RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-1407
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PBANKA_1438300; Gene model (P.falciparum): PF3D7_1223400; Gene product: phospholipid-transporting ATPase, putative (ATP8)
Name tag: mCherry-3xMyc
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: Not available; Gene product: Not available
Replacement locus: Gene model: PBANKA_1438300; Gene product: phospholipid-transporting ATPase, putative (ATP8)
Phenotype Asexual bloodstage;
Last modified: 5 April 2016, 12:02
  *RMgm-1407
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene tagging, Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 26796412
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/ResearcherKooij TWA; Kenthirapalan S
Name Group/DepartmentMolecular Parasitology - Departement of Medical Microbiology
Name InstituteRadboud University Medical Centre
CityNijmegen
CountryThe Netherlands
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Name of the mutant parasite
RMgm numberRMgm-1407
Principal nameatp8::tag
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
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Phenotype
Asexual blood stageModerate mCherry expression at the parasite-host interface (as visualized by fluorescence microscopy).
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses a C-terminal mCherry-3xMyc-tagged version of ATP8

Protein (function)
Membrane transport proteins (MTP) transfer compounds across biological membranes and encompass diverse gene families, namely ion channels, ATP-dependent pumps and secondary active porters including those of the major facilitator superfamily. In contrast to bacteria, archaea and fungi, parasitic protozoa such as Trypanosoma brucei and the malaria parasite Plasmodium falciparum allocate only a small proportion of their genomes (2–3%) to membrane transport. P. falciparum encodes at least 122 MTPs. For 39 gene products, functional or subcellular localization predictions remain elusive, rendering them orphan MTPs.
In this study a comprehensive genetic analysis is reported of 35 orphan transport proteins of Plasmodium berghei during its life cycle in mice and Anopheles mosquitoes.

Phenotype
Moderate mCherry expression at the parasite-host interface (as visualized by fluorescence microscopy).

Additional information

Other mutants

  Tagged: Mutant parasite with a tagged gene
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Details of the target gene
Gene Model of Rodent Parasite PBANKA_1438300
Gene Model P. falciparum ortholog PF3D7_1223400
Gene productphospholipid-transporting ATPase, putative
Gene product: Alternative nameATP8
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Details of the genetic modification
Name of the tagmCherry-3xMyc
Details of taggingC-terminal
Additional remarks: tagging
Commercial source of tag-antibodies
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 markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationFragments of 400–650 bp in the 3' flanking regions of the genes of interest were amplified by using the 3' fragment forward and reverse primer combinations (3'MTP-F-AvrII and 3'MTP-R-KpnI). The 3' fragments were cloned using the indicated restriction sites into the pBAT-SIL6 vector, resulting in intermediate vectors (pMTP-IM).
For completion of the tagging vectors (pMTP-tag), 400–650 bp fragments at the carboxy-terminal (CT) ends of the coding sequences were amplified using the CT fragment forward and reverse primer combinations (CT-MTP-F-SacII and CTMTP-R-HpaI) and cloned into the pMTP-IM vectors using SacII and HpaI resulting in in-frame fusions of the CT coding regions with the mCherry-3xMyc tag.
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 markerpbdhfr
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 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_1438300
Gene productphospholipid-transporting ATPase, putative
Gene product: Alternative nameATP8
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