RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5356
Malaria parasiteP. berghei
Genotype
Transgene
 Transgene Plasmodium: Gene model: PBANKA_1128300; Gene model (P.falciparum): PF3D7_0629500; Gene product: amino acid transporter AAT1 (AAT1)
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;
Last modified: 24 May 2023, 14:27
  *RMgm-5356
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) : 36976018
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/ResearcherAnand A, Nagaraj VA
Name Group/DepartmentInfectious Disease Biology
Name InstituteInstitute of Life Sciences
CityBhubaneswar, Odisha
CountryIndia
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Name of the mutant parasite
RMgm numberRMgm-5356
Principal namePbWT(+GFP-AAT1)
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
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Phenotype
Asexual blood stageLive fluorescence imaging performed with PbWT(+GFP-AAT1) parasites showed a faint signal probably due to the loss of GFP fluorescence in the acidic environment of food vacuoles (FVs). Indirect immunofluorescence using GFP antibodies showed the colocalization of GFP-AAT1 with discrete FVs containing hemozoin (Hz) in PbWT(+GFP-AAT1) parasites, suggesting the FV localization of PbAAT1.
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses an N-terminal GFP-tagged version of AAT1. This transgene is introduced by single cross-over into the small subunit ribosomal rna gene (c-type unit). 

Protein (function)
The P. berghei genome encodes a putative AAT1 (PBANKA_1128300) consisting of 614 amino acids. Its orthologue in P. falciparum, PfAAT1 (PF3D7_0629500), has been shown to localize in the food vacuole (FV). It contains 10 to 11 predicted transmembrane domains.

Phenotype
Analysis of a mutant lacking AAT1 (RMgm-5357) provided evidence for the following: 
- A slight growth delay of asexual blood stages in mice
- Reduction in hemozoin production, and the hemozoin crystals display a thin morphology compared with wild-type parasites.
- Reduced sensitivity to chloroquine and amodiaquine.
- Mice infected with the knockout parasites show reduced neuronal inflammation and cerebral complications 

Live fluorescence imaging performed with PbWT(+GFP-AAT1) parasites showed a faint signal probably due to the loss of GFP fluorescence in the acidic environment of food vacuoles (FVs). Indirect immunofluorescence using GFP antibodies showed the colocalization of GFP-AAT1 with discrete FVs containing hemozoin (Hz) in PbWT(+GFP-AAT1) parasites, suggesting the FV localization of PbAAT1.

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_1128300
Gene Model P. falciparum ortholog PF3D7_0629500
Gene productamino acid transporter AAT1
Gene product: Alternative nameAAT1
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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 modificationGeneration of PbWT(+GFP-AAT1) parasites: pL0027 plasmid containing a GFP-Luc-expressing cassette and TgDHFR-TS as a selectable marker was used to generate PbWT1GFP-AAT1 transgenic parasites by replacing luciferase in the plasmid with AAT1. For this, PbAAT1 was amplified from PbWT genomic DNA using Phusion DNA polymerase with the following forward and reverse primers, respectively: 59-GCCAGGATCCTGATGGCTAATAATATAGATATATTAGATTATTGTC-39 and 59-CCCTCTAGACTAGTATATTATAATAAATGCGGTAACTATAGATG-39. The PCR product was digested with the respective enzymes and cloned into pL0027 plasmid digested with BamHI and XbaI to excise the luciferase sequence. The recombinant plasmid was then linearized by digesting with ApaI and SacII, and the transfection was performed in PbWT parasites.
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