RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-1590
Malaria parasiteP. berghei
Genotype
Transgene
 Transgene Plasmodium: Gene model: PBANKA_0501600; Gene model (P.falciparum): PF3D7_1017300; Gene product: golgi re-assembly stacking protein 1 (GRASP)
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/d-type unit))
Phenotype Liver stage;
Last modified: 15 September 2016, 18:36
  *RMgm-1590
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) : 27566438
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/ResearcherKaiser G; Heussler VT; Stanway RR
Name Group/DepartmentInstitute of Cell Biology
Name InstituteUniversity of Bern
CityBern
CountrySwitzerland
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Name of the mutant parasite
RMgm numberRMgm-1590
Principal namePbcGRASP-GFP
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
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Phenotype
Asexual blood stageNot tested
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageSee additional remarks phenotype
Additional remarks phenotype

Mutant/mutation
The mutant expresses an N-terminal GFP-tagged version of P. berghei GRASP. The transgene is integrated into the silent c/d-type rrna gene unit under control of the constitutive eef1a promoter.

Protein (function)
In the cells of most organisms, the cis-Golgi is found juxtaposed to the tER, to allow effective COPII-mediated protein transport between the ER and Golgi apparatus. As Plasmodium parasites possess a non-classical unstacked Golgi, the trans-face of the Golgi is found in close proximity to the cis-Golgi. The Golgi reassembling stacking protein (GRASP) and Rab6 are well-established marker proteins of the cis-Golgi and trans-Golgi, respectively, and have been used in P. falciparum for visualisation of both Golgi faces in the parasite’s blood stage.

Phenotype
In the paper another mutant expressing GFP-tagged GRASP is described and characterized ( PblsGRASP-GFP; RMgm-1590). In this mutant the grasp gene is under control of the promoter of lisp2, a liver stage specific gene. PblsGRASP-GFP parasites were fixed at different time points of liver stage development and the ER was stained with anti-PbBiP antiserum. Cis-Golgi faces are found at distinct ER sites, with the cis-Golgi being mostly found at ER accumulations.

See also  RMgm-1588 for a mutant expresses an N-terminal GFP-tagged version of P. berghei Sec61β. Sec61β is a known marker protein for endoplasmic reticulum (ER). 

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: Plasmodium
Gene Model of Parasite PBANKA_0501600
Gene Model P. falciparum ortholog PF3D7_1017300
Gene productgolgi re-assembly stacking protein 1
Gene product: Alternative nameGRASP
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Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct(Linear) plasmid 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 construct was based on pL0017 (Malaria Research and Reference Reagent Resource Center-MR4, as part of the BEI Resources Repository, MRA-786), which facilitates constitutive expression of GFP under the eef1αa promoter in P. berghei. Integration takes place by single cross-over homologous recombination into the c-ssu or d-ssu-RNA locus.

To generate PbcGRASP-GFP parasites, cDNA of Pbgrasp (PBANKA_0501600) was amplified using the primers 5’-ATA TGG ATC CAT GGG AGC AGG ACA AAC G-3’ and 5’-ATA TGG ATC CTT TAT CCC CAA TCA ATT CAT TAT AGC-3’. The fragment was cloned into pL0017 upstream of the gfp using BamHI to generate pcSec13p-GFP.
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/d-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: StudioDrie StudioDrie; S. Mededovic and A. van Wigcheren