RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5100
Malaria parasiteP. berghei
Genotype
MutatedGene model (rodent): PBANKA_0501200; Gene model (P.falciparum): PF3D7_1016900; Gene product: early transcribed membrane protein 10.3 | protein of early gametocyte 4 (UIS4, up-regulated in infective sporozoites, ETRAMP10.3)
Details mutation: a truncated version of UIS4 with only an amino-terminal portion (amino acids 1-76)
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 Liver stage;
Last modified: 8 November 2021, 16:25
  *RMgm-5100
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene mutation, Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 34715088
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 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).
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The mutant parasite was generated by
Name PI/ResearcherBrandsma AM, Montagna GN
Name Group/DepartmentParasitology Unit
Name InstituteMax Planck Institute for Infection Biology
CityBerlin
CountryGermany
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Name of the mutant parasite
RMgm numberRMgm-5100
Principal nameuis4Δc
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
SporozoiteNot different from wild type
Liver stageNormal formation of the parasitophorous vacuole membrane. A slight reduction in numbers of liver stages at 42 hours. No blood-stage infection after infection of mice by mosquito bite or intravenous injection of sporozoites.
Additional remarks phenotype

Mutant/mutation
In order to study the importance of the carboxy-terminal domain of UIS4, a P. berghei transgenic parasite was generated where the endogenous uis4 gene was replaced with a DNA fragment coding for a truncated version of UIS4, residues 1-76 only, employing a double cross-over strategy

Protein (function)
UIS4  is expressed throughout liver stage development and localizes to the parasitophorous vacuole. UIS4 has a total length of 220 amino acid residues and can be divided into three domains: an amino-terminal domain of 54 residues including the signal peptide encompassing 25 residues, the trans-membrane region and a positively charged carboxy-terminal domain of 143 residues. Inside an infected hepatocyte, UIS4 is distributed at the PVM and the tubovesicular network (TVN), which forms vesicular protrusions into the host cell cytoplasm.

Phenotype
Normal formation of the parasitophorous vacuole membrane. A slight reduction in numbers of liver stages at 42 hours. No blood-stage infection after infection of mice by mosquito bite or intravenous injection of sporozoites. However, in vitro developed and isolated merosomes are infectious to mice after intravenous inoculation.

Additional information

Other mutants

  Mutated: Mutant parasite with a mutated gene
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Details of the target gene
Gene Model of Rodent Parasite PBANKA_0501200
Gene Model P. falciparum ortholog PF3D7_1016900
Gene productearly transcribed membrane protein 10.3 | protein of early gametocyte 4
Gene product: Alternative nameUIS4, up-regulated in infective sporozoites, ETRAMP10.3
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Details of the genetic modification
Short description of the mutationa truncated version of UIS4 with only an amino-terminal portion (amino acids 1-76)
Inducable system usedNo
Short description of the conditional mutagenesisNot available
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 parasitetgdhfr
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationTo generate parasites expressing a truncated version of UIS4 protein, a fragment of UIS4 comprising the UIS4 promoter and the amino-terminal portion of the UIS4 open reading frame (ORF) (amino acids 1-76) was amplified from P. berghei genomic DNA (gDNA) using the primers UIS4 5’UTRfd and UIS4-COOHSpeI. The resulting fragment was cloned into the P. berghei transfection vector B3D+, which contains the 3’ untranslated region (UTR) of GAPDH and the Toxoplasma gondii DHFR/TS-positive selectable marker. Then, we added the UIS4 3’UTR region, which was amplified using the primers UIS4 3’UTRfd and UIS4 3’UTRrv, leading to plasmid uis4Δc.
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 (gfp-mu3)
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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 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.
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Other details transgene
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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
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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
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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