RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-4935
Malaria parasiteP. yoelii
Genotype
DisruptedGene model (rodent): PY17X_0405400; Gene model (P.falciparum): PF3D7_0304600; Gene product: circumsporozoite (CS) protein (CS; CSP)
Transgene
 Transgene not Plasmodium: GFP-Luciferase
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: PY17X_0306600; Gene product: 6-cysteine protein (P230p; 230)
Phenotype Oocyst; Sporozoite;
Last modified: 3 December 2020, 17:43
  *RMgm-4935
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) Not published (yet)
MR4 number
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Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. yoelii
Parent strain/lineP. y. yoelii 17XNL
Name parent line/clone RMgm-689
Other information parent lineThis mutant (1971cl1; RMgm-689) expresses the fusion protein GFP-Luciferase under the control of the constitutive P. berghei eef1a promoter. The GFP-Luc reporter cassette has been introduced into the silent p230p locus. The mutant does not contain a drug-selectable marker.
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The mutant parasite was generated by
Name PI/ResearcherFranke-Fayard B, Janse C.J
Name Group/DepartmentLeiden Malaria Group, Department of Parasitology
Name InstituteLeiden University Medical Center, LUMC
CityLeiden
CountryThe Netherlands
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Name of the mutant parasite
RMgm numberRMgm-4935
Principal name3194cl1
Alternative namePyXNL-CS GIMO
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
OocystNormal numbers of oocysts are produced in Anopheles stephensi mosquitoes. Sporozoite formation within the oocysts is profoundly inhibited: up to day eight after feeding of the mosquitoes the morphology of oocysts is normal; after day 10, the oocysts display a highly vacuolated structure. No sporozoite formation.
SporozoiteNo sporozoite formation in oocysts; No salivary gland sporozoites
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of CSP. In the mutant the endogenous P. yoelii csp gene has deleted by replacing the csp gene with the drug-selectable marker cassette hdhfr-yfcu. It also expresses GFP and luciferase under the constitutive eef1a promoter.

Protein (function)
The CS protein is the major protein on the surface of sporozoites and is critical for development of sporozoites within the oocysts and is involved in motility and invasion of both the salivary gland of the mosquito and the liver cells. The protein is also found on the oocyst plasma membrane and on the inner surface of the oocyst capsule. Specific motifs in CS are involved in sporozoite binding to mosquito salivary glands and in sporozoite attachment to heparan sulfate proteoglycans in the liver of the mammalian host. During substrate-dependent locomotion of sporozoites, CS is secreted at the sporozoite anterior pole, translocated along the sporozoite axis and released on the substrate at the sporozoite posterior pole. Following sporozoite invasion of hepatocytes, the CS is released in the host cell cytoplasm.

Phenotype
Normal numbers of oocysts are produced in Anopheles stephensi mosquitoes. Sporozoite formation within the oocysts is profoundly inhibited: up to day eight after feeding of the mosquitoes the morphology of oocysts is normal; after day 10, the oocysts display a highly vacuolated structure. No sporozoite formation. No sporozoite formation in oocysts; No salivary gland sporozoites.

Additional information

Other mutants

  Disrupted: Mutant parasite with a disrupted gene
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Details of the target gene
Gene Model of Rodent Parasite PY17X_0405400
Gene Model P. falciparum ortholog PF3D7_0304600
Gene productcircumsporozoite (CS) protein
Gene product: Alternative nameCS; CSP
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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/yfcu
Promoter of the selectable markerP. yoelii hsp70
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationWe deleted the Pycsp coding open reading frame (ORF) and replaced it with the positive-negative selectable marker, to create a P. yoelii csp deletion GIMO line (PyXNL-CS GIMO). In order to do this, we generated the pL2329 construct that is based on the standard GIMO DNA construct pL1980. This construct contains the positive-negative (hdhfr::yfcu) selection marker (SM) cassette and was used to insert both the Pycsp 5’ and 3’ gene targeting regions (TR), encompassing the full length promoter and transcription terminator sequences respectively. First we replaced the eef1α of the hdhfr::yfcu selectable marker (SM) cassette of the standard GIMO DNA construct pL0034 (MRA-849, www.beiresources.org) with the PCR amplified P. yoelii hsp70 promoter to create pL2137. Next we replaced the eef1α-hdhfr::yfcu selectable marker (SM) cassette from pL1980 with the hsp70-hdhfr::yfcu cassette from pL2137 by digestion of the plasmids with PstI and AgeI. Finally, the Pycsp 5’ and 3’ gene targeting regions were PCR amplified and ligated using KpnI/EcoRI and HindIII/PstI restriction sites to generate pL2329.
The construct was linearized using SacII and ScaI restriction sites outside of the 5’ and 3’ TRs before transfection. The construct pL2329 was used to transfect Py-GFP-Luceef1α (1971cl1) parasites using standard methods of GIMO-transfection. Transfected parasites were selected in mice by applying positive selection by providing pyrimethamine in the drinking water. Positive selection results in selection of transgenic parasites where the csp gene of Py-GFP-Luceef1α parasites is replaced by the hdhfr::yfcu SM cassette, resulting in line 3194. Transfected parasites of line 3194 were cloned by limiting dilution, resulting in the PyXNL-CS GIMO line (line 3194cl1)
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-Luciferase
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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 markereef1a
Selection (positive) procedureNo
Selection (negative) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modification
Additional remarks selection procedureThis reporter mutant expressing GFP-Luciferase does not contain a drug-selectable marker.
The mutant has been generated in the reference line GIMOPy17x (RMgm-688). The 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_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
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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 PY17X_0306600
Gene product6-cysteine protein
Gene product: Alternative nameP230p; 230
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