RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5190
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PBANKA_0701900; Gene model (P.falciparum): PF3D7_0828800; Gene product: GPI-anchored micronemal antigen (GAMA, Putative Secreted Ookinete Protein 9, PSOP9)
Name tag: mCherry
Transgene
Transgene not Plasmodium: A fusion of GFP (gfp-mu3) and Luciferase Firefly (LucIAV)
Promoter: Gene model: PBANKA_1133300; Gene model (P.falciparum): PF3D7_1357100; Gene product: elongation factor 1-alpha (eef1a)
3'UTR: Gene model: PBANKA_0719300; Gene product: bifunctional dihydrofolate reductase-thymidylate synthase, putative (dhfr/ts)
Replacement locus: Gene model: PBANKA_0306000; Gene product: 6-cysteine protein (230p)
Phenotype Asexual bloodstage; Fertilization and ookinete; Oocyst; Sporozoite;
Last modified: 15 June 2023, 15:57
  *RMgm-5190
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene tagging, Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 37314965
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 676m1cl1 (RMgm-29)
Other information parent line676m1cl1 (RMgm-29) is a reference ANKA mutant line which expresses GFP-luciferase under control of a constitutive promoter. This reference line does not contain a drug-selectable marker (PubMed: PMID: 16242190).
The mutant parasite was generated by
Name PI/ResearcherJennison C, Vaughan AM
Name Group/DepartmentCenter for Global Infectious Disease Research
Name InstituteSeattle Children’s Research Institute
CitySeattle
CountryUSA
Name of the mutant parasite
RMgm numberRMgm-5190
Principal nameGAMA::mCherry
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageImaging of mature blood-stage schizonts revealed mCherry signal predominantly localized in puncta toward the exterior facing end of merozoites, typical of an apical localization
Gametocyte/GameteNot tested
Fertilization and ookineteInternal localization of mCherry staining throughout ookinetes, frequently accumulated towards the wider apical end.
OocystAt day seven, using a long exposure time, weak mCherry signal was visible in GAMA-mCherry oocysts. No significant increase in mCherry signal was observed until day 11, when a subset of oocysts became brightly mCherry positive. mCherry was first localized within the nascent apical tip of developing sporozoites inside oocysts. mCherry localization becomes less restricted to the apical tip as sporozoites matured replicating the tip-backwards maturation of micronemes within developing sporozoites.
SporozoiteLive-imaging of midgut sporozoites recovered from mechanically ruptured mosquito midguts found GAMA-mCherry to be localized within sporozoites, occupying the same space as cytoplasmic GFP, with a somewhat punctate localization.
Salivary gland sporozoites exhibited a localization of GAMA-mCherry similar to that observed in midgut sporozoites; however, there was an increase in the abundance of protein.
Evidence is presented that GAMA is concentrated at the apical tip of sporozoites where it is then translocated to the sporozoite surface and shed during the process of gliding motility.
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses a C-terminal mCherry-tagged version of GAMA and expresses the reporter GFP-Luciferase under the constitutive eefia promoter.

Protein (function)
GAMA is a micronemal protein with a C-terminal GPI-anchor. P. berghei GAMA consists of 625 aa with a predicted molecular mass of ~71 kDa, (signal peptide cleavage results in a 69 kDa mass). In P. falciparum, two additional asparagine rich repeat regions result in a larger 738 aa protein (predicted molecular weight ~85 kDa, 83 kDa after cleavage of the signal peptide). For P. berghei, SignalP-5.0 predicts a signal peptide with a cleavage site at S21/L22. For P.falciparum, cleavage is predicted at the same locus, A21/L22.
P. berghei and P. falciparum share 44.8% identity across the full-length sequence, with areas of higher conservation found towards the N-terminus (residues 64-347 = 60% identity) and C-terminus (residues 501-700 = 60% identity) split by a central repeat region of low shared identity (residues 348-500, with predicted disorderly structure. The C-terminal transmembrane domain is predicted to be cleaved and appended with a GPI anchor. Downstream of signal peptide cleavage, P. falciparum contains seven cysteine residues prior to the central repeat region and one downstream which are conserved across all Plasmodium species investigated. P. berghei and other rodent infective species of Plasmodium bear an additional cysteine at position 152 in the alignment. GAMA is present in all Plasmodium species and indeed all other intracellular apicomplexans.

