RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-296
Malaria parasiteP. berghei
Genotype
Transgene
 Transgene not Plasmodium: mCherry (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, 17:02
  *RMgm-296
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
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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
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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
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Name of the mutant parasite
RMgm numberRMgm-296
Principal nameP. berghei-mCherry
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
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Phenotype
Asexual blood stagemCherry expression in blood stages
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocysttdTomato expression in blood stages
SporozoitemCherry expression in blood stages
Liver stagemCherry expression in blood stages
Additional remarks phenotype

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

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

Phenotype
mCherry 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 (RMgm-295) and mCherry  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
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Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene namemCherry (RFP)
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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 mCherry was generated by subcloning the mCherry 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 mCherry.
Additional remarks selection procedure
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Other details transgene
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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
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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
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Conceptual design of the database: Dr. C.J. Janse (Leiden Malaria Research Group, Leiden, The Netherlands).
Technical design and realization: S. Mededovic and A. van Wigcheren