RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-1147
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_0720900; Gene model (P.falciparum): PF3D7_0418800; Gene product: MOLO1 domain-containing protein, putative
Phenotype Oocyst; Sporozoite; Liver stage;
Last modified: 6 April 2016, 10:15
  *RMgm-1147
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption
Reference (PubMed-PMID number) Reference 1 (PMID number) : 25418785
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 820cl1m1cl1 (RMgm-164)
Other information parent lineP. berghei ANKA 820cl1m1cl1 (RMgm-164) is a reference ANKA mutant line which expresses GFP under control of a male and RFP under control of a female gametocyte specific promoter. This reference line does not contain a drug-selectable marker (PubMed: PMID: 19438517).
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The mutant parasite was generated by
Name PI/ResearcherGuerreiro A; Mair GR
Name Group/DepartmentInstituto de Medicina Molecular
Name InstituteFaculdade de Medicina da Universidade de Lisboa
CityLisbon
CountryPortugal
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Name of the mutant parasite
RMgm numberRMgm-1147
Principal name2099cl1m7; 2100cl1m1
Alternative namePBANKA_072090 null mutant-a,b
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
OocystOocyst are produced in normal numbers. Although oocysts in the mutant were established in high numbers, they never produced sporozoites and remained empty throughout the course of the infection. On a molecular level, mutant oocysts are characterized by a lack of DNA replication and expression of the sporozoite surface marker circumsporozoite protein (CSP).
SporozoiteNo sporozoite formation
Liver stageNo sporozoite formation
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of PBANKA_072090 and expresses GFP under control of a male and RFP under control of a female gametocyte specific promoter.

Protein (function)
This gene was identified in a screen of mRNAs in female gametocytes that are bound to the translational repressor proteins DOZI and CITH.
These genes are transcribed in female gametecytes but are translationally repressed. Translation occurs after fertilisation of the female gamete.

Phenotype
Oocyst are produced in normal numbers. Although oocysts in the mutant were established in high numbers, they never produced sporozoites and remained empty throughout the course of the infection. On a molecular level, mutant oocysts are characterized by a lack of DNA replication and expression of the sporozoite surface marker circumsporozoite protein (CSP).

Analysis of a mutant expressing a C-terminal GFP-tagged version of this protein (RMgm-1146) showed: mRNA presence in gametocytes but absence of the GFP-tagged protein (translationally repressed). Expression of the GFP-tagged protein occurs in developing zygotes/ookinetes. The fluorescence pattern showed evidence for localisation of the protein in the crystalloid body of ookinetes.

Additional information
In this study 5 (translationally repressed) genes were C-terminally tagged with GFP (PBANKA_082120, PBANKA_133470, PBANKA_111410, PBANKA_010770, PBANKA_072090. All genes are transcribed in gametocytes but expression of the GFP-tagged proteins only occurs after fertilization in the developing zygote/ookinete

Other mutants
A mutant expressing a C-terminal GFP-tagged version of this protein (RMgm-1146)

  Disrupted: Mutant parasite with a disrupted gene
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Details of the target gene
Gene Model of Rodent Parasite PBANKA_0720900
Gene Model P. falciparum ortholog PF3D7_0418800
Gene productMOLO1 domain-containing protein, putative
Gene product: Alternative name
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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 modificationTo disrupt PBANKA_072090, the replacement construct pLIS0092 was constructed that contains the pyrimethamine resistant Toxoplasma gondii (tg) dhfr/ts as a selectable marker cassette. Target sequences for homologous recombination were PCR-amplified from P. berghei WT genomic DNA using primers specific for the 5′ or 3′ flanking regions. The PCR-amplified target sequences were cloned upstream or downstream of the selectable marker to allow for integration of the linearized construct into the genomic locus by homologous recombination. DNA construct used for transfection was obtained after digestion of the replacement construct with the appropriate restriction enzymes.
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 1aaaggtaccTTGTTATTCTTTGCAAG
Additional information primer 1g0711 (5'UTR)
Sequence Primer 2aaaaagcttTGAATAATTAAAAAAAAGC
Additional information primer 2g0712 (5'UTR)
Sequence Primer 3aaagaattcTTATTTGATGATCAACC
Additional information primer 3g0713 (3'UTR)
Sequence Primer 4aaagcggccgcAATATATATAGTTATTTCC
Additional information primer 4g0714 (3'UTR)
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6
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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