RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-910
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PBANKA_0822300; Gene model (P.falciparum): PF3D7_0921400; Gene product: NifU-like scaffold protein (NifU; NFUapi)
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: PBANKA_1340000; Gene product: dihydrofolate synthase/folylpolyglutamate synthase, putative (PbDHFS-FPGS)
Replacement locus: Gene model: PBANKA_0822300; Gene product: NifU-like scaffold protein (NifU; NFUapi)
Phenotype Oocyst; Liver stage;
Last modified: 2 July 2013, 12:51
  *RMgm-910
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) : 23805304
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/ResearcherJ.M. Haussig, K. Matuschewski, T.W.A. Kooij
Name Group/DepartmentParasitology Unit
Name InstituteMax Planck Institute for Infection Biology
CityBerlin
CountryGermany
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Name of the mutant parasite
RMgm numberRMgm-910
Principal nameNFU::tag
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
OocystLive parasites only produced faint undefined signals in developing midgut-associated oocysts and cultured liver stage parasites. Immunofluorescence analyses of mature liver stages using anti-cMyc antibodies showed the extended branched structure reminiscent of the apicoplast.
SporozoiteNot different from wild type
Liver stageLive parasites only produced faint undefined signals in developing midgut-associated oocysts and cultured liver stage parasites. Immunofluorescence analyses of mature liver stages using anti-cMyc antibodies showed the extended branched structure reminiscent of the apicoplast.
Additional remarks phenotype

Mutant/mutation
The mutant expresses a mCherry-3xMyc tagged version of NFUapi and expresses GFP under the control of the constitutive and strong hsp70 promoter. The construct to tag the nfuapi gene contains a GFP expression cassette. In this cassette the gfp gene is under the control of the hsp70 promoter (see for more information on the basic plasmid pBAT-SIL6 the mutant RMgm-757)

Protein (function)
Iron-sulfur [Fe-S] clusters are inorganic cofactors that constitute one of the most ancient and ubiquitous prosthetic groups. Proteins containing [Fe-S] clusters have diverse functions. In eukaryotes, different biogenesis machineries have been identified that assemble [Fe-S] clusters in various cellular compartments, namely the cytoplasmic iron-sulfur protein assembly (CIA), the mitochondrial ironsulfur cluster (ISC), and the plastid-localized sulfur utilization factor (SUF).
Plasmodium species harbor genes that could be involved in all three [Fe-S] cluster biosynthetic pathways found in eukaryotes. Malaria parasites are equipped with components of the SUF system, which are predicted to target to the vestigial plastid. [Fe-S] cluster-containing proteins in the apicoplast are central to several biosynthesis pathways, including mevalonate independent isoprenoid biosynthesis, lipoic acid metabolism, and biogenesis of [Fe-S] clusters itself.
NFUapi is a NifUlike domain containing protein (NFU) in the [Fe-S] biosynthesis pathway of the Plasmodium. Malaria parasite genomes encode three NifU-like domain containing proteins, related to the bacterial NIF system. Two of these were predicted to target to the mitochondrion, one ISU/IscU ortholog (PBANKA_131820) and one NFU1/NfuA ortholog (PBANKA_083170). The third protein, that was termed NFUapi, is expected to localize to the apicoplast (PBANKA_082230).

Phenotype
Phenotype analyses of a mutant lacking NFUapi (RMgm-909) indicate that NFUapi has no essential role throughout the complete life cycle. Only the final maturation of liver stgae development was 'slightly' affected.
Phenotype analyses of liver stages of the mutant expressing a C-terminal mCherry-3xMyc tagged version of NFUapi provide evidence for a localisation in the apicoplast. Live parasites only produced faint undefined signals in developing midgut-associated oocysts and cultured liver stage parasites. Immunofluorescence analyses of mature liver stages using anti-cMyc antibodies showed the extended branched structure reminiscent of the apicoplast.

Additional information

Other mutants
A mutant lacking NFUapi (RMgm-909)

  Tagged: Mutant parasite with a tagged gene
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Details of the target gene
Gene Model of Rodent Parasite PBANKA_0822300
Gene Model P. falciparum ortholog PF3D7_0921400
Gene productNifU-like scaffold protein
Gene product: Alternative nameNifU; NFUapi
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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/constructPlasmid 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 modificationThe construct to tag the nfuapi gene contains a GFP expression cassette. In this cassette the gfp gene is under the control of the hsp70 promoter (see for more information on the basic plasmid pBAT-SIL6 the mutant RMgm-757).


PCR fragments were cloned into the berghei adaptable transfection vector pBAT-SIL6. First, the 3'UTR homologous sequences were cloned following restriction digestion of vector and insert with HindIII and KpnI (see RMgm-909).
Next the carboxy-terminal part of NFUapi was PCR amplified using gene-specific primers. After restriction digestion with SacII and EcoRV, the fragment was cloned into the SacII and HpaI digested pBAT-SIL6 vector already containing the 3'UTR sequence of NFUapi, thus fusing the NFUapi carboxyterminal sequence in frame with the mCherry-3xMyc tag sequence. The resulting plasmid was linearized with SalI and used to transfect P. berghei strain ANKA (WT) parasites.
Additional remarks selection procedureCloned by FACS sorting of GFP-expressing parasites
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/constructPCR construct 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 modificationThe construct to tag the nfuapi gene contains a GFP expression cassette. In this cassette the gfp gene is under the control of the hsp70 promoter (see for more information on the basic plasmid pBAT-SIL6 the mutant RMgm-757).


PCR fragments were cloned into the berghei adaptable transfection vector pBAT-SIL6. First, the 3'UTR homologous sequences were cloned following restriction digestion of vector and insert with HindIII and KpnI (see RMgm-909).
Next the carboxy-terminal part of NFUapi was PCR amplified using gene-specific primers. After restriction digestion with SacII and EcoRV, the fragment was cloned into the SacII and HpaI digested pBAT-SIL6 vector already containing the 3'UTR sequence of NFUapi, thus fusing the NFUapi carboxyterminal sequence in frame with the mCherry-3xMyc tag sequence. The resulting plasmid was linearized with SalI and used to transfect P. berghei strain ANKA (WT) parasites.
Additional remarks selection procedureCloned by FACS sorting of GFP-expressing parasites
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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_1340000
Gene productdihydrofolate synthase/folylpolyglutamate synthase, putative
Gene product: Alternative namePbDHFS-FPGS
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_0822300
Gene productNifU-like scaffold protein
Gene product: Alternative nameNifU; NFUapi
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