RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5156
Malaria parasiteP. berghei
Genotype
MutatedGene model (rodent): PBANKA_0931200; Gene model (P.falciparum): PF3D7_1116800; Gene product: heat shock protein 101 (HSP101)
Details mutation: The promoter of hsp101 replaced by the promoter of ptex150
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_1340000; Gene product: dihydrofolate synthase/folylpolyglutamate synthase, putative (PbDHFS-FPGS)
Replacement locus: Gene model: Not available; Gene product: Not available
PhenotypeNo phenotype has been described
Last modified: 4 February 2022, 14:47
  *RMgm-5156
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene mutation, Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 34956312
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/ResearcherKreutzfeld O, Matuschewski K
Name Group/DepartmentMolecular Parasitology, Institute of Biology/Faculty for Life Sciences
Name InstituteHumboldt Universität zu Berlin
CityBerlin
CountryGermany
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Name of the mutant parasite
RMgm numberRMgm-5156
Principal nameHSP101:5′ptex150
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 tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot different from wild type
Liver stageNot different from wild type
Additional remarks phenotype

Mutant/mutation
In the 'promoter swap mutant': the promoter of hsp101 has been replaced with the asexual blood stage promoter ptex150 (PBANKA_1008500). The hsp101promoter is only active in blood stages and not in liver stages. The ptex150 promoter is  active in both blood stages and in liver stages.
In addition the mutant expreses GFP under control of the hsp70 promoter.

Protein (function)
Plasmodium parasites remodel their vertebrate host cells by translocating hundreds of proteins across an encasing membrane into the host cell cytosol via a putative export machinery termed PTEX (Plasmodium Translocon of EXported protein). HSP101 (PbANKA_094120), PTEX150 (PbANKA_100850), EXP2 (PbANKA_133430), PTEX88 (PbANKA_094130) and TRX2 (PbANKA_135800) have been identified as members of the PTEX complex. In contrast to most PTEX proteins, HSP101 is only expressed in blood stages and not in liver stages.  PTEX150 is expressed in both blood and liver stages.

Phenotype
Normal (wild type) development throughout the complete life cycle (see also 'Additional information' below).  

Additional information
Evidence is presented that  expression of HSP101 in liver stages (by the promoter-swap with the promoter of ptex150) did not result in export of PEXEL-containing proteins during liver stage development, indicating that HSP101 is not a limiting factor for PEXEL-dependent protein export during this life cycle stage.

In this paper  additional mutants are generated that express an additional copy of PbHSP101 (PbANKA_0931200) under the constitutive eef1a promoter inserted into a silent locus on chromosome 6 (RMgm-5155). The additional copy of HSP101 is C-terminally tagged with either 3xcmyc (mutant ef1α:HSP101-myc) or mCherry (mutant ef1α:HSP101-mCherry).

Other mutants

  Mutated: Mutant parasite with a mutated gene
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Details of the target gene
Gene Model of Rodent Parasite PBANKA_0931200
Gene Model P. falciparum ortholog PF3D7_1116800
Gene productheat shock protein 101
Gene product: Alternative nameHSP101
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Details of the genetic modification
Short description of the mutationThe promoter of hsp101 replaced by the promoter of ptex150
Inducable system usedNo
Short description of the conditional mutagenesisNot available
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/yfcu
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationFor swap of the endogenous promoter with the 5′ region of PTEX150 a pBARTbased plasmid containing the PTEX150 promoter linking HSP101 ORF was generated. In brief, the amino-terminal portion of the HSP101 ORF (primers HSP101_amino_F, HSP101_amino_R) was integrated under the control of PTEX150 promoter (primers PTEX150-5′-PrimerF, PTEX150-5′-PrimerR) integrated with SacII and HpaI. The 5′UTR of HSP101 amplified with HSP101-5′-F and HSP101-5′-R. The 5′UTR together with the HSP101 amino-terminus served as homologous recombination sites.
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/yfcu
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationFor swap of the endogenous promoter with the 5′ region of PTEX150 a pBARTbased plasmid containing the PTEX150 promoter linking HSP101 ORF was generated. In brief, the amino-terminal portion of the HSP101 ORF (primers HSP101_amino_F, HSP101_amino_R) was integrated under the control of PTEX150 promoter (primers PTEX150-5′-PrimerF, PTEX150-5′-PrimerR) integrated with SacII and HpaI. The 5′UTR of HSP101 amplified with HSP101-5′-F and HSP101-5′-R. The 5′UTR together with the HSP101 amino-terminus served as homologous recombination sites.
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_1340000
Gene productdihydrofolate synthase/folylpolyglutamate synthase, putative
Gene product: Alternative namePbDHFS-FPGS
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 Not available
Gene productNot available
Gene product: Alternative name
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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P.berghei model of malaria
Technical design and realization: S. Mededovic and A. van Wigcheren