RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-4944
Malaria parasiteP. yoelii
Genotype
MutatedGene model (rodent): PY17X_0405400; Gene model (P.falciparum): PF3D7_0304600; Gene product: circumsporozoite (CS) protein (CSP)
Details mutation: The P. yoelii csp gene replaced by P. falciparum 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)
PhenotypeNo phenotype has been described
Last modified: 8 December 2020, 16:48
  *RMgm-4944
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) 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-4935
Other information parent lineIn this mutant (3194cl1; RMgm-4935; PyXNL-CS GIMO) the endogenous P. yoelii csp gene has been 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.
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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-4944
Principal name3226 (after mosquito passage)
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationNo
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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
SporozoiteNot different from wild type
Liver stageNot different from wild type
Additional remarks phenotype

Mutant/mutation
In the mutant the endogenous P. yoelii csp gene has been replaced by the P. falciparum csp gene. This has been performed by the GIMO method of transfection. The P. falciparum csp gene is under control of the 5'- and 3'-UTR regions of the P. yoelii csp gene. The mutant does not contain a drug-selectable marker. The mutant also expresses the fusion protein GFP-Luciferase under control of the constitutive eefia 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 development throughout the complete life cycle showing complementation of P. yoelii CSP by P. falciparum CSP

Additional information

Other mutants

  Mutated: Mutant parasite with a mutated 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 nameCSP
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Details of the genetic modification
Short description of the mutationThe P. yoelii csp gene replaced by P. falciparum csp
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 markereef1a
Selection (positive) procedureNo
Selection (negative) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modificationTo generate chimeric parasites where the P. yoelii csp coding sequence (CDS; PY17X_0405400) has been replaced by the P. falciparum csp CDS (Pfcsp; PF3D7_0304600), we used a 2-step GIMO transfection protocol.

In the first step we 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).

In the second step we replaced the positive-negative SM in the PyXNL-CS GIMO genome with a Pfcsp gene by GIMO transfection, using construct pL2310. To generate pL2310, we exchanged the Pbcsp 5’ and 3’ UTR regions of pL1972 by the Pycsp 5’ and 3’ UTR regions (using HindIII/BamHI and EcoRI/NotI). The construct was linearized using SacII and ScaI restriction sites before transfection and used to transfect parasites of the PyXNL-CS GIMO line using standard methods of GIMO-transfection. Transfected parasites were selected in mice by applying negative selection by providing 5-fluorocytosine (5-FC) in the drinking water of mice. Negative selection results in selection of transgenic parasites where the hdhfr::yfcu SM in the csp locus of the PyXNL-CS GIMO line is replaced by the Pfcsp gene, resulting in line 3226. This line has been passed through mosquitoes to collect only the complemented parasites.
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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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