RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-4987
Malaria parasiteP. berghei
Genotype
MutatedGene model (rodent): PBANKA_0403200; Gene model (P.falciparum): PF3D7_0304600; Gene product: circumsporozoite (CS) protein (CSP)
Details mutation: The P. berghei csp gene replaced by P. falciparum csp
MutatedGene model (rodent): PBANKA_1349800; Gene model (P.falciparum): PF3D7_1335900; Gene product: thrombospondin-related anonymous protein | sporozoite surface protein 2 (TRAP; SSP2)
Details mutation: The P. berghei trap gene replaced by P. falciparum trap
Transgene
 Transgene not Plasmodium: A fusion of GFP (gfp-mu3) and Luciferase Firefly (LucIAV)
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: PBANKA_0306000; Gene product: 6-cysteine protein (230p)
PhenotypeNo phenotype has been described
Last modified: 13 May 2021, 20:02
  *RMgm-4987
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene mutation, Gene mutation, Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 29195810
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 RMgm-4110
Other information parent lineIn the mutant the endogenous P. berghei csp gene has been replaced by the P. falciparum csp gene.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. berghei 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.
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The mutant parasite was generated by
Name PI/ResearcherSalman AM, Khan SM, Janse CJ
Name Group/DepartmentLeiden Malaria Research Group
Name InstituteLeiden University Medical Center
CityLeiden
CountryThe Netherlands
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Name of the mutant parasite
RMgm numberRMgm-4987
Principal name2644cl8 cl3-6
Alternative namePbANKA-PfCSP(r)PbCSP-PFTRAP(r)PbTRAP
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
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. berghei csp gene has been replaced by the P. falciparum csp gene.
- The endogenous P. berghei trap gene has been replaced by the P. falciparum trap gene
This has been performed by the GIMO method of transfection.

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)
CSP: 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.
TRAP: TRAP is a type 1 transmembrane protein, containing two adhesive domains in its extracellular portion, an A-domain of von Willebrand factor and a thrombospondin type I repeat (TSR, TSP). The cytoplasmic part (tail) of the protein is postulated to interact with actin–myosin motor proteins giving the force needed for motility.
TRAP is located in the micronemes of sporozoites. The protein plays a role in the gliding motility of sporozoites and invasion of host cells as has been shown by analysis of mutant parasites lacking expression of TRAP

Phenotype
Normal development throughout the complete life cycle showing complementation of P. berghei CSP by P. falciparum CSP and P. berghei TRAP by P. falciparum TRAP

Additional information
In the mutant the endogenous P. berghei trap gene has been replaced by the P. falciparum trap gene. This has been performed by the GIMO method of transfection. In the 2257cl2 parent line (expressing P. falciparum CSP) the trap open reading frame (orf) was first replaced with the hdhfr-yfcu selectable marker (SM) using plasmid pL2111 (RMgm-4112), resulting in mutant line 2560cl4. Subsequently, the SM was replaced by the P. falciparum trap orf by transfection and negative selection. The P. falciparum trap gene is under control of the 5'- and 3'-UTR regions of the P. berghei trap gene.

Other mutants

  Mutated: Mutant parasite with a mutated gene
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Details of the target gene
Gene Model of Rodent Parasite PBANKA_0403200
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. berghei 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 the chimeric parasites where the P. berghei csp coding sequence (CDS; Pbcsp; PBANKA_0403200) 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 Pbcsp CDS and replaced it with the positive-negative selectable marker, to create a P. berghei csp deletion GIMO line (PbANKA-CSP GIMO). In order to do this, we generated a construct (pL1929) that is based on the standard GIMO DNA construct pL0034. This construct contains the positive-negative (hdhfr::yfcu) selection marker (SM) cassette, and was used to insert both the Pbcsp 5’ and 3’ gene targeting regions (TR), encompassing the full length promoter and transcription terminator sequences respectively. The linear DNA construct was introduced into PbGFP-Luccon parasites (676m1cl1) using standard methods of transfection. Transfected parasites were selected in mice by applying positive selection by providing pyrimethamine in the drinking water. Transfected parasites were cloned by limiting dilution, resulting in the PbANKA-CSP GIMO line (line 2151cl1).

In the second step we replaced the positive-negative SM in the PbANKA-CSP GIMO genome with the Pfcsp CDS by GIMO transfection to create the P. berghei chimeric CSP replacement line. This was achieved by modifying the construct used in the first step (pL1929); specifically, the hdfhr::yfcu SM cassette was removed and replaced with Pfcsp CDS sequence, generating plasmid pL1972. The Pfcsp CDS was amplified from genomic DNA of the NF54 strain of P. falciparum. The pL1972 construct was sequenced to ensure there were no mutations in the Pfcsp CDS. The constructs was linearized using ApaI and NotI restriction enzymes outside of the 5’ and 3’ TRs before transfection. The construct was used to transfect parasites of the PbANKA-CSP GIMO line (line 2151cl1) 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 chimeric parasites where the hdhfr::yfcu SM in the csp locus of PbANKA-CSP GIMO line is replaced by the CDS of Pfcsp. Selected chimeric parasites were cloned by the method of limiting dilution. Correct integration of the constructs into the genome of chimeric parasites was analysed by diagnostic PCR-analysis on gDNA and Southern analysis of pulsed field gel (PFG) separated chromosomes. This method creates chimeric ‘gene replacement’ P. berghei parasites that lack the Pbcsp CDS but express PfCSP (PbANKA-PFCSP(r)PbCSP; line 2257cl2) under the control of the Pbcsp regulatory sequences.
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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  Mutated: Mutant parasite with a mutated gene
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Details of the target gene
Gene Model of Rodent Parasite PBANKA_1349800
Gene Model P. falciparum ortholog PF3D7_1335900
Gene productthrombospondin-related anonymous protein | sporozoite surface protein 2
Gene product: Alternative nameTRAP; SSP2
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Details of the genetic modification
Short description of the mutationThe P. berghei trap gene replaced by P. falciparum trap
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 modificationIn the mutant the endogenous P. berghei trap gene has been replaced by the P. falciparum trap gene. This has been performed by the GIMO method of transfection. In the 2257cl2 parent line (expressing P. falciparum CSP) the trap open reading frame (orf) was first replaced with the hdhfr-yfcu selectable marker (SM) using plasmid pL2111 (RMgm-4112), resulting in mutant line 2560cl4. Subsequently, the SM was replaced by the P. falciparum trap orf by transfection and negative selection. The P. falciparum trap gene is under control of the 5'- and 3'-UTR regions of the P. berghei trap gene.
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 nameA fusion of GFP (gfp-mu3) and Luciferase Firefly (LucIAV)
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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 parasitegfp (FACS)
Promoter of the selectable markereef1a
Selection (positive) procedureFACS (flowsorting)
Selection (negative) procedureNo
Additional remarks genetic modification
Additional remarks selection procedure
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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 PBANKA_0306000
Gene product6-cysteine protein
Gene product: Alternative name230p
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