RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-1023
Malaria parasiteP. berghei
Genotype
Transgene
 Transgene not Plasmodium: TN-XXL, a fluorescence resonance energy transfer (FRET) biosensor
Promoter: Gene model: PBANKA_0711900; Gene model (P.falciparum): PF3D7_0818900; Gene product: heat shock protein 70 (HSP70)
3'UTR: Gene model: Not available; Gene product: Not available
Insertion locus: Gene model: Not available; Gene product: Not available (integration into intergenic region of chromosome 12)
Phenotype Sporozoite;
Last modified: 13 April 2014, 16:16
  *RMgm-1023
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) : 24617597
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 P. berghei ANKA cl15cy1
Other information parent lineA reference wild type clone from the ANKA strain of P. berghei (PubMed: PMID: 17406255).
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The mutant parasite was generated by
Name PI/ResearcherCarey, AF; Amino, R
Name Group/DepartmentUnité de Biologie et Genétique du Paludisme
Name InstituteInstitut Pasteur
CityParis
CountryFrance
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Name of the mutant parasite
RMgm numberRMgm-1023
Principal nameTN-XXL FRET
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
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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
SporozoiteThis parasite expresses TN-XXL (a fluorescence resonance energy transfer (FRET) biosensor) under control of the hsp70 promoter and was used to monitor Ca2+ dynamics in gliding sporozoites by time-lapse fluorescence imaging.
Liver stageNot different from wild type
Additional remarks phenotype

Mutant/mutation
The mutant expresses TN-XXL (a fluorescence resonance energy transfer (FRET) biosensor) under control of the hsp70 promoter. The transgene is integrated into an intergenic region on chromosome 12.

Protein (function)
TN-XXL, a fluorescence resonance energy transfer (FRET) biosensor which has been used to monitor Ca2+ dynamics in mammalian cells. The TN-XXL indicator consists of a mutagenized troponin C domain linking a yellow (YFP) and cyan fluorescent protein (CFP). When the troponin domain binds Ca2+, a conformational change occurs that places the two fluorescent proteins into close proximity such that the efficiency of FRET increases, mounting the ratio between YFP and CFP emission. A similar FRET-based sensor has been used to monitor intracellular Ca2+ during P. berghei sporozoite transformation (Doi, Shinzawa et al. 2011). This parasite was used to monitor Ca2+ dynamics in gliding sporozoites by time-lapse fluorescence imaging

Phenotype
This parasite was used to monitor Ca2+ dynamics in gliding sporozoites by time-lapse fluorescence imaging

Additional information

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 nameTN-XXL, a fluorescence resonance energy transfer (FRET) biosensor
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Details of the genetic modification
Inducable system usedNo
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 parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe TN-XXL FRET cassette (Mank, Santos et al. 2008) was cloned into the vector Pb237 (derived from b3D+) and flanked by the PbHsp70 5’ promoter region and the PbDHFS 3’ untranslated region (Deligianni, Morgan et al. 2011). The vector contains two homologous regions to target integration by double crossover at an intergenic locus on chromosome 12 (820541-820841 bp).
Chr12a and Chr12b (Chromosome 12, 820043 - 820541 bp and 820841 – 821272 bp respectively) are the homology regions used to integrate the linearized vector via double homologous recombination. The sequence between these homology regions is replaced with the vector, leading to expression of TN-XXL under the control of the PbHSP70 promotor region.
Additional remarks selection procedure
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Other details transgene
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Promoter
Gene Model of Parasite PBANKA_0711900
Gene Model P. falciparum ortholog PF3D7_0818900
Gene productheat shock protein 70
Gene product: Alternative nameHSP70
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 Not available
Gene productNot available
Gene product: Alternative name
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 nameintegration into intergenic region of chromosome 12
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