RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-4956
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_0506200; Gene model (P.falciparum): PF3D7_1022000; Gene product: RNA-binding protein, putative (UIS12)
Transgene
Transgene not Plasmodium: GFP (gfp-mu3)
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 Gametocyte/Gamete; Oocyst; Sporozoite;
Last modified: 24 March 2021, 10:01
  *RMgm-4956
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) : 33747980
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone P. berghei ANKA 507cl1 (RMgm-7)
Other information parent lineP.berghei ANKA 507cl1 (RMgm-7) is a reference ANKA mutant line which expresses GFP under control of a constitutive promoter. This reference line does not contain a drug-selectable marker (PubMed: PMID: 16242190).
The mutant parasite was generated by
Name PI/ResearcherMüller K, Matuschewski K
Name Group/DepartmentDept. of Molecular Parasitology, Institute of Biology
Name InstituteHumboldt University
CityBerlin
CountryGermany
Name of the mutant parasite
RMgm numberRMgm-4956
Principal nameuis12(-)
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteReduced production of gametocytes, reduced exflagellation of male gametocytes
Fertilization and ookineteNot tested
OocystReduced numbers of oocysts. Oocysts remain small and no sporozoite formation inside oocysts. While up until day 4 the oocyst size was comparable between mutant and wild type oocysts, over time uis12(-) oocysts displayed an abnormal morphology. Their size remained unchanged, whereas the wild type oocyst diameter increased ~10-fold over the following ten days.
SporozoiteNo midgut or salivary gland sporozoites
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of UIS12 and expresses GFP under the constitutive eef1a promoter

Protein (function)
The gene had been identified as a gene with upregulated transcription in sporozoites.
The gene two ~70 amino acid long RNA-recognition motifs (RRM), belonging to the class of RNA-binding domains (RBD). The UIS12 gene, and particularly the two RRM domains, are highly conserved between different Plasmodium UIS12 orthologs, while in a candidate Toxoplasma gondii ortholog, the conservation is limited to the two RRM domains only. In general, the amino- and carboxy- terminal regions are less conserved, and all Plasmodium ortholog proteins have similar sizes of ~1,300 amino acid residues.  The PbUIS12 coding region has a total length of 4,070 base pairs and consists of two exons. Of note, the two regions encoding the RRM domains are separated by the single intron, which also exhibits a degree of conservation.

Phenotype
Normal asexual growth/multiplication of asexual blood stages. Reduced production of gametocytes, reduced exflagellation of male gametocytes. Reduced numbers of oocysts. Oocysts remain small and no sporozoite formation inside oocysts. While up until day 4 the oocyst size was comparable between mutant and wild type oocysts, over time uis12(-) oocysts displayed an abnormal morphology. Their size remained unchanged, whereas the wild type oocyst diameter increased ~10-fold over the following ten days. No midgut or salivary gland sporozoites.

Additional information
We measured UIS12 transcript abundance throughout the P. berghei life cycle by quantitative RT-PCR. In good agreement with the previous notion of up-regulation in salivary gland sporozoites, UIS12 mRNA steady state levels are low in midgut oocysts and highest in salivary gland sporozoites. High UIS12 transcript levels were also detected in early (24h) liver stages. In mixed asexual blood stages UIS12 mRNA levels are low, whereas in schizonts, gametocytes and ookinetes, considerable expression was detected.

An independent mutant lacking uis12 has been made in the reference Bergreen P. berghei ANKA line (RMgm-757) expressing GFP under control of the constitutive hsp70 promoter

Other mutants


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0506200
Gene Model P. falciparum ortholog PF3D7_1022000
Gene productRNA-binding protein, putative
Gene product: Alternative nameUIS12
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 parasitetgdhfr
Promoter of the selectable markerpbdhfr
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-mu3)
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 parasitegfp (FACS)
Promoter of the selectable markereef1a
Selection (positive) procedureFACS (flowsorting)
Selection (negative) procedureNo
Additional remarks genetic modificationThe GFP gene (1 copy) has been inserted into the 230p locus (PBANKA_030600) by double cross-over integration
Additional remarks selection procedureThis reporter mutant expressing GFP does not contain a drug-selectable marker. This mutant has been selected by FACS sorting after transfection based on GFP fluorescence.
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