Phenotype
Imaging of mature blood-stage schizonts revealed mCherry signal predominantly localized in puncta toward the exterior facing end of merozoites, typical of an apical localization.
Internal localization of mCherry staining throughout ookinetes, frequently accumulated towards the wider apical end.
In oocysts, at day seven, using a long exposure time, weak mCherry signal was visible . No significant increase in mCherry signal was observed until day 11, when a subset of oocysts became brightly mCherry positive. mCherry was first localized within the nascent apical tip of developing sporozoites inside oocysts. mCherry localization becomes less restricted to the apical tip as sporozoites matured replicating the tip-backwards maturation of micronemes within developing sporozoites. 
Live-imaging of midgut sporozoites recovered from mechanically ruptured mosquito midguts found GAMA-mCherry to be localized within sporozoites, occupying the same space as cytoplasmic GFP, with a somewhat punctate localization. 
Salivary gland sporozoites exhibited a localization of GAMA-mCherry similar to that observed in midgut sporozoites; however, there was an increase in the abundance of protein.
Evidence is presented that GAMA is concentrated at the apical tip of sporozoites where it is then translocated to the sporozoite surface and shed during the process of gliding motility.

Additional information
Further phenotypic analysis of GAMA-mCherry revealed no detectable changes in the number of oocysts per midgut, the number of sporozoites per midgut, the number of sporozoites per salivary gland, and the infectiousness of these sporozoites to mice. In light of the strong phenotypes observed across these life cycle stages in a mutant lacking GAMA expression (ΔGAMA; RMgm-5188) parasites we concluded that the tagging strategy did not interfere with the function of GAMA

Other mutants


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0701900
Gene Model P. falciparum ortholog PF3D7_0828800
Gene productGPI-anchored micronemal antigen
Gene product: Alternative nameGAMA, Putative Secreted Ookinete Protein 9, PSOP9
Details of the genetic modification
Name of the tagmCherry
Details of taggingC-terminal
Additional remarks: tagging
Commercial source of tag-antibodies
Type of plasmid/constructCRISPR/Cas9 construct: integration through double strand break repair
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 modificationA CRISPR/Cas9 methodology was used to generate tagged parasites. For mCherry tagging, homology arms for the tagging construct were designed so that the tag would be inserted between bases 1818-1819 of the ORF, corresponding to an insertion 19 aa from the end of the GAMA protein, 5 residues downstream of the predicted GPI anchoring site. Four guides were designed close to the intended insertion site of the tag with multiple shield mutations built into the repair construct. Of these guides, guides 9 and 42 were used in combination. Repair constructs were synthesized by Genscript and ligated into the pYC plasmid by restriction cloning with KpnI and NotI.

Single stranded guide primers were annealed in a 50 μL volume (10 μL of each 100 μM primer with 25 μL water and 5 μL cutsmart buffer), by heating to 95 ºC for 5 minutes and allowing to cool to room temperature. Annealed guides were inserted into the pYC-L2 plasmid, via restriction cloning with the enzyme Esp3I. Guide integration was confirmed via sanger sequencing with the primer CJ059.
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

  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameA fusion of GFP (gfp-mu3) and Luciferase Firefly (LucIAV)
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
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 parasitegfp (FACS)
Promoter of the selectable markereef1a
Selection (positive) procedureFACS (flowsorting)
Selection (negative) procedureNo
Additional remarks genetic modificationThe GFP-Luciferase gene (1 copy) has been inserted into the 230p locus (PB000214.00.0) by double cross-over integration.
Additional remarks selection procedureThis reporter mutant expressing GFP-Luciferase does not contain a drug-selectable marker. This mutant has been selected by FACS sorting after transfection based on GFP fluorescence.
Other details transgene
Promoter
Gene Model of Parasite PBANKA_1133300
Gene Model P. falciparum ortholog PF3D7_1357100
Gene productelongation factor 1-alpha
Gene product: Alternative nameeef1a
Primer information details of the primers used for amplification of the promoter sequence  Click to view information
Primer information details of the primers used for amplification of the promoter sequence  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
3'-UTR
Gene Model of Parasite PBANKA_0719300
Gene productbifunctional dihydrofolate reductase-thymidylate synthase, putative
Gene product: Alternative namedhfr/ts
Primer information details of the primers used for amplification the 3'-UTR sequences  Click to view information
Primer information details of the primers used for amplification the 3'-UTR sequences  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
Insertion/Replacement locus
Replacement / InsertionReplacement locus
Gene Model of Parasite PBANKA_0306000
Gene product6-cysteine protein
Gene product: Alternative name230p
Primer information details of the primers used for amplification of the target sequences  Click to view information
Primer information details of the 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