Summary

RMgm-295
Malaria parasiteP. berghei
Genotype
Transgene
Transgene not Plasmodium: tdTomato (RFP)
Promoter: Gene model: PBANKA_1133300; Gene model (P.falciparum): PF3D7_1357100; Gene product: elongation factor 1-alpha (eef1a)
3'UTR: Gene model: PBANKA_0719300; Gene product: bifunctional dihydrofolate reductase-thymidylate synthase, putative (dhfr/ts)
Insertion locus: Gene model: Not available; Gene product: Not available (small subunit ribosomal rna gene (c-type unit))
Phenotype Asexual bloodstage; Oocyst; Sporozoite; Liver stage;
Last modified: 26 June 2009, 17:04
  *RMgm-295
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) : 19492329
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/ResearcherS. Graewe, V.T. Heussler
Name Group/DepartmentDepartment of Parasitology
Name InstituteBernhard Nocht Institute for Tropical Medicine
CityHamburg
CountryGermany
Name of the mutant parasite
RMgm numberRMgm-295
Principal nameP. berghei-tdTomato
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stagetdTomato expression in blood stages
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocysttdTomato expression in oocysts
SporozoitetdTomato expression in sporozoites
Liver stagetdTomato expression in liver stages
Additional remarks phenotype

Mutant/mutation
The mutant expresses tdTomato under the control of the constitutive promoter of the elongation factor 1 alpha (eef1a) gene of P. berghei.

Protein (function)
tdTomato is a red fluorescent protein (RFP).

Phenotype
tdTomato is expressed in blood, mosquito and liver stages and expression of the transgene has no influence on parasite viability and infectivity.

Additional information
Comparison of the absolute fluorescence intensity (measured as MFI) of mutants expressing  the red fluorescent proteins RedStar (RMgm-86), tdTomato and mCherry (RMgm-296) revealed that tdTomato and mCherry are significantly brighter even when imaged at a lower laser intensity than RedStar expressing parasites.
The observations of the photostability of all three RFP proteins show that in general, they bleach far more rapidly in intravital than in in vitro live imaging of different parasite stages..
In intravital imaging experiments mCherry is far more photostable than RedStar and tdTomato.

Other mutants


  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nametdTomato (RFP)
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 parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe transgenic line expressing tdTomato was generated by subcloning the tdTomato coding sequences from the supplied pRSET-B plasmid; these sequences were subsequently inserted into the Plasmodium berghei expression plasmid pL0017 (www.mr4.org), replacing the GFP coding sequence with tdTomato.
Additional remarks selection procedure
Other details transgene
Promoter
Gene Model of Parasite PBANKA_1133300
Gene Model P. falciparum ortholog PF3D7_1357100
Gene productelongation factor 1-alpha
Gene product: Alternative nameeef1a
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_0719300
Gene productbifunctional dihydrofolate reductase-thymidylate synthase, putative
Gene product: Alternative namedhfr/ts
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 Not available
Gene productNot available
Gene product: Alternative namesmall subunit ribosomal rna gene (c-type unit)
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