RMgmDB - Rodent Malaria genetically modified Parasites


Malaria parasiteP. berghei
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, lacking region II
Phenotype Oocyst; Sporozoite; Liver stage;
Last modified: 26 January 2011, 14:37
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
Name of the mutant parasite
RMgm numberRMgm-71
Principal nameCSP(RII-)-7
Alternative nameCSP(RII-)-7 (replacement with PfCS without region II)
Standardized name
Is the mutant parasite cloned after genetic modificationYes
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 290-fold reduction in the number of salivary gland sporozoites. Sporozoites do not show gliding motility.
Liver stageSporozoites failed to infective mice (C57Bl/6) after intravenous injection of sporozoites or after infection by mosquito bite.
Additional remarks phenotype

In the mutant the endogenous P. berghei CS is replaced by a mutated form of P. falciparum CS (circumsporozoite protein) . The mutated form lacks Region II (WSPCSVTCGNGIQVRIK).

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.

Analyses of a P. berghei mutant in which the endogenous P. berghei CS gene is replaced with the 'wild type' CS gene of P. falciparum (RMgm-69) show  that the CS protein of P. falciparum can complement the activity of the endogenous P. berghei CS protein efficiently (although the mutant sporozoites invade less well the salivary glands). The phenotype analysis of the mutant expressing the mutated PfCS protein lacking Region II, indicates that Region-II is involved in gliding motility. The lack of Region-II affects both salivary gland invasion and infectivity of sporozoites to the mammalian host. In comparison with the P. berghei mutant expressing 'wild type' PfCS, the mutants sporozoites expressing PfCS lacking Region II show a >95% reduction in salivary gland invasion.
A. P. berghei mutant has been described expressing a mutated from of CS lacking Region II-plus (RMgm-68). These mutants show a defect in egress of the oocysts. These sporozoites are also not infective to the mammalian host but they show normal gliding behavior. A difference between the mutated CS genes is that the Region II-plus deletion in RMgm-68 does not affect the thrombospondin (TSR) domain whereas in the mutant described here two of the four cysteines of the TSR domain are deleted.

Other mutants
P. berghei mutant has been generated that express the 'wild type' form of P. falciparum CS (RMgm-69).
P. berghei mutant has been generated that express another mutated form of P. falciparum CS (RMgm-70 with a mutated Region I).
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).
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. 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 endogenous P. berghei gene replaced with the ortholog of P. falciparum, lacking region II
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 CS coding sequence(1600bp) from P. falciparum (Wellcome strain)lacking region II was introduced into the P. berghei genome.
Deletion of coding region II (WSPCSVTCGNGIQVRIK) was introduced by site-directed mutagenesis.
The mutated PfCS coding sequence linked to 250 nucleotides of its 3'-UTR was introduced into the genome, thereby replacing the endogenous P. berghei CS gene. The pfCS gene is under control of the PbCS regulatory sequences: i) a 5'-UTR of the PbCS gene encompassing nucleotides 1-1130 immediately upstream of the start codon and ii) the 3'-UTR encompassing nucleotides 1-1150 downstream of the stop codon.
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