RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-4460
Malaria parasiteP. berghei
Genotype
Transgene
 Transgene Plasmodium: Gene model: PF3D7_1346700; Gene model (P.falciparum): PF3D7_1346700; Gene product: 6-cysteine protein; transmission blocking target antigen precursor (P48/45; Pfs48/45)
Promoter: Gene model: PBANKA_0831000; Gene model (P.falciparum): PF3D7_0930300; Gene product: merozoite surface protein 1 (MSP1)
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 Asexual bloodstage;
Last modified: 14 August 2018, 17:55
  *RMgm-4460
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 30053393
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 GIMO-PbANKA (RMgm-687)
Other information parent lineGIMO-PbANKA (RMgm-687) contains as a selectable marker (SM) the fusion gene of hdhfr (human dihydrofolate reductase; positive SM) and yfcu (yeast cytosine deaminase and uridyl phosphoribosyl transferase; negative SM) stably integrated into the 230p locus (PBANKA_030600) through double cross-over recombination. The SM is under control of the P. berghei eef1α promoter. This reference line of P. berghei ANKA line is used for rapid introduction of transgenes free of drug-resistance genes (PubMed: PMID: 22216235).
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The mutant parasite was generated by
Name PI/ResearcherOthman AS, Janse CJ, Khan SM
Name Group/DepartmentLeiden Malaria Research Group, Parasitology
Name InstituteLeiden University Medical Center (LUMC)
CityLeiden
CountryThe Netherlands
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Name of the mutant parasite
RMgm numberRMgm-4460
Principal name1807cl2
Alternative namePfs48/45@PbMSP1
Standardized name
Is the mutant parasite cloned after genetic modificationYes
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Phenotype
Asexual blood stagePfs48/45@PbMSP1 schizont-infected red blood cells produced full length Pfs48/45
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The P48/45 gene of P. falciparum is introduced as a transgene into the neutral 230p locus under control of the P. berghei (schizont-specific) msp1 promoter

Protein (function)
The P48/45 protein is a member of a small family of proteins, the 6-cysteine (cys) family of (surface) proteins. P48/45 is a surface protein of both male and female gametes and is considered as a major candidate antigen for development of a transmission blocking vaccine. P. berghei P48/45 plays an important role in fertilization (male fertility factor). Motile males fail to attach to and penetrate female gametes.

Phenotype
Pfs48/45@PbMSP1 schizont-infected red blood cells produced full length Pfs48/45

Additional information
The structural integrity of Pfs48/45 was confirmed using a panel of conformation-specific monoclonal antibodies that bind to different Pfs48/45 epitopes. Sera from mice immunized with transgenic Pfs48/45@PbMSP1 schizonts showed strong transmission-reducing activity in mosquitoes infected with P. falciparum using standard membrane feeding. These results demonstrate that transgenic rodent malaria parasites expressing human malaria antigens may be used as means to evaluate immunogenicity and functionality of difficult to express malaria vaccine candidate antigens.

Other mutants

  Transgene: Mutant parasite expressing a transgene
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Type and details of transgene
Is the transgene Plasmodium derived Transgene: Plasmodium
Gene Model of Parasite PF3D7_1346700
Gene Model P. falciparum ortholog PF3D7_1346700
Gene product6-cysteine protein; transmission blocking target antigen precursor
Gene product: Alternative nameP48/45; Pfs48/45
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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 parasiteNo selectable marker
Promoter of the selectable markerNo
Selection (positive) procedureNo
Selection (negative) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modificationTo introduce the Pfs48/45 gene (PF3D7_1346700) into the redundant p230p gene locus (PBANKA_0306000) of the Pb genome, we generated DNA construct pL1706. The basic gene insertion construct pL0046 was used, which contains the 5’ and 3’ 230p targeting regions, the tgdhfr/ts selectable marker (SM) cassette and an mCherry expression cassette under the control of the eef1α promoter with 3’ terminal sequence of pbdhfr/ts. The eef1α promoter was replaced by the msp1 promoter (PBANKA_0831000) using AflII and BamHI digestion. The msp1 promoter was amplified from genomic Pb ANKA DNA using primers 6145 and 6146. In addition the mCherry coding sequence (CDS) was replaced by the Pfs48/45 CDS using BamHI and SgrAI digestion. The Pfs48/45 CDS was amplified from genomic DNA of the Pf NF54 strain using primers 5583 and 5584. This resulted in construct pL1706. In order to introduce the expression construct in the genome of the parent GIMO PbANKA line (1596cl1) , we next removed the tgdhfr/ts SM by digestion of the plasmid with SbfI and AflII. The ends of the linearized constructs were then rendered blunt using Klenow enzyme treatment, and re-ligated. This final construct (pL1707) were analyzed via restriction
digestions to confirm correct assembly. Before transfection, the construct pL1707 was linearized by digesting the plasmid with KspI.
Additional remarks selection procedureThe GIMO mother line is used for introduction of transgenes into the modified 230p locus through transfection with constructs that target the 230p locus. These constructs insert into the 230p locus (‘gene insertion’), thereby removing the hdhfr::yfcu selectable marker (‘marker out’) from the genome of the mother lines. Transgenic parasites that are marker-free are subsequently selected by applying negative drug selection using 5-FC. This selection procedure is performed in vivo in mice.
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Other details transgene
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Promoter
Gene Model of Parasite PBANKA_0831000
Gene Model P. falciparum ortholog PF3D7_0930300
Gene productmerozoite surface protein 1
Gene product: Alternative nameMSP1
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_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
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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