RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-279
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_1024600; Gene model (P.falciparum): PF3D7_1418100; Gene product: liver specific protein 1, putative (LISP1, liver specific protein 1)
Transgene
 Transgene not Plasmodium: GFP (gfp-mu3)
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 Liver stage;
Last modified: 8 December 2009, 10:41
  *RMgm-279
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption, Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 19438514
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 507cl1 (RMgm-7)
Other information parent lineP.berghei ANKA 507cl1 (RMgm-7) is a reference ANKA mutant line which expresses GFP under control of a constitutive promoter (PubMed: PMID: 16242190).
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The mutant parasite was generated by
Name PI/ResearcherT. Ishino, P. Baldacci
Name Group/DepartmentBiologie et Génétique du Paludisme
Name InstituteInstitut Pasteur
CityParis
CountryFrance
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Name of the mutant parasite
RMgm numberRMgm-279
Principal nameLisp1ΔGreen
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
OocystNot different from wild type
SporozoiteNot different from wild type
Liver stageBy analysis of blood stage parasitemia after intravenous injection of sporozoites, the mutant sporozoites showed a 15-fold decrease in infectivity.
The number and size of liver schizonts in rats and in HepG2 cells of the mutant was comparable to those of wild type parasites. The number of merozoites in mutant liver schizonts was similar to the merozoite number in wild type schizonts. The number of merozoites released from liver schizonts was 10-fold reduced compared to wild type. Released mutant merozoites showed an infectivity to erythrocytes that was comparable to that of wild type merozoites.
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of LISP1 (liver specific protein 1) and constitutivily expresses GFP. The lisp1 gene has been disrupted in mutant RMgm-7, This mutant contains a gfp gene under the control of the eef1a promoter, stably introduced into the genome witthout a drug-selectable marker.

Protein (function)
Phenotype analyses of liver stage development of this and additional mutants lacking expression of LISP1 (RMgm-237, RMgm-280)  indicate that LISP plays a role in egress of merozoites from liver cells (see also 'Phenotype')

Phenotype
The phenotype analyses indicate a normal development of the sporozoites and liver stages, up to the mature liver schizont. Normal numbers of merozoites are formed that show a morphology that is comparable to wild type when analysed by transmission electron microscopy. The presence of the PVM and the 10-fold reduction in release of merozoites indicate that the mutants are defective in disrupting the PVM.

Additional information
Using polyclonal antibody expression was detected in 48h liver schizonts in livers from rats and in infected HepG2 cells at 36, 48 and 65h post infection. No signal was detected in blood stages and sprorozoites. IFA analysis indicated a location of LISP in the parasitophorous vacuole membrane (PVM).
Four gene models exist: PB000708.00.0; PB000682.00.0; PB001247.00.0; PB000250.00.0.
Gen Bank accession no: AB231328.

Other mutants
RMgm-237: An independent mutant lacking expression of LISP1 (NK65 strain)
RMgm-280: An independent mutant lacking expression of LISP1 (ANKA strain)

  Disrupted: Mutant parasite with a disrupted gene
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Details of the target gene
Gene Model of Rodent Parasite PBANKA_1024600
Gene Model P. falciparum ortholog PF3D7_1418100
Gene productliver specific protein 1, putative
Gene product: Alternative nameLISP1, liver specific protein 1
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Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct usedPlasmid double cross-over
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid
Partial or complete disruption of the geneComplete
Additional remarks partial/complete disruption The lisp1 gene has been disrupted in mutant RMgm-7, This mutant contains a gfp gene under the control of the eef1a promoter, stably introduced into the genome witthout a drug-selectable marker.
Selectable marker used to select the mutant parasitehdhfr
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modification
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 15’- CGATGCGGGCCCGAGAATACAACTTACTAGGAAATGCAC-3’
Additional information primer 1ApaI - 630bp
Sequence Primer 25’- AGCTGGCGCGCCCTTGCATCTTCAGACATGTTATTTTCGA-3’
Additional information primer 2SmaI - 630bp
Sequence Primer 35’-
CCAGTGAGTGCGGCCGCGGCTAAAGCTCGATGTCGTATTCAAGAA-3’
Additional information primer 3NotI - 600bp
Sequence Primer 45’- AGCTGGCGCGCCCTGGGAATTTTTCAATTTCTTCCATTTG-3’
Additional information primer 4AscI - 600bp
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 (gfp-mu3)
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Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/constructPlasmid 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 gene (1 copy) has been inserted into the 230p locus (PB000214.00.0) by double cross-over integration.
Additional remarks selection procedureThe gfp gene is introduced into the genome using a DNA construct without a drug-selectable marker. This reporter mutant (507cl1; RMgm-7) 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 15'-cccaagcttccgcgggtatatggtaaagaacctactaacac
Additional information primer 1primer L1345: 0.7kb 5'region of PB000214.00.0 (230p gene)
Sequence Primer 25'-cccaagcttgatgtgttttatttggatgtgc
Additional information primer 2primer L1346: 0.7kb 5'region of PB000214.00.0 (230p gene)
Sequence Primer 35'-ccggaattctcttgagcccgttaatg
Additional information primer 3primer L1347: 1kb 3'region of PB000214.00.0 (230p gene)
Sequence Primer 45'-tccccgcgggtatggaactacatctatatagg
Additional information primer 4primer L1348: 1kb 3'region of PB000214.00.0 (230p gene)
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Conceptual design of the database: Dr. C.J. Janse (Leiden Malaria Research Group, Leiden, The Netherlands).
Technical design and realization: StudioDrie StudioDrie; S. Mededovic and A. van Wigcheren