RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-4677
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_0311400; Gene model (P.falciparum): PF3D7_0214600; Gene product: serine/threonine protein kinase, putative (stk2)
Transgene
 Transgene not Plasmodium: GFP
Promoter: Gene model: PBANKA_0711900; Gene model (P.falciparum): PF3D7_0818900; Gene product: heat shock protein 70 (HSP70)
3'UTR: Gene model: PBANKA_0711900; Gene product: heat shock protein 70 (HSP70)
Replacement locus: Gene model: PBANKA_0311400; Gene product: serine/threonine protein kinase, putative (stk2)
Phenotype Sporozoite; Liver stage;
Last modified: 29 August 2019, 16:08
  *RMgm-4677
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) : 31444161
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/ResearcherJillapalli R, Kumar KA
Name Group/DepartmentDepartment of Animal Biology, School of Life Sciences
Name InstituteUniversity of Hyderabad
CityHyderabad
CountryIndia
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Name of the mutant parasite
RMgm numberRMgm-4677
Principal namePbstk2KO
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
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Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNot different from wild type
Fertilization and ookineteNot different from wild type
OocystNot different from wild type
SporozoiteNormal sporozoite production. Pbstk2 KO sporozoites exhibit normal gliding motility, cell traversal activity, and infection of hepatocytes in vitro.
Liver stageNormal sporozoite production. Pbstk2 KO sporozoites exhibit normal gliding motility, cell traversal activity, and infection of hepatocytes in vitro. Prolonged prepatent period after infection of mice by intravenous injection of sporozoites. Evidence is presented for reduced hepatic schizogony and merozoite maturation (reduced MSP1 expression).
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of stk2 and expresses GFP under the constitutive hsp70 promoter

Protein (function)
Serine/threonine protein kinase, putative.
Bioinformatic analysis predicted one or more calmodulin binding domains in PbSTK2 suggesting that it belongs to the family of CaMK. CaMKs are activated by an increase in intracellular concentration of Ca2+ ions leading to the transfer of phosphates from ATP to defined serine or threonine residues of the substrate proteins.

Phenotype
Normal sporozoite production. Pbstk2 KO sporozoites exhibit normal gliding motility, cell traversal activity, and infection of hepatocytes in vitro. Prolonged prepatent period after infection of mice by intravenous injection of sporozoites. Evidence is presented for reduced hepatic schizogony and merozoite maturation (reduced MSP1 expression).

Additional information

Other mutants 
See also PF3D7_0214600 for other mutants and negative attempts to disrupt this gene

  Disrupted: Mutant parasite with a disrupted gene
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Details of the target gene
Gene Model of Rodent Parasite PBANKA_0311400
Gene Model P. falciparum ortholog PF3D7_0214600
Gene productserine/threonine protein kinase, putative
Gene product: Alternative namestk2
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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
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationDouble homologous recombination strategy was used to delete Pbstk2 locus. To achieve this, Pbstk2 KO targeting construct was generated using pBC-GFP-hDHFR vector. Plasmodium berghei genomic DNAwas used as a template to amplify 725 bp of 5′ upstream region of Pbstk2 using Pbstk2 5′ forward primer-FP1 and Pbstk2 5′ reverse primer-RP1. A 619 bp of 3′ downstream region of Pbstk2 was also amplified using Pbstk2 3′ forward primer-FP2 and Pbstk2 3′ reverse primer-RP2. The PCR product corresponding to 5′ and 3′ regions were confirmed by sequencing. The 5′ and 3′ PCR amplified products were cloned into the pBC-GFP-hDHFR vector at XhoI/ClaI and NotI/AscI sites, respectively. The knockout plasmid was digested with XhoI/AscI and gel purified.
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 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 PBANKA_0711900
Gene Model P. falciparum ortholog PF3D7_0818900
Gene productheat shock protein 70
Gene product: Alternative nameHSP70
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_0711900
Gene productheat shock protein 70
Gene product: Alternative nameHSP70
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_0311400
Gene productserine/threonine protein kinase, putative
Gene product: Alternative namestk2
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