Summary

RMgm-951
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PBANKA_1358000; Gene model (P.falciparum): PF3D7_1345100; Gene product: thioredoxin 2 (TRX2)
Name tag: mCherry-3xMyc
Transgene
Transgene not Plasmodium: GFP
Promoter: Gene model: PBANKA_0711900; Gene model (P.falciparum): PF3D7_0818900; Gene product: heat shock protein 70 (HSP70)
3'UTR: Gene model: PBANKA_0719300; Gene product: bifunctional dihydrofolate reductase-thymidylate synthase, putative (dhfr/ts)
Insertion locus: Gene model: PBANKA_1358000; Gene product: thioredoxin 2 (TRX2)
Phenotype Asexual bloodstage; Gametocyte/Gamete;
Last modified: 11 October 2013, 18:35
  *RMgm-951
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene tagging, Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 24076174
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 cl15cy1
Other information parent lineA reference wild type clone from the ANKA strain of P. berghei (PubMed: PMID: 17406255).
The mutant parasite was generated by
Name PI/ResearcherTWA. Kooij: JM Matz
Name Group/DepartmentParasitology Unit
Name InstituteMax Planck Institute for Infection Biology
CityBerlin
CountryGermany
Name of the mutant parasite
RMgm numberRMgm-951
Principal nametrx2::tag
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageTRX2::mCherry expression in asexual blood stages
Gametocyte/GameteTRX2::mCherry expression in gametocytes
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses a C-terminal mCherry-tagged version of of TRX2 and expresses GFP under the control of the HSP70 promoter

Protein (function)
Plasmodium parasites remodel their vertebrate host cells by translocating hundreds of proteins across an encasing membrane into the host cell cytosol via a putative export machinery termed PTEX (Plasmodium Translocon of EXported protein). HSP101 (PbANKA_094120), PTEX150 (PbANKA_100850), EXP2 (PbANKA_133430), PTEX88 (PbANKA_094130) and TRX2 (PbANKA_135800) have been identified as members of the PTEX complex.
In this study HSP101, PTEX150, EXP2 could not be genetically deleted in P. berghei. In contrast, the putative thioredoxin-like protein TRX2 and PTEX88 could be deleted. See RMgm-918 and RMgm-943 for  mutants lacking expression of PTEX88. These mutants show a (slightly) reduced growth rate of asexual blood stages.

Phenotype
Mutants lacking expression of TRX2 (RMgm-918; RMgm-942) show a (slightly) reduced growth rate of asexual blood stages.
Analysis of the mutant expressing mCherry-tagged TRX2 shows expression in all blood stages (see 'Additional information')

Additional information
TRX2::mCherry bloodstages grew indistinguishable from wild type parasites, thus providing evidence for a functional complementation and simultaneously ruling out aberrant localization of the tagged protein.

Parasites expressing TRX2::tag exhibited a very consistent staining throughout blood stage development. The protein localizes to a distinct peripheral focus in free merozoites and mature schizonts. The punctate staining remains throughout ring and trophozoite development and in gametocytes. The number of TRX2::tag-positive structures varies and appears loosely associated with the developmental stage of the parasite. Ring stages usually display one or two of these structures, whereas the number of foci in trophozoites is more variable.

The standard pBAT vector was used to generate the gene deletion constructs. This vector contains: (i) a drug-selectable cassette, (ii) a high expressing GFP cassette, (iii) a C-terminal red fluorescent (mCherry) and triple epitope (3xMyc) tag, (iv) two extensive multiple cloning sites (see RMgm-757 for details of this vector). The 3' fragment of trx2 was amplified from gDNA using XhoI and KpnI to generate the intermediate construct pPTEX-IM.
A fragment of the carboxy-terminal coding region was amplified from gDNA and fused in frame to the mCherry-3xMyc tag in ptrx2-IM using SacII and HpaI. The carboxy-terminal coding sequences cloned into the tagging plasmids were verified by commercial Sanger sequencing. All vectors targeting the putative PTEX translocon components were linearized with AhdI and ApaLI before transfection into wild-type P. berghei strain ANKA parasites.

See mutant RMgm-923 for the localization of TRX2 in a mutant expressing HA-tagged version of TRX2.

Other mutants
See RMgm-918 and RMgm-943 for  mutants lacking expression of TRX2
See mutant RMgm-923 for the localization of TRX2 in a mutant expressing HA-tagged version of TRX2.


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1358000
Gene Model P. falciparum ortholog PF3D7_1345100
Gene productthioredoxin 2
Gene product: Alternative nameTRX2
Details of the genetic modification
Name of the tagmCherry-3xMyc
Details of taggingC-terminal
Additional remarks: tagging
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
Selectable marker used to select the mutant parasitehdhfr/yfcu
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe standard pBAT vector was used to generate the gene deletion constructs. This vector contains: (i) a drug-selectable cassette, (ii) a high expressing GFP cassette, (iii) a C-terminal red fluorescent (mCherry) and triple epitope (3xMyc) tag, (iv) two extensive multiple cloning sites (see RMgmDB-757 for details of this vector). The 3' fragment of trx2 was amplified from gDNA using XhoI and KpnI to generate the intermediate construct pPTEX-IM.
A fragment of the carboxy-terminal coding region was amplified from gDNA and fused in frame to the mCherry-3xMyc tag in ptrx2-IM using SacII and HpaI. The carboxy-terminal coding sequences cloned into the tagging plasmids were verified by commercial Sanger sequencing. All vectors targeting the putative PTEX translocon components were linearized with AhdI and ApaLI before transfection into wild-type P. berghei strain ANKA parasites.
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
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
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
Selectable marker used to select the mutant parasitehdhfr/yfcu
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe standard pBAT vector was used to generate the gene deletion constructs. This vector contains: (i) a drug-selectable cassette, (ii) a high expressing GFP cassette, (iii) a C-terminal red fluorescent (mCherry) and triple epitope (3xMyc) tag, (iv) two extensive multiple cloning sites (see RMgmDB-757 for details of this vector). The 3' fragment of trx2 was amplified from gDNA using XhoI and KpnI to generate the intermediate construct pPTEX-IM.
A fragment of the carboxy-terminal coding region was amplified from gDNA and fused in frame to the mCherry-3xMyc tag in ptrx2-IM using SacII and HpaI. The carboxy-terminal coding sequences cloned into the tagging plasmids were verified by commercial Sanger sequencing. All vectors targeting the putative PTEX translocon components were linearized with AhdI and ApaLI before transfection into wild-type P. berghei strain ANKA parasites.
Additional remarks selection procedure
Other details transgene
Promoter
Gene Model of Parasite PBANKA_0711900
Gene Model P. falciparum ortholog PF3D7_0818900
Gene productheat shock protein 70
Gene product: Alternative nameHSP70
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 / InsertionInsertion locus
Gene Model of Parasite PBANKA_1358000
Gene productthioredoxin 2
Gene product: Alternative nameTRX2
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