Summary

RMgm-69
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. falciparum
Phenotype Oocyst; Sporozoite; Liver stage;
Last modified: 26 January 2011, 14:32
  *RMgm-69
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) : 12244064
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, 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-69
Principal nameCSP-9
Alternative nameCSP-9 (replacement with PfCS)
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 at day 14 after infection of A. stephensi mosquitoes.
SporozoiteCompared to wild type, the mutant showed a 10 fold reduction in the number of salivary gland sporozoites. Sporozoites show a normal gliding motility.
Liver stageInfectivity of sporozoites to mice (C57Bl/6) was comparable to wild type sporozoites as shown by similar prepatent periods after intravenous injection of sporozoites or after infection by mosquito bite.
Additional remarks phenotype

Mutant/mutation
In the mutant the endogenous P. berghei CS is replaced by P. falciparum CS (circumsporozoite protein) .

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 show that the CS protein of P. falciparum can complement the activity of the endogenous P. berghei CS protein (although the mutant sporozoites invade less well the salivary glands). 

See also mutant RMgm-342 for an independent mutant in which the endogenous P. berghei CS is replaced by P. falciparum CS using the same construct. This mutant has been generated in a reference reporter line that expresses the fusion protein GFP-luciferase under control of a constitutive promoter. This mutant showed, in addition to a reduced invasion of the salivary glands, a reduced sporozoite infectivity to the mammalian host.

Other mutants
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 lacks expression of CS (RMgm-9) .
A. P. berghei mutant has been generated that produces reduced levels of CS (RMgm-72).
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 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 is replaced with P. gallinaceum CS (RMgm-74).
A P. berghei mutant has been generated in which the endogenous P. berghei CS is replaced with P. yoelii CS (RMgm-75).
RMgm-342: A P. berghei ANKA  mutant  in which the endogenous P. berghei CS is replaced by P. falciparum CS. The construct used to generate the mutant is the same construct used for mutant described here.


  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. falciparum
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 (1660bp) from P. falciparum (Wellcome strain) linked to 250 nucleotides of its 3'-UTR was introduced into the genome, thereby replacing the endogenous P. berghei CS gene. The introduced pfCS gene is under control of the PbCS 5'-regulatory sequences (a fragment of the 5'-UTR of the PbCS gene encompassing nucleotides 1-1130 immediately upstream of the start codon). The 3'-UTR region consists of 250 nucleotides of the pfCS 3'UTR followed by 302 nucleotides of the PbCS 3'UTR region.
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