Summary

RMgm-137
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_1006300; Gene model (P.falciparum): PF3D7_0408700; Gene product: perforin-like protein 1 | sporozoite micronemal protein essential for cell traversal (PPLP1; Plasmodium perforin like protein 1; Membrane attack Complex; SPECT2)
Transgene
Transgene not Plasmodium: GFP
Promoter: Gene model: PBANKA_0711900; Gene model (P.falciparum): PF3D7_0818900; Gene product: heat shock protein 70 (HSP70)
3'UTR: Gene model: PBANKA_0711900; Gene product: heat shock protein 70 (HSP70)
Replacement locus: Gene model: PBANKA_0719300; Gene product: bifunctional dihydrofolate reductase-thymidylate synthase, putative (dhfr/ts)
Phenotype Sporozoite; Liver stage;
Last modified: 28 May 2009, 20:55
  *RMgm-137
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) : 18312843
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone Not applicable
Other information parent lineThe mutant is the result of a crossing between the transgenic GFP-expressing mutant RMgm-136 (ConF) and the mutant RMgm-135 that lacks expression of SPECT2/PPLP1. Both mutants have been generated in an ANKA background parent line.
The mutant parasite was generated by
Name PI/ResearcherR. Amino, R. Menard
Name Group/DepartmentUnité de Biologie et Génétique du Paludisme
Name InstituteInstitut Pasteur
CityParis
CountryFrance
Name of the mutant parasite
RMgm numberRMgm-137
Principal nameSpect2F
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
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
SporozoiteNormal numbers of midgut- and salivary gland sporozoites are formed. Sporozoites showed wild type gliding motility. Sporozoites had lost cell passage ability (membrane-damaging capacity) as shown in the cell-wounding assay. Compared to wild type sporozoites, the mutant sporozoites were rapidly immobilized in the dermis. Mutant sporozoites were shown to be arrested by and invade dermal fibroblasts.
Liver stageNo difference was noticed between wild type and mutant parasites in number, size, and fluorescence intensity of EEF at 4, 12, 24, or 48 hr in rat or mouse primary hepatocytes. Sporozoites show in vitro a 'rapid invader' phenotype, as a result of the lack of cell traversal. Mutant sporozoites show an adhesion/attachment phenotype (to CRL-2017 cells in vitro) which is comparable to wild type sporozoites.
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of SPECT2/PPLP1 (Membrane Attack Complex; MAC/perforin, putative; Plasmodium perforin like protein 1, PPLP1;  sporozoite protein essential for cell traversal, SPECT2). In addition to the disrupted spect2/pplp1 gene, this mutant contains gfp stably integrated into the genome (see below). The mutant is the result of a crossing between the transgenic GFP-expressing mutant RMgm-136 (ConF) and the mutant RMgm-135 that lacks expression of SPECT2/PPLP1.

Protein (function)
The spect2/pplp1 gene is a member of a small, conserved family of proteins encoding perforin-like proteins containing membrane-attack complex/perforin domains (MACPF)(Kaiser, K et al., 2004, Mol. Biochem. Parasitol. 133, 15-26). SPECT2/PPLP1 is specifically expressed in salivary gland sporozoites (not in midgut sporozoites) and has a micronemal location (micronemes).

Phenotype
See also the description of the phenotype of mutant RMgm-135, which lacks expression of SPECT2/PPLP1. Analyses of the phenotype of sporozoites lacking SPECT2/PPLP1 indicate a role of SPECT2/PPLP1 in traversal through host cells. For example, host cells in the dermis (for freely moving until endothelial barriers are reached and for resisting attacks by phagocytic cells) and in the liver sinusoids, presumably for resisting destruction by Kupffer cells.

Other mutants
Mutants that lack expression of other members of the PPLP protein family:
RMgm-135: Another mutant lacking expression of SPECT2/PPLP1
RMgm-123: A mutant lacking expression of PPLP5
RMgm-134: A mutant lacking expression of PPLP3/MAOP
See also RMgm-92 for an unsuccessful attempt to disrupt PPLP4

See also RMgm-138 and RMgm-139 for mutants lacking the expression of SPECT which show a comparable defect in cel traversal capacity, whereas the capabilty to invade hepatocytes is not affected.


 


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1006300
Gene Model P. falciparum ortholog PF3D7_0408700
Gene productperforin-like protein 1 | sporozoite micronemal protein essential for cell traversal
Gene product: Alternative namePPLP1; Plasmodium perforin like protein 1; Membrane attack Complex; SPECT2
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
Selectable marker used to select the mutant parasitepbdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationIn addition to a disrupted spect2/pplp1 gene, this mutant contains gfp stably integrated into the genome (see below).
The mutant is the result of a crossing between the transgenic GFP-expressing mutant RMgm-136 (ConF) and the mutant RMgm-135 that lacks expression of SPECT2/PPLP1.
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'-CGCGAGCTCGCTAACACATAGCGAAACCATGTTGTC-3'
Additional information primer 1
Sequence Primer 25'-CGCGGATCCTATAATCGTCATAATCATCTTCATCA TCACC-3'
Additional information primer 2
Sequence Primer 35'-CCGCTCGAGAAAGATGAAGAACAAAATGAGCA TATAGATA-3'
Additional information primer 3
Sequence Primer 45'-CGGGGTACCGCCAATTGTGTATTTTATGC AGTTTGACT-3'
Additional information primer 4
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6

  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameGFP
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 parasitepbdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationIn addition to a stably integrated gfp gene, this mutant contains a disrupted spect2/pplp1 gene (see above).
The mutant is the result of a crossing between the transgenic GFP-expressing mutant RMgm-136 (ConF) and the mutant RMgm-135 that lacks expression of SPECT2/PPLP1.
ConF parasites were generated using a construct that integrates by double crossover integration of the gfp expression cassette at the dhfr-ts locus. The gfp coding region was fused to the upstream and downstream regions of the hsp70 gene and the resulting expression cassette was inserted between the pyrimethamine-resistant, mutant dhfr-ts gene and its downstream region (targeting construct).
Additional remarks selection procedure
Other details transgene
Promoter
Gene Model of Parasite PBANKA_0711900
Gene Model P. falciparum ortholog PF3D7_0818900
Gene productheat shock protein 70
Gene product: Alternative nameHSP70
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
3'-UTR
Gene Model of Parasite PBANKA_0711900
Gene productheat shock protein 70
Gene product: Alternative nameHSP70
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_0719300
Gene productbifunctional dihydrofolate reductase-thymidylate synthase, putative
Gene product: Alternative namedhfr/ts
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