RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-906
Malaria parasiteP. berghei
Genotype
MutatedGene model (rodent): PBANKA_0403200; Gene model (P.falciparum): PF3D7_0304600; Gene product: circumsporozoite (CS) protein (CS; CSP)
Details mutation: Chimeric cs gene: the repeat region of cs is replaced with the repeat region of P. vivax (VK210) cs
PhenotypeNo phenotype has been described
Last modified: 16 June 2013, 13:11
  *RMgm-906
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) : 23716612
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone Not applicable
Other information parent line
The mutant parasite was generated by
Name PI/ResearcherEspinosa DA, Zavala F
Name Group/DepartmentJohns Hopkins Malaria Research Institute
Name InstituteJohns Hopkins Bloomberg School of Public Health, Johns Hopkins University
CityBaltimore, Maryland
CountryUS
Name of the mutant parasite
RMgm numberRMgm-906
Principal namePb-Pv
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
OocystNot different from wild type
SporozoiteNot different from wild type
Liver stageNot different from wild type
Additional remarks phenotype

Mutant/mutation
A mutant with the endogenous cs gene replaced by a chimeric cs gene: the central repeat region of P. berghei cs is replaced by the repeat region of P. vivax (VK210) cs (CS is comprised of an immunodominant central repeat region, diverse between Plasmodium species, flanked by two conserved domains: Region I at the N-terminus of the repeats, and a type I thrombospondin repeat (TSR) motif C-terminal to the 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
Normal numbers of chimeric salivary gland sporozoites are produced and sporozoites show normal infectivity to mice.

Additional information
The chimeric sporozoites are recognized by a monoclonal antibody, 2F2, specific for the P. vivax VK210 CS repeats

Using this transgenic parasite, it is demonstrated that monoclonal and polyclonal antibodies against P. vivax CS strongly inhibit sporozoite infectivity and thus support the notion that these antibodies play an important role in protective immunity. The chimeric parasites reported represent a model for evaluating protective immune responses against P. vivax vaccines based on CS.

Other mutants


  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 nameCS; CSP
Details of the genetic modification
Short description of the mutationChimeric cs gene: the repeat region of cs is replaced with the repeat region of P. vivax (VK210) cs
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 transfection plasmid, pR-CSPv, carrying the repeat region of P. vivax was derived from plasmid pR-CSwt that encodes for the CS gene of P. berghei (wildtype) and results from the combination of plasmids pIC-CSwt and a synthetic gene pPv (Retrogen Inc., CA) encoding the VK210 repeat motif based on the Salvador I (Sal I) strain of P. vivax. Briefly, a PmlI-SexAI 567-bp fragment was excised from pIC-CSwt and replaced with a PmlI-SexAI 675-bp fragment, comprising the P. vivax (VK210) CSP repeat region that was codon harmonized to optimize protein synthesis and folding in the P. berghei transgenic host and was excised from the engineered pPv. From the resulting intermediate plasmid, pIC-CSPv, the entire CSP gene was excised as a KpnI-XhoI fragment and inserted into the transfection plasmid, pR-CSPv. KpnI and SacI were used to linearize pR-CSPv prior to transfection.
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