RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-4879
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_0614300; Gene model (P.falciparum): PF3D7_0716600; Gene product: cysteine desulfurase (SufS)
Transgene
Transgene not Plasmodium: GFP (gfp-mut3)
Promoter: Gene model: PBANKA_1133300; Gene model (P.falciparum): PF3D7_1357100; Gene product: elongation factor 1-alpha (eef1a)
3'UTR: Gene model: PBANKA_0719300; Gene product: bifunctional dihydrofolate reductase-thymidylate synthase, putative (dhfr/ts)
Replacement locus: Gene model: PBANKA_0306000; Gene product: 6-cysteine protein (230p)
Phenotype Asexual bloodstage;
Last modified: 30 September 2020, 16:02
  *RMgm-4879
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) Not published (yet)
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone PQ(r)
Other information parent lineThe P. berghei ANKA reference line 507cl1 (RMgmDB-7) which is selected for piperaquine resistance and expresses GFP from the constitutive eef1a promoter (described in PMID 19318094)
The mutant parasite was generated by
Name PI/ResearcherBara FD, Kiboi D
Name Group/DepartmentDepartment of Biochemistry
Name InstituteJomo Kenyatta University of Agriculture and Technology (JKUAT)
CityNairobi
CountryKenya
Name of the mutant parasite
RMgm numberRMgm-4879
Principal namePQ(R)_SufS_KD
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageReduced asexual growth/multiplication in mice (33%)
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of SufS and expresses GFP under the constitutive eef1a promoter.
(bioRxiv preprint doi: https://doi.org/10.1101/833145. this version posted September 14, 2020)

Protein (function)
Iron-sulfur [Fe-S] clusters are inorganic cofactors that are found on proteins from a range of biological processes and are present in most organisms. The assembly of [Fe-S] clusters on apoproteins is not a spontaneous process but is mediated by distinct pathways that mobilise sulfur atoms from L-cysteines, and assemble them onto scaffold components. The scaffold also receives iron from iron donors and the assembled [Fe-S] clusters are transferred to the target apoprotein via carrier protein(s). In bacteria, three distinct sets of factors/enzymes encoded as operons assemble [Fe-S] clusters; the bacterial isc, suf and nif operons encode proteins that are components of the ISC (iron-sulfur cluster formation), SUF (sulfur mobilization) and NIF (nitrogen fixation) assembly systems. The ISC system is the major [Fe-S] biogenesis system with the SUF system being activated under iron starvation or oxidative stress.
In eukaryotes, [Fe-S] assembly systems are thought to have evolved from bacterial endosymbionts. The SUF pathway is found in plastid-containing protozoa and plants. The e apicoplast, the relict plastid of the malaria parasite, harbours a functional SUF pathway. Except apicoplast-encoded SufB in Plasmodium spp., all constituent proteins of the SUF pathway are nuclear-encoded and targeted to the apicoplast. These include the cysteine desulfurase SufS and its interacting partner SufE [18], SufC and SufD that are predicted to constitute the scaffold complex with SufB, and putative carrier proteins SufA and Nfu. Components of the SUF pathway in P. berghei have also been reported to be refractory to gene deletion. Apicoplast proteins that likely require [Fe-S] clusters include ferredoxin, two enzymes (IspG and IspH) of the DOXP pathway of isoprenoid biosynthesis, LipA (lipoic acid synthase), and MiaB (tRNA methylthiotransferase).

Phenotype
Reduced asexual growth/multiplication in mice (33%)

Additional information
Deletion of the SufS in the PQ(R) parasites abolished the impact of the chemosensitizers probenecid, verapamil, or cyproheptadine on lumefantrine (LM) activity, and restored the susceptibility of the PQ(R) parasites to LM.

Other mutants


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0614300
Gene Model P. falciparum ortholog PF3D7_0716600
Gene productcysteine desulfurase
Gene product: Alternative nameSufS
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 usedYes
Name of PlasmoGEM construct/vectorPbGEM-018972
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
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
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

  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameGFP (gfp-mut3)
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct(Linear) plasmid single 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 parasitegfp (FACS)
Promoter of the selectable markereef1a
Selection (positive) procedureFACS (flowsorting)
Selection (negative) procedureNo
Additional remarks genetic modification
Additional remarks selection procedure
Other details transgene
Promoter
Gene Model of Parasite PBANKA_1133300
Gene Model P. falciparum ortholog PF3D7_1357100
Gene productelongation factor 1-alpha
Gene product: Alternative nameeef1a
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
3'-UTR
Gene Model of Parasite PBANKA_0719300
Gene productbifunctional dihydrofolate reductase-thymidylate synthase, putative
Gene product: Alternative namedhfr/ts
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_0306000
Gene product6-cysteine protein
Gene product: Alternative name230p
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