Summary

RMgm-77
Malaria parasiteP. berghei
Genotype
Transgene
Transgene not Plasmodium: GFPmut2
Promoter: Gene model: PBANKA_0403200; Gene model (P.falciparum): PF3D7_0304600; Gene product: circumsporozoite (CS) protein (CSP)
3'UTR: Gene model: PBANKA_0403200; Gene product: circumsporozoite (CS) protein (CSP)
Insertion locus: Gene model: PBANKA_0403200; Gene product: circumsporozoite (CS) protein (CSP)
Phenotype Oocyst; Sporozoite; Liver stage;
Last modified: 21 August 2011, 15:55
  *RMgm-77
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 11422080
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/ResearcherR. Natarajan, R. Menard, K.D. Vernick
Name Group/DepartmentDepartment of Medical and Molecular Parasitology
Name InstituteNew York University School of Medicine
CityNew York
CountryUSA
Name of the mutant parasite
RMgm numberRMgm-77
Principal namePbFluspo; GFP P. berghei
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
OocystOocysts first displayed strong GFP-fluorescence beginning on day 5-6 post blood-meal. Oocyst-derived and salivary gland sporozoites show strong (bright) GFP-fluorescence.
SporozoiteOocyst-derived and salivary gland sporozoites show strong (bright) GFP-fluorescence with a cytoplasmic localization of GFP. GFP expression did not affect salivary gland invasion and invasion of HepG2 cells in vitro.
Liver stageGFP expression in the sporozoites did not affect salivary gland invasion and invasion of HepG2 cells in vitro. HepG2 cells still showed fluorescence 6 houtrs after invasion by the sporozoites.
Additional remarks phenotype

Mutant/mutation
The mutant expresses GFP under the control of the regulatory sequences of the gene encoding the circumsporozoite protein (CS).

Phenotype
Gametocytes, ookinetes and young oocysts show a very weak GFP fluorescence whereas maturing oocysts, sporozoites and young liver stages show strong GFP expression, correlating with expression of the CS protein.

GFP expression has no influence on sporozoite vaibility and infectivity.
This mutant has been used to analyse sporozoite biology. See for example Frevert, U. et al., 2005, PloS Biol. 3, e192.


  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameGFPmut2
Details of the genetic modification
Inducable system usedNo
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 GFP gene is introduced in the 5' untranslated region of the cs gene (1.4 kb upstream of the start codon) using an insertion vector that integrates by single cross-over homologous recombination. The GFP gene is under control of the regulatory sequences of the cs gene: 2.2 kb of cs 5'UTR and 321 bp of CS 3'UTR.
Additional remarks selection procedure
Other details transgene
Promoter
Gene Model of Parasite PBANKA_0403200
Gene Model P. falciparum ortholog PF3D7_0304600
Gene productcircumsporozoite (CS) protein
Gene product: Alternative nameCSP
Primer information details of the primers used for amplification of the promoter sequence  Click to view information
Primer information details of the primers used for amplification of the promoter sequence  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
3'-UTR
Gene Model of Parasite PBANKA_0403200
Gene productcircumsporozoite (CS) protein
Gene product: Alternative nameCSP
Primer information details of the primers used for amplification the 3'-UTR sequences  Click to view information
Primer information details of the primers used for amplification the 3'-UTR sequences  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
Insertion/Replacement locus
Replacement / InsertionInsertion locus
Gene Model of Parasite PBANKA_0403200
Gene productcircumsporozoite (CS) protein
Gene product: Alternative nameCSP
Primer information details of the primers used for amplification of the target sequences  Click to view information
Primer information details of the 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