RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-1243
Malaria parasiteP. berghei
Genotype
Transgene
 Transgene not Plasmodium: dtTomato
Promoter: Gene model: PBANKA_0711900; Gene model (P.falciparum): PF3D7_0818900; Gene product: heat shock protein 70 (HSP70)
3'UTR: Gene model: PBANKA_1340000; Gene product: dihydrofolate synthase/folylpolyglutamate synthase, putative (PbDHFS-FPGS)
Replacement locus: Gene model: Not available; Gene product: Not available
Phenotype Asexual bloodstage;
Last modified: 5 April 2015, 16:49
  *RMgm-1243
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) : 25831536
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/ResearcherSturm A; McFadden GI
Name Group/DepartmentPlant Cell Biology Research Centre, School of BioSciences
Name InstituteUniversity of Melbourne
CityMelbourne
CountryAustralia
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Name of the mutant parasite
RMgm numberRMgm-1243
Principal nameG6Tm
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
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Phenotype
Asexual blood stage(Strong) dtTomato expression in all stages.
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutants (strongly) express dtTomato in blood stages under the control of the constitutive and strong hsp70 promoter (other stages have not been analysed). The G6Tm mutant does not contain a drug-selectable marker. The dtTomato-gene has been integrated by double cross-over integration into a silent intergenic region on chromosome 6.

A modified pBAT-SIL6 plasmid (see RMgm-757), was used to generate the transgenic parasites line G6Tm. The GFP gene of the pBATSIL6 plasmid was swapped with the dtTomato gene.

The plasmid is integrated by double cross-over integration in a locus on P. berghei chromosome 6 between two hypothetical open reading frames (PBANKA_061210 and PBANKA_061220), both displaying only limited gene expression in the stages studied thus far (see RMgm-757).

After negative selection of G6 parasites the G6Tm which lacks the  hdhfr/yfcu selectable marker

Protein (function)

Phenotype
The mutants (strongly) express dtTomato in blood stages under the control of the constitutive and strong hsp70 promoter (other stages have not been analysed).

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 namedtTomato
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Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct(Linear) plasmid 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 parasitehdhfr/yfcu
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modificationAfter transfection with the modified pBATSIL6 vector (RMgm-757), where the gfp gene was swapped with the dtTomato gene, trangenic parasites were selected and cloned. After cloning the hdhfr/yfcu selectable marker was removed by applying negative selection.
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 PBANKA_1340000
Gene productdihydrofolate synthase/folylpolyglutamate synthase, putative
Gene product: Alternative namePbDHFS-FPGS
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 / InsertionReplacement locus
Gene Model of Parasite Not available
Gene productNot available
Gene product: Alternative name
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