RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5157
Malaria parasiteP. berghei
Genotype
Transgene
Transgene not Plasmodium: murine IL6
Promoter: Gene model: PBANKA_1003000; Gene model (P.falciparum): PF3D7_0405300; Gene product: liver specific protein 2, putative | sequestrin | 6-cysteine protein (LISP2)
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 (P230p)
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_0719300; Gene product: bifunctional dihydrofolate reductase-thymidylate synthase, putative (dhfr/ts)
Replacement locus: Gene model: PBANKA_0306000; Gene product: 6-cysteine protein (P230p)
Phenotype Liver stage;
Last modified: 4 February 2022, 17:00
  *RMgm-5157
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Introduction of a transgene, Introduction of a transgene
Reference (PubMed-PMID number) Not published (yet)
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone RMgm-1026
Other information parent lineThe mutant expresses GFP under the control of the HSP70 promoter. The reporter gene is introduced into the silent 230p locus using the GOMO method of transfection and is drug-selectable marker-free.
The mutant parasite was generated by
Name PI/ResearcherBelhimeur S, Silvie S, Mecheri S
Name Group/DepartmentInstitut Pasteur, Université de Paris
Name InstituteCNRS
CityParis
CountryFrance
Name of the mutant parasite
RMgm numberRMgm-5157
Principal nameIL-6 Tg-PbANKA/LISP2 line1,2
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationNo
Phenotype
Asexual blood stageNot tested
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot different from wild type
SporozoiteNot different from wild type
Liver stageNo blood-stage infection developed in mice after infecting with isolated sporozoites.
Mutant sporozoites invade and develop in cultured hepatocytes (complete liver stage development and egress) but in mice arrest during liver stage development. In mice the mutant parasite liver load remained low over the course of infection, except at the 48-h time point, when the amount of mutant parasites rose but at levels significantly lower than those obtained for wild-type parasites.
Additional remarks phenotype

Mutant/mutation
The mutant expresses murine IL6. a codon optimized IL-6 gene comprising amino acids 25-211 of murine IL-6 was fused to the signal peptide of P. berghei liver-specific protein 2 (LISP2), to enable secretion of the protein. The IL-6 gene is under control of the LISP2 promoter. The IL6 expression cassette is integrated into the neutral p230p locus. In addition the mutant expresses GFP under the hsp70 promoter.

Published in: bioRxiv preprint doi: https://doi.org/10.1101/2021.11.16.468835 

Protein (function)

IL-6 may act as a critical pro-inflammatory signal from the host in response to the parasite infection

Phenotype
No blood-stage infection developed in mice after infecting with isolated sporozoites.
Mutant sporozoites invade and develop in cultured hepatocytes (complete liver stage development and egress) but in mice arrest during liver stage development. In mice the mutant parasite liver load remained low over the course of infection, except at the 48-h time point, when the amount of mutant parasites rose but at levels significantly lower than those obtained for wild-type parasites.

Additional information
Evidence is presented that:
- IL-6 Tg-PbANKA/LISP2 parasites express IL-6 mRNA and secrete the murine IL-6 cytokine
- Immunization of mice with IL-6 Tg-PbANKA/LISP2 sporozoites elicited a long-lasting CD8+ T cell-mediated protective immunity against a subsequent infectious sporozoite challenge.

Other mutants


  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene namemurine IL6
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 markerunknown
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationTo generate transgenic P. berghei parasites expressing the murine IL6 transgene, we used a selection-linked integration strategy. We assembled a plasmid construct, named GFP-SLI-IL6, containing two cassettes. The first cassette includes a 3’ terminal sequence of the GFP coding sequence, fused to a 2A skip peptide and the human dihydrofolate reductase (hDHFR) gene, followed by the 3’ UTR of P. berghei calmodulin (CAM) gene. The second cassette corresponds to a codon-optimized version of murine IL6, under control of the promoter of P. berghei LISP2, and followed by the 3’ UTR of P. berghei DHFR. To ensure IL6 secretion, the peptide signal peptide of mIL6 was replaced by the signal peptide of P. berghei LISP2.
Additional remarks selection procedure
Other details transgene
Promoter
Gene Model of Parasite PBANKA_1003000
Gene Model P. falciparum ortholog PF3D7_0405300
Gene productliver specific protein 2, putative | sequestrin | 6-cysteine protein
Gene product: Alternative nameLISP2
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 nameP230p
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

  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameGFP
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 markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationTo generate transgenic P. berghei parasites expressing the murine IL6 transgene, we used a selection-linked integration strategy. We assembled a plasmid construct, named GFP-SLI-IL6, containing two cassettes. The first cassette includes a 3’ terminal sequence of the GFP coding sequence, fused to a 2A skip peptide and the human dihydrofolate reductase (hDHFR) gene, followed by the 3’ UTR of P. berghei calmodulin (CAM) gene. The second cassette corresponds to a codon-optimized version of murine IL6, under control of the promoter of P. berghei LISP2, and followed by the 3’ UTR of P. berghei DHFR. To ensure IL6 secretion, the peptide signal peptide of mIL6 was replaced by the signal peptide of P. berghei LISP2.
Additional remarks selection procedure
Other details transgene
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
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 nameP230p
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