RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5235
Malaria parasiteP. berghei
Genotype
MutatedGene model (rodent): PBANKA_0403200; Gene model (P.falciparum): PF3D7_0304600; Gene product: circumsporozoite (CS) protein (CS; CSP)
Details mutation: P. berghei csp replaced with the 'P. vivax like' csp (locus PVU09738).
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)
Phenotype Oocyst; Sporozoite;
Last modified: 2 September 2022, 17:45
  *RMgm-5235
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) : 34504134
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-2151
Other information parent lineThe mutant lacks expression of CSP. In the mutant the endogenous P. berghei 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
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The mutant parasite was generated by
Name PI/ResearcherGiminez, AM, Reyes-Sandoval A
Name Group/DepartmentInstituto Politécnico Nacional, IPN
Name InstituteInstituto Politécnico Nacional, IPN
CityMexico City
CountryMexico
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Name of the mutant parasite
RMgm numberRMgm-5235
Principal name2710cl1,2
Alternative name
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
OocystWild-type numbers of mature oocysts are formed. However, no visible spz inside oocysts, and only very few salivary gland spz were observed.
Sporozoiteno visible spz inside oocysts, and only very few salivary gland spz were observed.
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
In the mutant the endogenous P. berghei csp gene has been replaced with the with the 'P. vivax like' csp (Gabon Clone G10; Locus PVU09738, Accession U09738). 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
Wild-type numbers of mature oocysts are formed. However, no visible spz inside oocysts, and only very few salivary gland spz were observed. these result indicate that the "P vivax like' csp gene of P. vivax cannot complement P. berghei 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 PBANKA_0403200
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
Short description of the mutationP. berghei csp replaced with the 'P. vivax like' csp (locus PVU09738).
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 P. berghei replacement line, we replaced the P. berghei csp coding sequence (CDS; Pbcsp; PBANKA_0403200) with the PvCSP-P. vivax-like CDS (Locus PVU09738, Accession U09738) using a 2-step GIMO transfection protocol.
In the first step, we deleted the P. berghei csp CDS and replaced it with a positive–negative selectable marker to create a P. berghei csp deletion GIMO line (PbANKA-CSP GIMO). The construct (pL1929) used and the generation of the PbANKA-CSP GIMO line (line 2251cl1). This construct contains the positive–negative (hdhfr::yfcu) SM cassette and was used to insert both the Pbcsp 5′ and 3′ gene targeting regions (TRs), encompassing the full-length promoter and transcription terminator sequences, respectively, and was transfected into PbGFP-Luccon parasites (676m1cl1) using standard transfection methods. Transfected parasites were selected in mice by applying positive selection by providing pyrimethamine in the drinking water. Transfected parasites were cloned using the limiting dilution method, resulting in the PbANKA-CSP GIMO line (line 2251 cl1). In the second step, we replaced the positive–negative SM in the PbANKA-CSP GIMO genome with the PvCSP-P. vivax-like CDS by GIMO transfection to create the P. berghei chimeric Pb-PvG10 replacement line Pb-PvG10(r). This line was obtained by modifying the construct used in the first step (pL1929); specifically, the hdfhr::yfcu SM cassette was removed and replaced with the PvCSP-P. vivax-like CDS, generating plasmid pL2161. The PvCSP-P. vivax-like CDS was ordered from GeneArt Gene Synthesis—Thermo Fisher Scientific. The pL2161 construct was sequenced to ensure that no mutations were present in the PvCSP-P. vivax-like CDS during the cloning process. The construct was linearized using AflII and SacI 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 2251 cl1) using standard methods of GIMO transfection to generate a single replacement gene chimeric parasite. Transfected parasites were selected in mice by applying negative selection by providing 5-fluorocytosine (5-FC) in the drinking water. Negative selection results in the selection of chimeric parasites where the hdhfr::yfcu SM in the csp locus on chromosome 4 of the PbANKA-CSP GIMO line is replaced with the PvCSP-P. vivax-like CDS. Selected chimeric parasites were cloned using the limiting dilution method.
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 modificationThe GFP-Luciferase gene (1 copy) has been inserted into the 230p locus by double cross-over integration.
Additional remarks selection procedureThis reporter mutant expressing GFP-Luciferase does not contain a drug-selectable marker. This mutant has been selected by FACS sorting after transfection based on GFP fluorescence.
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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