RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-9
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_0403200; Gene model (P.falciparum): PF3D7_0304600; Gene product: circumsporozoite (CS) protein (CSP)
Phenotype Oocyst; Sporozoite; Liver stage;
Last modified: 19 February 2009, 20:33
  *RMgm-9
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) : 9002517
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei NK65
Name parent line/clone Not applicable
Other information parent line
The mutant parasite was generated by
Name PI/ResearcherR. Menard; R.S Nussenzweig; V. Nussenzweig
Name Group/DepartmentMichael Heidelberger Division of Immunology
Name InstituteNew york University Medical Center
CityNew York
CountryUSA
Name of the mutant parasite
RMgm numberRMgm-9
Principal nameCS(-)
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
OocystNormal numbers of oocysts are produced in Anopheles stephensi mosquitoes. Sporozoite formation within the oocysts is profoundly inhibited: up to day ten after feeding of the mosquitoes the morphology of oocysts is normal; after day 10, the oocysts display a highly vacuolated structure
SporozoiteSee the description of the phenotype of the oocyst
Liver stageFeeding on mice of mosquitoes that were infected with CS(-) parasites did not result in infection of these mice
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of the circumsporozoite protein (CS).

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. The protein is also found on the oocyst plasma membrane and on the inner surface of the oocyst capsule. Specific motifs in CS are involved in sporozoite binding to mosquito salivary glands and in sporozoite attachment to heparan sulfate proteoglycans in the liver of the mammalian host. 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.

Phenotype
The phenotype analyzes demonstrate the essential role of CS in the formation of sporozoites within the oocyst. See also the study of Thathy V. et al. (2002, EMBO Journal 21, 1586) for an analysis of the morphology of the aberrant CS(-) oocysts by transmission electron microscopy. The results of this study indicate that CS is essential for establishing polarity in oocysts by affecting the development of the inner membrane and associated microtubules underneath the oocyst outer membrane, which normally demarcate focal budding sites of daughter cells.

Other mutants
A. P. berghei mutant has been generated that produces reduced levels of CS (RMgm-72).
A P. berghei mutant has been generated with a mutated Region II-plus (RMgm-68).
P. berghei mutants have been generated with a mutated GPI-anchor addition sequence (RMgm-73).
A P. berghei mutant has been generated in which the endogenous P. berghei CS was replaced with P. falciparum CS (RMgm-69).
Two P. berghei mutants have been generated that express mutated forms of P. falciparum CS (RMgm-70 with a mutated Region I; RMgm-71 with a mutated Region II).
P. berghei mutant has been generated that express a hybrid form of CS of P. berghei and  P. falciparum (the P. berghei CS repeat region  is exchanged with the P. falciparum CS repeat region)(RMgm-76).
A P. berghei mutant has been generated in which the endogenous P. berghei CS was replaced with P. gallinaceum CS (RMgm-74).
A P. berghei mutant has been generated in which the endogenous P. berghei CS was replaced with P. yoelii CS (RMgm-75).


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0403200
Gene Model P. falciparum ortholog PF3D7_0304600
Gene productcircumsporozoite (CS) protein
Gene product: Alternative nameCSP
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 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
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6