMMS: Plasmodium Infection of the Mosquito Midgut Screen)

PbPIMMS01 and PbPIMMS57 transcripts were highly abundant in purified mature ookinetes and not detectable in mixed blood stages and purified gametocytes. While PbPIMMS57 appears to be specific for ookinetes, low levels of PbPIMMS01 transcripts are also detected in mature oocysts and midgut sporozoites at 10 days and 15 days post blood-feeding. Like PfPIMMS22, expression of PbPIMMS22 starts in gametocytes and continues at high levels at 24 hour and peaks in midgut sporozoites at day 15.

Western blot analysis using the c22::gfp transgenic parasite line and a-GFP antibody revealed high levels of the PbPIMMS22::GFP fusion protein in ookinete and oocyst derived sporozoites, at the expected molecular weight of 71 kDa. This band was not detected in the ANKA 2.34 background parasite line. IFAs on the c22::gfp parasites allowed us to analyze the sub-cellular localization of the fusion protein. In Triton X-100 permeabilized blood stage gametocytes, PbPIMMS22::GFP was found to be localized on the cell periphery with the majority of the protein localizing in the cytoplasm. Non-Triton X-100 permeabilized blood stage gametocytes are visibly less fluorescent than their Triton X-100 permeabilized counterparts. In Triton X100 permeabilized in vitro ookinetes, PbPIMMS22::GFP was clearly observed on the ookinete periphery, a result that is inconsistent with the predicted absence of a signal peptide or transmembrane domain. Additional staining on non-Triton X-100 permeabilized ookinetes show some peripheral signal albeit much weaker than its Triton X-100 permeabilized counterpart suggesting that PbPIMMS22::GFP is mainly localized on the inner surface of the ookinete and not on the plasma membrane. IFAs on in vivo midgut epithelium invading ookinetes at 26 hpbf also show surface localization of PbPIMMS22::GFP. In midgut sporozoites, PbPIMMS22::GFP is found on the surface of both Triton X-100 and non-Triton X-100 permeabilized sporozoites.

From the abstract:
PIMMS01 and PIMMS57 are specifically and highly expressed in ookinetes, while PIMMS22 transcription starts already in gametocytes and peaks in sporozoites. All three genes show strong phenotypes associated with the ookinete to oocyst transition, as their disruption leads to very low numbers of oocysts and complete abolishment of transmission. PIMMS22 has a secondary essential function in the oocyst. 

Other mutants


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1422900
Gene Model P. falciparum ortholog PF3D7_0814600
Gene productPIMMS22 protein, putative
Gene product: Alternative namePIMMS22
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 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 parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationFor GFP tagging of Pbc01 in the 2.34 line, a 603 bp ApaI/HindIII 5’ and a 770 bp EcoRI/BamHI 3’ homology arm region were amplified from P. berghei 2.34 genomic DNA using the primer pairs P1/P2 and P3/P4, respectively. For GFP tagging of Pbc57 in the 2.34 line, a 919 bp ApaI/SacII 5’ homology arm corresponding to the most 3’ region of the CDS without the stop codon and a 359 bp XhoI/XmaI 3’ homology arm region corresponding to the 3’ UTR of the gene were amplified using the primer pairs P5/P6 and P7/P8, respectively. For GFP tagging of Pbc22 in the 2.34 line, a 753 bp ApaI/HindIII 5’ homology arm corresponding to the most 3’ region of the CDS without the stop codon and a 530 bp EcoRI/BamHI 3’ homology arm region corresponding to the 3’ UTR of the gene were amplified using the primer pairs P9/P10 and P11/P12, respectively. The Pbc01 and Pbc22 fragments were cloned into the pBS-TgDHFR vector which carries a modified Toxoplasma gondii dihydrofolate gene (TgDHFR/TS) cassette that confers resistance to pyrimethamine (Dessens et al., 1999). The Pbc57 fragments were cloned into plasmid pL0035 which carries the hDHFR selection cassette. Finally, to put GFP tag in frame with the 3’ region of the CDS, a HindIII or SacII GFP- P. berghei DHFR 3’UTR fragment was amplified from the pL00018 vector (MRA787, MR4) using primers P13/P14 or P15/P16, respectively.
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