Summary

RMgm-666
Malaria parasiteP. yoelii
Genotype
Transgene
Transgene not Plasmodium: T. cruzi CD8+ T cell epitope, ANYNFTLV fused to GFP
Promoter: Gene model: PBANKA_0711900; Gene model (P.falciparum): PF3D7_0818900; Gene product: heat shock protein 70 (HSP70, HSP70-1)
3'UTR: Gene model: PY04370; Gene product: thymidylate synthase, putative (dhfr/ts)
Insertion locus: Gene model: PY04370; Gene product: thymidylate synthase, putative (dhfr/ts)
PhenotypeNo phenotype has been described
Last modified: 6 October 2012, 13:57
  *RMgm-666
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 22902783
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. yoelii
Parent strain/lineP. y. yoelii 17X
Name parent line/clone Not applicable
Other information parent line17X is a lethal strain of P. yoelii (17XL)
The mutant parasite was generated by
Name PI/ResearcherT. Ono; Y. Miyahira
Name Group/DepartmentDepartment of Global Infectious Diseases and Tropical Medicine
Name InstituteNational Defense Medical College
CityTokorozawa City
CountryJapan
Name of the mutant parasite
RMgm numberRMgm-666
Principal namepyDH7GFPTSSA
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses a well-defined Trypanosoma cruzi-derived, H-2Kb-restricted CD8+ T cell epitope (ANYNFTLV, from TSSA) fused to GFP under control of the P. berghei hsp70 promoter.

Protein (function)

Phenotypee
The mutant has been used to analyse blood-stage specific CD8+ T cells in protective immunity taking advantage of the H-2Kb-restricted, T. cruzi-derived CD8+ T cell-inducing  epitope, ANYNFTLV

Additional information
The lack of MHC molecules on red blood cells (RBCs) has led to questions regarding the immunological function of CD8+ T cells against malarial blood-stage (MBS). However, several recent reports contradicting with this concept have suggested that they play an important role in the course of MBS infection. The present study generated genetically engineered murine malaria, P. yoelii, which expresses a well-defined T. cruzi-derived, H-2Kb-restricted CD8+ T cell epitope, ANYNFTLV. Prime/boost vaccination by the use of recombinant adenovirus and recombinant modified vaccinia virus Ankara (MVA), which induced an enhanced number of ANYNFTLV-specific CD8+ T cells, failed to prevent a pathological outcome to occur upon ANYNFTLV-expressing murine MBS infection.
In contrast, the pre-infection of mice with T. cruzi, which intrinsically bears the same CD8+ T cell epitope significantly improved the survival of ANYNFTLV-expressing malaria-infected mice but not that of control malaria-infected ones. This protective effect was abrogated by the use of a CD8+ T cell-depleting monoclonal antibody.

Three other mutants were generated in this study: pyDH7TSSA (expressing theTSSA epitope not fused to GFP under the control of the hsp70  promoter) and two control mutants named pyDH7 (only the hsp70  promoter) and pyDH7GFP (only GFP under the control of the hsp70  promoter). These mutants are not included in RMgmDB.

Other mutants

 


  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameT. cruzi CD8+ T cell epitope, ANYNFTLV fused to GFP
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 parasitetgdhfr
Promoter of the selectable markerpydhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe sequence encoding the peptide MANYNFTLV was amplified by PCR and inserted into plasmid vectors to construct a mini gene encoding the peptide MANYNFTLV. The specific primers used for the amplification of the TSSA epitope were: Sense; 5’-atGATATCaccataatatatacccccttttg-3’ , Antisense; 5’- atgcggccgcTTACACAAGAGTGAAGTTGTAGTTCGCCATctttttttaattgtaattgt-3’. The GFP encoding gene was amplified by using the primers: Sense; 5’-ggGGATCCatgagtaaaggagaagaac-3’ , Antisense; 5’-ttGCGGCCGCTTATATTTGTTCCGCGCTTGGGACATAAGAtttgtatagttcatccatgc-3’ to construct a GFP-fused epitope. These PCR products were cloned into the pyDH7 vector, which was designed to confer resistance against pyrimethamine and to be integrated into the P. yoelii dihydrofolate reductase (DHFR) encoding region.
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, HSP70-1
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 PY04370
Gene productthymidylate 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 PY04370
Gene productthymidylate synthase, putative
Gene product: Alternative namedhfr/ts
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