Summary

RMgm-72
Malaria parasiteP. berghei
Genotype
MutatedGene model (rodent): PBANKA_0403200; Gene model (P.falciparum): PF3D7_0304600; Gene product: circumsporozoite (CS) protein (CSP)
Details mutation: truncation of the 3'UTR of the gene
Phenotype Oocyst; Sporozoite; Liver stage;
Last modified: 4 March 2010, 23:39
  *RMgm-72
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) : 11927543
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/ResearcherV. Thathy, V. Nussenzweig, R. Menard
Name Group/DepartmentMichael Heidelberger Division of Immunology
Name InstituteNew york University Medical Center
CityNew York
CountryUSA
Name of the mutant parasite
RMgm numberRMgm-72
Principal namePINA270-1; PINA270-2
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 affected. Oocysts display intermediary levels of extension of the inner membranes and microtubules and at least 50% of the oocyst-sporozoites had abnormal and variable sizes and shapes.
SporozoiteThe (aberrantly shaped) sporozoites did not invade salivary glands as shown by the greatly reduced ratio of salivary gland to midgut sporozoites and did not show normal gliding motility. Sporozoites were not infective.
Liver stageSee the description of the phenotype of the sporozoite.
Additional remarks phenotype

Mutant/mutation
The mutant expresses CS with a truncated 3'-UTR, resulting in reduced levels of CS production (see below).

Protein (function)
The 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
This mutant expresses CS with a truncated 3'-UTR, resulting in reduced levels of CS production. Cloning and sequencing of CS transcripts in P. berghei sporozoites have revealed numerous 3' ends. The 3' ends of the CS transcripts are heterogeneous, located between nucleotides 88 and 337 after the CS stop codon. The majority of the CS 3' ends are clustered 300 bp past the stop codon, just upstream of a 16 bp GU-rich element. In the mutant the CS gene is followed by 270 bp of its 3'-UTR. The mutant sporozoites reproducibly contained 5x less CS protein than wild-type sporozoites. They still produced a normal ratio of the 44 and 54 kDa forms of CS, which are thought to represent the surface molecule and intracellular precursor of CS, respectively.

The phenotype analyzes of this mutant and the mutant lacking expression of CS (RMgm-9)demonstrate the essential role of CS in the formation of sporozoites within the oocyst. The results of these analyzes 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 lacks expression of CS (RMgm-9) .
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).
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).
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 mutationtruncation of the 3'UTR of the gene
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 parasitepbdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe insertion construct used results in introduction replacement of the wild type cs gene with a copy of the cs gene that is under control of the wild type 5'UTR regulatory sequence but contains a truncated 3'UTR sequence.

Cloning and sequencing of CS transcripts in P.berghei sporozoites have revealed numerous 3' ends. The 3' ends of the CS transcripts are heterogeneous, located between nucleotides 88 and 337 after the CS stop codon. The majority of the CS 3' ends are clustered 300 bp past the stop codon, just upstream of a 16 bp GU-rich element. In the mutant the CS gene is followed by only 270 bp of its 3'-UTR. The mutant sporozoites reproducibly contained 5x less CS protein than wild-type sporozoites. They still produced a normal ratio of the 44 and 54 kDa forms of CS, which are thought to represent the surface molecule and intracellular precursor of CS, respectively.
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