RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-4480
Malaria parasiteP. berghei
Genotype
Transgene
 Transgene Plasmodium: Gene model: PF3D7_0304600; Gene model (P.falciparum): PF3D7_0304600; Gene product: circumsporozoite (CS) protein (CSP)
Promoter: Gene model: PBANKA_0501200; Gene model (P.falciparum): PF3D7_1016900; Gene product: early transcribed membrane protein 10.3 | protein of early gametocyte 4 (UIS4; ETRAMP10.3)
3'UTR: Gene model: PBANKA_0501200; Gene product: early transcribed membrane protein 10.3 | protein of early gametocyte 4 (UIS4; ETRAMP10.3)
Replacement locus: Gene model: PBANKA_0306000; Gene product: 6-cysteine protein (230p)
Phenotype Sporozoite; Liver stage;
Last modified: 30 August 2018, 12:16
  *RMgm-4480
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 30155278
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 GIMO-PbANKA (RMgm-687)
Other information parent lineGIMO-PbANKA (RMgm-687; 1596cl1) contains as a selectable marker (SM) the fusion gene of hdhfr (human dihydrofolate reductase; positive SM) and yfcu (yeast cytosine deaminase and uridyl phosphoribosyl transferase; negative SM) stably integrated into the 230p locus (PBANKA_030600) through double cross-over recombination. The SM is under control of the P. berghei eef1α promoter. This reference line of P. berghei ANKA line is used for rapid introduction of transgenes free of drug-resistance genes (PubMed: PMID: 22216235).
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The mutant parasite was generated by
Name PI/ResearcherMendes AM, Khan SM, Janse CJ, Prudencio M
Name Group/DepartmentInstituto de Medicina Molecular, Faculdade de Medicina
Name InstituteUniversidade de Lisboa
CityLisbon
CountryPortugal
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Name of the mutant parasite
RMgm numberRMgm-4480
Principal name2266cl1
Alternative namePb ANKA-PfCSP(Pb uis4); PbVac
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
SporozoiteExpresses CSP from both P. berghei and P. falciparum
Liver stageExpresses CSP from both P. berghei and P. falciparum
Additional remarks phenotype

Mutant/mutation
The mutant (PbVac) expresses P. falciparum CSP (PfCSP). The Pfcsp gene is introduced as an 'additional' copy in the neutral p230p locus and is under control of the uis4 promoter and 3'UTR regions. The mutant is drug-selectable marker free.

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. 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. CSP is a major vaccine target.

Phenotype
The mutant expresses CSP from both P. berghei and P. falciparum in sporozoites and liver stages. Evidence is presented for a surface localisation of PfCSP in sporozoites and the protein is shed by motile sporozoites. Wild type sporozoite production and sporozoites show wild type infectivity to hepatocytes.

Additional information
From the Abstract of the paper:
'.... we describe a novel WSp malaria vaccine that employs transgenic sporozoites of rodent P. berghei (Pb) parasites as cross-species immunizing agents and as platforms for expression and delivery of PfCS (PbVac). We show that both wild-type Pb and PbVac sporozoites unabatedly infect and develop in human hepatocytes while unable to establish an infection in human red blood cells. In a rabbit model, similarly susceptible to Pb hepatic but not blood infection, we show that PbVac elicits cross-species cellular immune responses, as well as PfCS-specific antibodies that efficiently inhibit Pf sporozoite liver invasion in human hepatocytes and in mice with humanized livers. Thus, PbVac is safe and induces functional immune responses in preclinical studies, warranting clinical testing and development.'

Other mutants
See also mutant RMgm-1316 that expresses PfCSP and has the Pfcsp gene is introduced as an 'additional' copy in the neutral p230p locus and is under control of the uis4 promoter. This line also contains a GFP-Luciferase expression cassette.

See mutant RMgm-1339 for a mutant expressing mCherry under control of the uis4 promoter and 3'UTR regions.

 

  Transgene: Mutant parasite expressing a transgene
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Type and details of transgene
Is the transgene Plasmodium derived Transgene: Plasmodium
Gene Model of Parasite PF3D7_0304600
Gene Model P. falciparum ortholog PF3D7_0304600
Gene productcircumsporozoite (CS) protein
Gene product: Alternative nameCSP
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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 modificationA transgenic Pb parasite line containing a Pfcs expression cassette in the neutral 230p locus was generated using the ‘gene insertion/marker out’ (GIMO) technology as previously described. The Pfcs expression cassette was introduced into the neutral 230p locus of the GIMO mother line 1596cl1, using construct pL1988. The pL1988 construct contains the Pfcs coding sequence (CDS) under the control of the Pbuis4 5′ and 3′ UTR regulatory sequences flanked by the 5′ and 3′ targeting sequences for the 230p locus. This construct integrates by double crossover homologous recombination and replaces the positive–negative selectable marker (SM) (human dihydrofolate reductase:: yeast cytosine deaminase and uridyl phosphoribosyl transferase (hdhfr::yfcu)) cassette in the GIMO mother line 1596cl1 with the Pfcs expression cassette. The expression cassette contains the Pfcs CDS, which was amplified by PCR from Pf NF54 genomic DNA.
The CDS is flanked by the 5′ and 3′ promoter and transcription terminator sequences of Pb UIS4, which were amplified from Pb ANKA WT genomic DNA. The coding sequence of the Pfcs gene was confirmed by sequencing. The construct pL1988 was linearized by digestion with by SacII before introduction into parasites of the GIMO mother line 1596cl1 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 230p locus is replaced by the Pfcs expression cassette.
Additional remarks selection procedureThe construct containing the PfCSP expression cassette is integrated into the p230p locus and thereby removing the yfcu::hdhfr (negative) selectable marker cassette
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Other details transgene
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Promoter
Gene Model of Parasite PBANKA_0501200
Gene Model P. falciparum ortholog PF3D7_1016900
Gene productearly transcribed membrane protein 10.3 | protein of early gametocyte 4
Gene product: Alternative nameUIS4; ETRAMP10.3
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_0501200
Gene productearly transcribed membrane protein 10.3 | protein of early gametocyte 4
Gene product: Alternative nameUIS4; ETRAMP10.3
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