Summary

RMgm-75
Malaria parasiteP. berghei
Genotype
MutatedGene model (rodent): PBANKA_0403200; Gene model (P.falciparum): PF3D7_0304600; Gene product: circumsporozoite (CS) protein (CSP)
Details mutation: The endogenous P. berghei gene replaced with the ortholog of P. yoelii
PhenotypeNo phenotype has been described
Last modified: 26 January 2011, 14:37
  *RMgm-75
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) : 15839899
Reference 2 (PMID number) : 19890387
Reference 3 (PMID number) : 20194600
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone P. berghei ANKA 2.34
Other information parent lineP. berghei ANKA 2.34 is a cloned, gametocyte producer line of the ANKA strain (PubMed: PMID: 15137943).
The mutant parasite was generated by
Name PI/ResearcherR. Tewari, D. Rathore, A. Crisanti
Name Group/DepartmentImperial College of Science, Technology and Medicine
Name InstituteImperial College of Science
CityLondon
CountryUK
Name of the mutant parasite
RMgm numberRMgm-75
Principal namePyCS-5
Alternative namePyCS-5 (replacement with PyCS)
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
SporozoiteNot different from wild type
Liver stageNot different from wild type
Additional remarks phenotype

Mutant/mutation
In the mutant the endogenous P. berghei CS is replaced by P. yoelii CS (circumsporozoite protein; PY03168) . In the paper no information is provided on the primers used to amplify the P. yoelii cs gene

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 analyses indicate that the CS protein of P. yoelii (PyCS) can  complement the activity of the endogenous P. berghei CS protein. 

Additional information
In the paper no information is provided on the primers used to amplify the P. yoelii cs gene and only minimal information is provided on the genotype analysis of the mutant.
The primer information shown below has been obtained from Dr. R Tewari.

This mutant has been used in studies investigating immune responses in mice against the CS protein after immunization with irradiated, attenuated sporozoites (PubMed, PMID 1989038; 20194600).

Other mutants
A P. berghei mutant has been generated in which the endogenous P. berghei CS was replaced with  P. falciparum CS (RMgm-69)
A P. berghei mutant has been generated in which the endogenous P. berghei CS was replaced with  P. gallinaceum (RMgm-74)
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 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 that lacks expression of CS (RMgm-9) .
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). 


  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 endogenous P. berghei gene replaced with the ortholog of P. yoelii
Inducable system usedNo
Short description of the conditional mutagenesisNot available
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 modificationThe complete CS coding sequence from P. yoelii was introduced into the genome, thereby replacing the endogenous P. berghei CS gene. The PyCS gene is under control of the PbCS regulatory sequences. In the paper no details are provided on the generation of this mutant (!) and only the analyses of the phenotype are presented. The primer information shown below has been obtained from Dr. R Tewari.
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'CCCCgaattcATGAAGAAGTGTACCATTTTAGTTGTAGCG
Additional information primer 1PyCS F (small font: EcoR1)
Sequence Primer 25'GGGGggattcTTAATTAAAGAATACTAATAC
Additional information primer 2PyCS R (small font BamH1)
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