Summary

RMgm-46
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_1432300; Gene model (P.falciparum): PF3D7_1216600; Gene product: cell traversal protein for ookinetes and sporozoites (CelTOS; cell traversal protein for ookinetes and sporozoites)
Phenotype Fertilization and ookinete; Oocyst; Sporozoite; Liver stage;
Last modified: 19 February 2009, 20:40
  *RMgm-46
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption
Reference (PubMed-PMID number) Reference 1 (PMID number) : 16468982
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 line
The mutant parasite was generated by
Name PI/ResearcherT. Kariu, M. Yuda
Name Group/DepartmentSchool of Medicine
Name InstituteMie University
CityMie
CountryJapan
Name of the mutant parasite
RMgm numberRMgm-46
Principal nameceltos(-)1; celtos(-)2; celtos(-)3
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 ookineteNormal production of gametocytes. Normal fertilization rate and ookinete production. ookinete infectivity is decreased (200 fold reduction in oocyst production). Ookinetes penetrate the epithelial cells of the mosquito midgut but are affected in migration through the cytoplasm of the epithelial cell to the basal lamina.
OocystThe number of oocysts is decreased by ~200 fold compared to wild type as a result of decreased infectivity of ookinetes (see phenotype 'Fertilization and ookinete'). Oocysts produce normal numbers of sporozoites that are not affected in their infectivity to salivary glands.
SporozoiteThe number of oocysts is decreased by ~200 fold compared to wildtype as a result of decreased infectivity of ookinetes (see phenotype 'Fertilization and ookinete'). Oocysts produce normal numbers of sporozoites that are not affected in their infectivity to salivary glands.
Motility of sporozoites was normal. Infectivity of sporozoites as measured by infection of rats (Wistar) by intravenous inoculation of sporozoites was reduced (~1/20 to ~1/50 of wild type sporozoites). Infectivity to HepG2 cells in vitro was not affected (similar numbers of EEFs were formed). However, cell traversal (cell-passage) was strongly impaired (as measured by the 'cell wound assay').
Infectivity of sporozoites to rats (Wistar) was restored in Kupffer cell-depleted rats.
Liver stageInfectivity of sporozoites as measured by infection of rats (Wistar) by intravenous inoculation of sporozoites was reduced (~1/20 to ~1/50 of wildtype sporozoites). Infectivity to HepG2 cells in vitro was not affected (similar numbers of EEFs were formed). However, cell traversal (cell-passage) was strongly impaired (as measured by the 'cell wound assay').
Infectivity of sporozoites to rats (Wistar) was restored in Kupffer cell-depleted rats.
Additional remarks phenotype

Mutant/mutation
This mutant lacks expression of CelTOS (cell-traversal protein for ookinetes and sporozoites)

Protein (function) 
CelTOS is localized to the micronemes of ookinetes and (salivary gland) sporozoites and is secreted.

Phenotype
The phenotype analyses of ookinetes and sporozoites indicate a role in migration through host cells. The ookinetes are affected in migration through the epithelial cells of the midgut. Sporozoites showed a normal invasion of salivary glands but have a strongly reduced hepatocyte traversal capacity. Infectivity of sporozoites as measured by HepG2 infection in vitro was not affected. In vivo liver stage infection however was strongly reduced. The results that show that sporozoite infectivity is restored in Kupffer cell-depleted rats indicate a role in sporozoite traversal through the sinusoidal cell layer.


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1432300
Gene Model P. falciparum ortholog PF3D7_1216600
Gene productcell traversal protein for ookinetes and sporozoites
Gene product: Alternative nameCelTOS; cell traversal protein for ookinetes and sporozoites
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 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′-GGCGAGCTCTATCTTTAATTTTTATTTTGTA-3′
Additional information primer 1
Sequence Primer 25′-GCCGGATCCACATAGAACATTAAAAAAGCAAAA-3′
Additional information primer 2
Sequence Primer 3GGCCTCGAGTTGAGAGGCAAAAATGGATCAGAA-3′
Additional information primer 3
Sequence Primer 45′-GCCGGTACCTTACGATTATCACTTAATGTTGTT-3′
Additional information primer 4
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6