RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5163
Malaria parasiteP. yoelii
Genotype
Transgene
 Transgene Plasmodium: Gene model: PY17X_1462100; Gene model (P.falciparum): PF3D7_1246400; Gene product: myosin light chain 1 | myosin A tail domain interacting protein (MTIP)
Promoter: Gene model: PBANKA_1324300; Gene model (P.falciparum): PF3D7_1460600; Gene product: inner membrane complex sub-compartment protein 3 (ISP3)
3'UTR: Gene model: Not available; Gene product: Not available
Phenotype Asexual bloodstage;
Last modified: 4 July 2022, 13:18
  *RMgm-5163
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) : 35775739
MR4 number
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Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. yoelii
Parent strain/lineP. y. yoelii 17XNL
Name parent line/clone Not applicable
Other information parent line
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The mutant parasite was generated by
Name PI/ResearcherQuian P, Yuan J
Name Group/DepartmentState Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signal Network, School o
Name InstituteXiamen University
CityXiamen
CountryChina
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Name of the mutant parasite
RMgm numberRMgm-5163
Principal namesee below
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationNo
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Phenotype
Asexual blood stageIMC location in blood stages
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The gene was tagged with a 6HA at the N- or C-terminus and driven by the promoter of gene isp3 (PBANKA_1324300) for episomal expression in the asexual blood stages.

Published in: bioRxiv preprint doi: https://doi.org/10.1101/2022.01.28.478263

Protein (function)
Biotin ligase (TurboID)-based proximity labelling was used to compile the proteome of the schizont Inner Membrane Complex (IMC) of rodent malaria parasite Plasmodium yoelii. In total, 300 TurboID-interacting proteins were identified. 19 of the 22 selected candidates were confirmed to localize in the IMC. To assess whether the identified proteins are indeed localized in the IMC, 22 candidates were selected among the 300 Tb-IMC interacting proteins for subcellular localization analysis. 

Phenotype
Biotin ligase (TurboID)-based proximity labelling was used to compile the proteome of the schizont Inner Membrane Complex (IMC) of rodent malaria parasite Plasmodium yoelii. In total, 300 TurboID-interacting proteins were identified. 19 of the 22 selected candidates were confirmed to localize in the IMC. To assess whether the identified proteins are indeed localized in the IMC, 22 candidates were selected among the 300 Tb-IMC interacting proteins for subcellular localization analysis. 

Among these proteins, the orthologues of 8 proteins have been experimentally validated to be IMC-residing or association in the schizonts of P. berghei or P.  falciparum,
PY17X_ 0314700 (CDPK1),
PY17X_0617900 (CDPK4),
PY17X_1440500 (PKAr),
PY17X_0839000 (PKAc),
PY17X_1420600 (Rab11A),
PY17X_1462100 (MTIP),
PY17X_0206000 (PhIL1),
PY17X_0525300 (GAPM2) 

IMC localization or association of the 14 other candidates have not been well characterized in the Plasmodium
PY17X_0207400 (no IMC location)
PY17X_0312400 (IMC)
PY17X_0417300 (no IMC location)
PY17X_0418000  (IMC)
PY17X_0812700  (IMC)
PY17X_0917100 (no IMC location)
PY17X_1131200  (IMC)
PY17X_1139700  (IMC)
PY17X_1220300  (IMC)
PY17X_1348200  (IMC)
PY17X_1359500  (IMC)
PY17X_1411000  (IMC)
PY17X_1441500  (IMC)
PY17X_1453100  (IMC).
Immunoblot assays were used to detect each protein, all displaying a band fitting their expected molecular weight. As expected, immunofluorescence assays (IFA) showed clear co-localization of the 8 known proteins (CDPK1, CDPK4, PKAr, PKAc, Rab11A, MTIP, PhIL1, 247 and GAPM2) with the IMC marker GAP45.

Among the 14 newly characterized candidates, 11 of them (PY17X_0312400, PY17X_0418000, PY17X_0812700, PY17X_1131200, PY17X_1139700, PY17X_1220300, PY17X_1348200, PY17X_1359500, PY17X_1411000, PY17X_1441500, and PY17X_1453100) displayed the IMC or IMC-like pellicle localization, while 3 other candidates (PY17X_0207400, PY17X_0417300 and PY17X_0917100) did not. Collectively, we confirmed the IMC or IMC-like localization of 19 proteins from the 22 candidates in the P. yoelii schizonts. 

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 PY17X_1462100
Gene Model P. falciparum ortholog PF3D7_1246400
Gene productmyosin light chain 1 | myosin A tail domain interacting protein
Gene product: Alternative nameMTIP
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Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/constructCircular plasmid
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 markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationCRISPR/Cas9 plasmid pYCm was used for parasite genomic modification.
To construct the plasmids for gene tagging, the DNA fragment encoding 6HA was inserted between the left and right arms in frame with the gene of interest. For each gene tagging, two sgRNAs were designed to target sites close to the N- or C-terminal part of the coding region.
Each candidate gene was tagged with a 6HA at the N- or C-terminus and driven by the promoter of gene isp3 (PBANKA_1324300) for episomal expression in the asexual blood stages.
Additional remarks selection procedure
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Other details transgene
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Promoter
Gene Model of Parasite PBANKA_1324300
Gene Model P. falciparum ortholog PF3D7_1460600
Gene productinner membrane complex sub-compartment protein 3
Gene product: Alternative nameISP3
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 / InsertionNot available
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