RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-161
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PBANKA_0911700; Gene model (P.falciparum): PF3D7_1136900; Gene product: subtilisin-like protease 2 (SUB2)
Name tag: c-myc
Phenotype Asexual bloodstage;
Last modified: 24 February 2009, 15:58
  *RMgm-161
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene tagging
Reference (PubMed-PMID number) Reference 1 (PMID number) : 14678331
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 cl15cy1
Other information parent line
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The mutant parasite was generated by
Name PI/ResearcherP. Uzureau; C.J. Janse; A.P. Waters; C. Braun Breton
Name Group/DepartmentUnité de Biologie des Interactions Hôte-Parasite
Name InstituteInstitut Pasteur
CityParis
CountryFrance
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Name of the mutant parasite
RMgm numberRMgm-161
Principal namesub2
Alternative namewt-TmDX
Standardized name
Is the mutant parasite cloned after genetic modificationYes
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Phenotype
Asexual blood stageNormal growth and multiplication of the asexual blood stages. IFA analysis using cmyc antibodies shows expression of SUB2 in schizonts/merozoites.
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses a c-myc tagged form of SUB2.

Protein (function)
SUB2 is a subtilisin-like serine protease. In Plasmodium three subtilisin-like proteases (SUB1-3) have been identified. SUB2 has been shown to be associated with dense granules and/or micronemes, the contents of which are released during erythrocyte invasion. It has been proposed to function as a 'sheddase', involved in the release of MSP1 and AMA1 during invasion (Harris et al., 2005, PloS Pathogens, 3, e29)
Attempts to disrupt the sub2 gene (in P. berghei and P. falciparum) were not successful, indicating an essential function in the blood stages (see RMgm-160).

Phenotype
The mutant shows normal growth and multiplication of the asexual blood stages, indicating that the cmyc-tagging does not affect the function of SUB2. IFA analysis using cmyc antibodies shows expression of SUB2 in schizonts/merozoites.

Additional information
Attempts to disrupt the sub2 gene were not successful, indicating an essential function in the blood stages (see RMgm-160).
See also GenBank accession numbers AJ242629 and AF145052 for the correct sequence of the gene encoding SUB2

Other mutants
RMgm-160: Unsuccessful attempts to disrupt sub2

  Tagged: Mutant parasite with a tagged gene
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Details of the target gene
Gene Model of Rodent Parasite PBANKA_0911700
Gene Model P. falciparum ortholog PF3D7_1136900
Gene productsubtilisin-like protease 2
Gene product: Alternative nameSUB2
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Details of the genetic modification
Name of the tagc-myc
Details of taggingC-terminal
Additional remarks: taggingMultiple epitope tag, called TrimycDuoXpress tag (TmDX tag). The TmDX tag consists of 56 amino acids corresponding to three c-myc epitopes separated by two Xpress epitopes . Each c-myc and Xpress epitope is separated by a hinge composed of two alanine or two valine residues
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 BsmI
Selectable marker used to select the mutant parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationA multiple epitope tag, called TrimycDuoXpress tag (TmDX tag) was used. The TmDX tag consists of 56 amino acids corresponding to three c-myc epitopes separated by two Xpress epitopes. Each c-myc and Xpress epitope is separated by a hinge composed of two alanine or two valine residues. The BsmI linearized pSub2-wt-TmDX construct was designed to integrate via a double crossing-over event, resulting in the insertion of the TmDX tag in frame with the C-terminus of the SUB2 protein and the insertion of the Toxoplasma gondii DHFR-TS selectable marker.
For more detailed information about plasmid construction and primer sequences, see 'Experimental Procedures' (Uzureau et.al., Cellular Microbiology (2003) 6:65-78)
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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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