Summary

RMgm-76
Malaria parasiteP. berghei
Genotype
MutatedGene model (rodent): PBANKA_0403200; Gene model (P.falciparum): PF3D7_0304600; Gene product: circumsporozoite (CS) protein (CSP)
Details mutation: The repeat region of the endogenous gene is exchanged with the P. falciparum repeat region
Phenotype Oocyst; Sporozoite; Liver stage;
Last modified: 4 March 2010, 23:48
  *RMgm-76
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene mutation
Reference (PubMed-PMID number) Reference 1 (PMID number) : 12471098
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei NK65
Name parent line/clone RMgm-9
Other information parent lineThe parent line used for the genetic modification is a mutant (RMgm-9) lacking the wild type CS gene
The mutant parasite was generated by
Name PI/ResearcherC. Persson, V. Nussenzweig, E. Nardin
Name Group/DepartmentDepartmentof Medical and Molecular Parasitology
Name InstituteNew York University School of Medicine
CityNew York
CountryUSA
Name of the mutant parasite
RMgm numberRMgm-76
Principal nameCS(pf)
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 of mature oocysts in A. stephensi mosquitoes. Salivary gland infectivity and the number of sporozoites per salivary gland was reduced compared to wild type (~30% of wild type).
SporozoiteSalivary gland infectivity and the number of sporozoites per salivary gland was reduced compared to wild type (~30% of wild type).
Mutant sporozoites display normal morphology and motility.
The sporozoites were found to invade and develop within HepG2 cells comparable to wt sporozoites.
Liver stageThe sporozoites were found to invade and develop within HepG2 cells comparable to wt sporozoites.
Additional remarks phenotype

Mutant/mutation
The mutant expresses a mutated form of the circumsporozoite protein (CS). It expresses a hybrid P. berghei/P. falciparum CS in which the P. berghei CS repeat region  was exchanged with the P. falciparum CS repeat region.

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 analysis shows that P. berghei CS bearing the P. falciparum CS repeat region can complement the wild type P. berghei CS protein, although the mutant sporozoites show a reduced salivary gland invasion.
Monoclonal and polyclonal Abs to the P. falciparum repeats neutralize the infectivity to HepG2 cells in vitro of the mutant sporozoites and infectivity to the C57Bl/6 mice was strongly reduced when mice were treated with mAbs against  the P. falciparum repeats. Mice immunized with the  P. falciparum (T1B)4 MAP vaccine  are protected against challenge with the mutant sporozoites.
This mutant has been used in a 'transgenic sporozoite neutralization assay (TSNA)'  to test the inhibition of sporozoite invasion of HepG2 cells by sera obtained from humans immunized with (T1B)4 MAP, a peptide vaccine containing P. falciparum CS repeats (Kumar et al., 2004, J. Immunol. Methods 292, 157-64).

Other mutants
A P. berghei mutant has been generated that lacks expression of CS (RMgm-9) .
A P. berghei mutant has been generated that produces reduced levels of CS (RMgm-72).
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).
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).
 


  Mutated: Mutant parasite with a mutated 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
Short description of the mutationThe repeat region of the endogenous gene is exchanged with the P.
falciparum repeat region
Inducable system usedNo
Short description of the conditional mutagenesisNot available
Additional remarks inducable system
Type of plasmid/constructPlasmid single 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 parasitehdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedureWR99210
Selection (negative) procedureNo
Additional remarks genetic modificationThe mutant expresses a hybrid P. berghei/P. falciparum CS in which the P. berghei CS repeat region was exchanged with the P. falciparum CS repeat region.
The P. falciparum CS repeat region was amplified from genomic DNA from the 7G8 isolate using primers that generated SpeI and XhoI sites: PfSpeI, 5'-GAGATGGAACTAGTAATAACGAAGACAACGAG-3'; PfXhoI, 5'-CTTGGTTCTTCCTCGAGGTTATTATTATTTTTTAC-3'. The CS repeat region within the CS gene of P. berghei in the construct used for transfection was exchanged with the P. falciparum CS repeat region using the SpeI and XhoI restriction sites
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