RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5194
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_1422900; Gene model (P.falciparum): PF3D7_0814600; Gene product: PIMMS22 protein, putative (SIMP, sporozoite structural integrity maintenance protein)
Transgene
 Transgene not Plasmodium: GFP
Promoter: Gene model: PBANKA_0711900; Gene model (P.falciparum): PF3D7_0818900; Gene product: heat shock protein 70 (HSP70)
3'UTR: Gene model: PBANKA_0711900; Gene product: heat shock protein 70 (HSP70)
Replacement locus: Gene model: PBANKA_1422900; Gene product: PIMMS22 protein, putative (SIMP, sporozoite structural integrity maintenance protein)
Phenotype Sporozoite; Liver stage;
Last modified: 7 April 2022, 17:24
  *RMgm-5194
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption, Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 35301756
MR4 number
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Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone Not applicable
Other information parent line
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The mutant parasite was generated by
Name PI/ResearcherSingh D, Kumar KA
Name Group/DepartmentDepartment of Animal Biology, School of Life Sciences
Name InstituteUniversity of Hyderabad
CityHyderabad
CountryIndia
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Name of the mutant parasite
RMgm numberRMgm-5194
Principal nameSIMP knockout mutant
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
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Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNot different from wild type
Fertilization and ookineteNot different from wild type
OocystNot different from wild type
SporozoiteNormal/wildtype oocyst development, morphology of oocysts sporozoites and numbers of salivary gland sporozoites. Salivary gland sporozoites showed aberrant morphology and did not show gliding motility (loss of regular shape, extensive membrane blebbing at multiple foci and membrane detachment while organization of cytoskeleton or inner membrane complex (IMC) were not affected).
Liver stageSporozoites were unable to infect mice (after intravenous injection of purified sporozoites).
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of PIMMS22/SIMP and expresses GFP under control of the constitutive hsp70 promoter

Protein (function)
PfPIMMS22 encodes a 393 amino acid (45 kDa) protein and PbPIMMS22/SIMP 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.

Phenotype
Normal/wildtype oocyst development, morphology of oocysts sporozoites and numbers of salivary gland sporozoites. Salivary gland sporozoites showed aberrant morphology and did not show gliding motility (loss of regular shape, extensive membrane blebbing at multiple foci and membrane detachment while organization of cytoskeleton or inner membrane complex (IMC) were not affected). Sporozoites were unable to infect mice (after intravenous injection of purified sporozoites).

See also mutants RMgm-4999 and RMgm-5127 lacking expression of SIMP (PIMS22/concavin) with (slightly) different phenotypes
 
Additional information
From the paper: 'To demonstrate that the observed phenotype was specific to SIMP depletion, we restored  the  gene  function  by  complementation.  Towards  this,  we  generated  a  PbSIMP GIMO line by replacing PbSIMPlocus with a positive-negative selectable marker cassette,hDHFR:yFCU. The GIMO line was further complemented with copy of PbSIMP open reading frame. The PbSIMP complemented line produced salivary gland sporozoites similar to WT and were motile'.

Evidence is presented for a location if SIMP at the sporozoite surface membrane (see also RMgm-5195).

Other mutants
See also mutants RMgm-4999 and RMgm-5127 lacking expression of SIMP (PIMS22/concavin) with slightly different phenotypes
 

  Disrupted: Mutant parasite with a disrupted gene
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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 nameSIMP, sporozoite structural integrity maintenance protein
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Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct used(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
Partial or complete disruption of the geneComplete
Additional remarks partial/complete disruption
Selectable marker used to select the mutant parasitehdhfr
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modification
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
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  Transgene: Mutant parasite expressing a transgene
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Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameGFP
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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 parasitehdhfr
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modification
Additional remarks selection procedure
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Other details transgene
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Promoter
Gene Model of Parasite PBANKA_0711900
Gene Model P. falciparum ortholog PF3D7_0818900
Gene productheat shock protein 70
Gene product: Alternative nameHSP70
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
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3'-UTR
Gene Model of Parasite PBANKA_0711900
Gene productheat shock protein 70
Gene product: Alternative nameHSP70
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_1422900
Gene productPIMMS22 protein, putative
Gene product: Alternative nameSIMP, sporozoite structural integrity maintenance protein
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
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Conceptual design of the database: Dr. C.J. Janse (Leiden Malaria Research Group, Leiden, The Netherlands).
Technical design and realization: S. Mededovic and A. van Wigcheren