RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-4404
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PbANKA_1308400; Gene model (P.falciparum): PF3D7_1444500; Gene product: eukaryotic initiation factor 2alpha kinase 1 (IK1; eIK1)
Transgene
 Transgene not Plasmodium: GFP
Promoter: Gene model: PbANKA_1308400; Gene model (P.falciparum): PF3D7_1444500; Gene product: eukaryotic initiation factor 2alpha kinase 1 (IK1; eIK1)
3'UTR: Gene model: Not available; Gene product: Not available
Replacement locus: Gene model: PbANKA_1308400; Gene product: eukaryotic initiation factor 2alpha kinase 1 (IK1; eIK1)
PhenotypeNo phenotype has been described
Last modified: 1 January 2018, 14:07
  *RMgm-4404
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) : 29241041
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 P. berghei ANKA cl15cy1
Other information parent lineA reference wild type clone from the ANKA strain of P. berghei (PubMed: PMID: 17406255).
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The mutant parasite was generated by
Name PI/ResearcherM. Zhang; V. Nussenzweig; W.J. Sullivan
Name Group/DepartmentDepartment of Pharmacology and Toxicology
Name InstituteIndiana University School of Medicine
CityIndianapolis
CountryUSA
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Name of the mutant parasite
RMgm numberRMgm-4404
Principal namePbeIK1(-)
Alternative namePbeIK1(-) ko
Standardized name
Is the mutant parasite cloned after genetic modificationYes
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Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of eukaryotic initiation factor- 2α (eIF2α) kinase (IK1). In addition it expresses GFP under control of the IK1 promoter

Protein (function)
One control mechanism of protein synthesis in eukaryotic cells involves the phosphorylation of the α-subunit of eukaryotic translation-initiation factor-2 (eIF-2α). During initiation of protein synthesis, a ternary complex of Met-tRNA, GTP, and the eukaryotic initiation factor 2 (eIF2) is delivered to the translation initiation machinery. During this translation process, eIF2-GTP is hydrolyzed to eIF2-GDP and released from the machinery. A guanine nucleotide exchange factor (eIF2B) facilitates the exchange of eIF2-GDP to eIF2-GTP, which is a requisite for a new round of translation. The phosphorylation of eIF2α blocks the recycling of eIF2-GTP and downregulates protein synthesis. The phosphorylation of eIF2α is associated with the appearance of stress granules in the cytoplasm of stressed cells.

Three eIF2α kinases, IK1 (PF14_0423; eukaryotic initiation factor 2alpha kinase 1), IK2 (PfA0380w; PBANKA_020580; serine/threonine protein kinase, putative), and PK4 (PFF1370w; PBANKA_112690; protein kinase PK4), have been identified in Plasmodium (Fenell C et al. 2009. Malar J. 8, 99).

Phenotype
Three eIF2a kinases have been identified in Plasmodium: eIK1, eIK2, and PK4. We determined whether parasites lacking each of these eIF2a kinases exhibited a defect in recrudescence following artemisinine (ART) treatment. We generated PbeIK1 (-) parasites, and our generation of PbeIK2 (-) parasites was described earlier (Zhang et al., 2010)(RMgm-482). PbeIK1 (-) and PbeIK2 (-) parasites proliferated normally in erythrocytes and no defect was observed in the enhanced eIF2a phosphorylation in response to ART treatment. Moreover, mice infected with either PbeIK1 (-) or PbeIK2 (-) parasites and treated with three or five doses of ART showed the same degree of recrudescence as wild-type. These results show that neither PbeIK1 nor PbeIK2 contributes to AS-induced latency.

Other mutants

  Disrupted: Mutant parasite with a disrupted gene
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Details of the target gene
Gene Model of Rodent Parasite PbANKA_1308400
Gene Model P. falciparum ortholog PF3D7_1444500
Gene producteukaryotic initiation factor 2alpha kinase 1
Gene product: Alternative nameIK1; eIK1
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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 genePartial
Additional remarks partial/complete disruption PbeIK1 knockouts were generated using the same method that we previously described for generation of PbeIK2 (-) parasites (Zhang et al., 2010). The 30- terminal of PbeIK1 coding sequence in the genome (PbANKA_1308400, 4.5kb) is adjacent (less than 500 bp) to PbANKA_130830. We deleted the 50- terminus (2.5-kb) of the PbeIK1 coding sequence to avoid interference with the downstream gene. The 2.5-kb of the 50-portion of this gene encodes the entire eIF2a kinase catalytic domain (Zhang et al., 2010).

The replacement plasmid pBC_PbeIK1KO contains a 1-kb DNA fragment encoding the 5’-UTR of PbeIK1, the GFP cassette, the mutated dihydrofolate reductase (DHFR) gene that confers resistance to pyrimethamine, and a 1-kb DNA fragment encoding the PbeIK1 CDS (position 2500bp – 3500bp).
Selectable marker used to select the mutant parasitehdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationPbeIK1 knockouts were generated using the same method that we previously described for generation of PbeIK2 (-) parasites (Zhang et al., 2010). The 30- terminal of PbeIK1 coding sequence in the genome (PbANKA_1308400, 4.5kb) is adjacent (less than 500 bp) to PbANKA_130830. We deleted the 50- terminus (2.5-kb) of the PbeIK1 coding sequence to avoid interference with the downstream gene. The 2.5-kb of the 50-portion of this gene encodes the entire eIF2a kinase catalytic domain (Zhang et al., 2010).

The replacement plasmid pBC_PbeIK1KO contains a 1-kb DNA fragment encoding the 5’-UTR of PbeIK1, the GFP cassette, the mutated dihydrofolate reductase (DHFR) gene that confers resistance to pyrimethamine, and a 1-kb DNA fragment encoding the PbeIK1 CDS (position 2500bp – 3500bp).
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
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationPbeIK1 knockouts were generated using the same method that we previously described for generation of PbeIK2 (-) parasites (Zhang et al., 2010). The 30- terminal of PbeIK1 coding sequence in the genome (PbANKA_1308400, 4.5kb) is adjacent (less than 500 bp) to PbANKA_130830. We deleted the 50- terminus (2.5-kb) of the PbeIK1 coding sequence to avoid interference with the downstream gene. The 2.5-kb of the 50-portion of this gene encodes the entire eIF2a kinase catalytic domain (Zhang et al., 2010).

The replacement plasmid pBC_PbeIK1KO contains a 1-kb DNA fragment encoding the 5’-UTR of PbeIK1, the GFP cassette, the mutated dihydrofolate reductase (DHFR) gene that confers resistance to pyrimethamine, and a 1-kb DNA fragment encoding the PbeIK1 CDS (position 2500bp – 3500bp).
Additional remarks selection procedure
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Other details transgene
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Promoter
Gene Model of Parasite PbANKA_1308400
Gene Model P. falciparum ortholog PF3D7_1444500
Gene producteukaryotic initiation factor 2alpha kinase 1
Gene product: Alternative nameIK1; eIK1
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_1308400
Gene producteukaryotic initiation factor 2alpha kinase 1
Gene product: Alternative nameIK1; eIK1
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