Summary

RMgm-139
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_1355600; Gene model (P.falciparum): PF3D7_1342500; Gene product: sporozoite protein essential for cell traversal (SPECT, SPECT1)
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: 23 September 2016, 14:20
  *RMgm-139
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-138 that lacks expression of SPECT. 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-139
Principal nameSpectF
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 SPECT (sporozoite protein essential for cell traversal, SPECT; SPECT1). In addition to the disrupted spect 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-138 that lacks expression of SPECT.

Protein (function)
SPECT has been identified by screening a sporozoite EST library and is a putative secretory protein of 241 amino acids with an expected molecular mass of 25 kDa for the N-terminal signal sequence-processed form of this protein.SPECT 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-138, which lacks expression of SPECT. Analyses of the phenotype of sporozoites lacking SPECT indicate a role of SPECT 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
RMgm-138: Another mutant lacking expression of SPECT.

See also RMgm-135 and RMgm-137 for mutants lacking the expression of SPECT2/PPLP1 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_1355600
Gene Model P. falciparum ortholog PF3D7_1342500
Gene productsporozoite protein essential for cell traversal
Gene product: Alternative nameSPECT, SPECT1
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct used(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
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 spect 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-138 that lacks expression of SPECT.
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′-CGCGAGCTCGCAATATGGTATTAAATTTTGGGCTAGCCA-3′
Additional information primer 1
Sequence Primer 25′-CGCGGATCCGGTATTTTCATTGTGTTAAACGATATGTGA-3′
Additional information primer 2
Sequence Primer 35′-CCGCTCGAGGTCCTATTTATCATTTTAAAATGTGTTTTATC-3′
Additional information primer 3
Sequence Primer 45′-CGGGGTACCAATCGTCATAAATAGGAGTTATGAAGT-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/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 parasitepbdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationNo PB gene model exists for SPECT, sporozoite protein essential for cell traversal, MAL13P1.212, hypothetical protein, conserved). The P. berghei GenBank Accession No. is AB125370.

In addition to a stably integrated gfp gene, this mutant contains a disrupted spect gene (see above).
The mutant is the result of a crossing between the transgenic GFP-expressing mutant RMgm-136 (ConF) and the mutant RMgm-138 that lacks expression of SPECT.
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