RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5549
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_1411400; Gene model (P.falciparum): PF3D7_1313000; Gene product: Ubiquitin-like protein nedd8 homologue, putative (NEDD8)
Transgene
 Transgene not Plasmodium: GFP
Promoter: Gene model: Not available; Gene model (P.falciparum): Not available; Gene product: Not available
3'UTR: Gene model: Not available; Gene product: Not available
Replacement locus: Gene model: PBANKA_1411400; Gene product: Ubiquitin-like protein nedd8 homologue, putative (NEDD8)
Phenotype Fertilization and ookinete; Oocyst; Sporozoite;
Last modified: 24 September 2024, 18:18
  *RMgm-5549
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption, Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 38411954
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/ResearcherNayak B, Mishra S
Name Group/DepartmentDivision of Molecular Microbiology and Immunology
Name InstituteCSIR-Central Drug Research Institute
CityLucknow
CountryIndia
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Name of the mutant parasite
RMgm numberRMgm-5549
Principal nameNedd8 KO
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationNo
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Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNot different from wild type
Fertilization and ookineteZygotes are produced but no mature ookinetes. Zygotes exhibited small apical protrusions and were found to be developmentally arrested during the stage I–II transition.
OocystNo oocyst formation, no sporozoite formation.
SporozoiteNo oocyst formation, no sporozoite formation
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of NEDD8 and expresses GFP (under a constitutive promoter).

Protein (function)
Neddylation is a type of posttranslational modification known to regulate a wide range of cellular processes by covalently conjugating the ubiquitin-like protein Nedd8 to target proteins at lysine residues. The potential neddylation substrates are cullin (CUL) family members, which are implicated in controlling the cell cycle. Cullin neddylation leads to the activation of cullin-RING ubiquitin ligases, which regulate a myriad of biological processes through target-specific ubiquitylation.

Phenotype
Normal asexual blood stage development and production of gametocytes an gametes.
Zygotes are produced but no mature ookinetes. Zygotes exhibited small apical protrusions and were found to be developmentally arrested during the stage I–II transition.
No oocyst formation, no sporozoite formation.

Additional information

Other mutants

  Disrupted: Mutant parasite with a disrupted gene
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Details of the target gene
Gene Model of Rodent Parasite PBANKA_1411400
Gene Model P. falciparum ortholog PF3D7_1313000
Gene productUbiquitin-like protein nedd8 homologue, putative
Gene product: Alternative nameNEDD8
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Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct used(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
Partial or complete disruption of the geneComplete
Additional remarks partial/complete disruption
Selectable marker used to select the mutant parasitehdhfr/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationTo determine the function of Nedd8 in the P. berghei life cycle, the gene was disrupted using double crossover homologous recombination. For this purpose, two homologous fragments, F1 and F2, were amplified and cloned into the pBC-GFP-hDHFR:yFCU vector. The vector was linearized and transfected into P. berghei ANKA schizonts.
Additional remarks selection procedure
Primer information: Primers used for amplification of the target sequences  Click to view information
Primer information: 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
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6
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  Transgene: Mutant parasite expressing a transgene
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Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameGFP
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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 markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modification
Additional remarks selection procedure
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Other details transgene
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Promoter
Gene Model of Parasite Not available
Gene Model P. falciparum ortholog Not available
Gene productNot available
Gene product: Alternative name
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 / InsertionReplacement locus
Gene Model of Parasite PBANKA_1411400
Gene productUbiquitin-like protein nedd8 homologue, putative
Gene product: Alternative nameNEDD8
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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P.berghei model of malaria
Technical design and realization: S. Mededovic and A. van Wigcheren