RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-4111
Malaria parasiteP. berghei
Genotype
MutatedGene model (rodent): PBANKA_1432300; Gene model (P.falciparum): PF3D7_1216600; Gene product: cell traversal protein for ookinetes and sporozoites (CelTOS)
Details mutation: The P. berghei celtos gene replaced by P. vivax (Sal I) celtos (PVX_123510)
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: 4 May 2021, 18:38
  *RMgm-4111
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) Reference 1 (PMID number) : 28179403
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 P. berghei ANKA 676m1cl1 (RMgm-29)
Other information parent line676m1cl1 (RMgm-29) is a reference ANKA mutant line which expresses GFP-luciferase under control of a constitutive promoter. This reference line does not contain a drug-selectable marker (PubMed: PMID: 16242190).
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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-4111
Principal name2321cl3
Alternative namePbANKA-PvCelTOS(r)PbCelTOS
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 celtos gene has been replaced by the P. vivax Sal I strain celtos gene (PVX_123510). This has been performed by the GIMO method of transfection. The P. vivax celtos gene is under control of the 5'- and 3'-UTR regions of the P. berghei celtos 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)
CelTOS is localized to the micronemes of ookinetes and (salivary gland) sporozoites and is secreted. It plays a role in migration of ookinetes through mosquito midgut cells and traversal of sporozoites through heptocytes

Phenotype
Normal development throughout the complete life cycle showing complementation of P. berghei CELTOS by P. vivax CELTOS

Additional information
This chimeric parasite line has been used for analysing (protective) immune responses in mice immunized with different vaccines targeting P. vivax CELTOS. Immunized mice were challenged with chimeric sporozoites expressing P. vivax CELTOS.

Other mutants

  Mutated: Mutant parasite with a mutated gene
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Details of the target gene
Gene Model of Rodent Parasite PBANKA_1432300
Gene Model P. falciparum ortholog PF3D7_1216600
Gene productcell traversal protein for ookinetes and sporozoites
Gene product: Alternative nameCelTOS
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Details of the genetic modification
Short description of the mutationThe P. berghei celtos gene replaced by P. vivax (Sal I) celtos (PVX_123510)
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 celtos coding sequence (CDS; Pbceltos; PBANKA_1432300) has been replaced by the P. vivax celtos CDS (Pvceltos; PVX_123510), we used a 2-step gene insertion/marker out transfection protocol (GIMO). In the first step we deleted the Pbceltos CDS and replaced it with the positive-negative selectable marker, to create a P. berghei celtos deletion GIMO line (PbANKA-CelTOS GIMO). In order to do this, we generated the pL1960 construct 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 Pbceltos 5’ and 3’ gene targeting regions (TR), encompassing the full length promoter and transcription terminator sequences respectively. The linear pL1960 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-CelTOS GIMO line (line 2217).

In the second step we replaced the positive-negative SM in the PbANKA-CelTOS GIMO genome with the Pvceltos CDS by GIMO transfection to create the P. berghei chimeric CelTOS replacement line. This was achieved by modifying the construct used in the first step (pL1960); specifically, the hdfhr::yfcu SM cassette was removed and replaced with Pvceltos CDS sequence, generating plasmid pL2017. TThe Pvceltos CDS was amplified from DNA of P. vivax Sal I strain (Pvceltos; PVX_123510). The pL2017 construct was sequenced to ensure there were no mutations in the Pvceltos CDS. The construct 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-CELTOS GIMO line (line 2217cl1) 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 celtos locus of PbANKA-CelTOS GIMO line is replaced by the CDS of Pvceltos (line 2320). Selected chimeric parasites were cloned by 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 Pbceltos CDS but express PvCelTOS (PbANKA-PvCelTOS(r)PbCelTOS; line 2320cl2) under the control of the Pbceltos 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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  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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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