RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-332
Malaria parasiteP. berghei
Genotype
Transgene
Transgene not Plasmodium: CD4+ and CD8+ T cell epitopes and B cell epitopes (OVA, HA, gB) fused to GFP
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)
Phenotype Asexual bloodstage;
Last modified: 21 July 2011, 12:54
  *RMgm-332
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) : 18799734
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone Not applicable
Other information parent line
The mutant parasite was generated by
Name PI/ResearcherR.J. Lundie; W.R. Heath
Name Group/DepartmentNot applicable
Name InstituteWalter and Eliza Hall Institute of Medical Research
CityParkville, Victoria 3050
CountryAustralia
Name of the mutant parasite
RMgm numberRMgm-332
Principal namePbTG
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationNo
Phenotype
Asexual blood stageIt was demonstrated that the 'polytope transgene' was functionally expressed, correctly processed, and presented by the host immune system for specific stimulation of CD4+ and CD8+ T cells during blood-stage infection (see 'Additional remarks phenotype').
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses model T and B cell epitopes (OVA, HA, gB; 'polytope') fused to GFP under the control of the constitutive P. berghei elongation factor (EF)-1α promoter. The 'polytope' fused to GFP was introduced and maintained in the transgenic parasites as circular plasmids (episomes) by in vivo pyrimethamine selection.

Protein (function)
The transgene consists of T and B cell epitopes ('polytope') fused to GFP.
The T cell epitopes chosen were MHC I- and MHC II-restricted epitopes presented in C57BL/6 (B6) and BALB/c mice, which are differentially sensitive to P. berghei-mediated experimental cerebral malaria (ECM). For B6 mice, which are susceptible to ECM, MHC I- and II-restricted epitopes from chicken ovalbumin and an MHC I-restricted epitope from glycoprotein B of herpes simplex virus-1 were included. For ECM-resistant BALB/c mice, MHC I- and II-restricted epitopes from hemagglutinin of the influenza virus PR8 were included, whereas the MHC II-restricted OVA epitope can also be presented on I-Ad of BALB/c mice.

Sequences of overlapping oligonucleotides designed for PCR amplification of the selected CD4+ and CD8+ T cell epitopes (OVA257–264 [SIINFEKL], H-2Kb restricted; OVA323–339 [KISQAVHAAHAEINEAG], I-Ab and IAd restricted; gB498–505 [SSIEFARL], H-2Kb restricted; HA518–526 [IYSTVASSL], H-2Kd restricted; and HA126–138 [HNTNGVTAACSHE], I-Ad restricted), B cell epitopes FLAG (DYKDDDK) and c-myc (EQKLISEEDL), and restriction sites to facilitate cloning are illustrated in the Figure shown below.

Phenotype
The mutant expresses a variety of model T cell epitopes for which T cell receptor (TCR) transgenic mice are available. This approach of transgenic expression was necessary as MHC class I-restricted antigens to blood-stage infection have not been defined. It was demonstrated that the 'polytope' transgene was functionally expressed, correctly processed, and presented by the host immune system for specific stimulation of CD4+ and CD8+ T cells during blood-stage infection. Using these mutants it was demonstrated that antigens expressed by blood-stage P. berghei parasites are captured and cross-presented by CD8α DC to stimulate naive CD8+ T cell proliferation and lytic function  (see also 'Additional information').

Additional information
Expression of the 39-kDa polytope-GFP fusion protein by PbTG was confirmed by Western blot analysis using antibodies to GFP, FLAG, or c-myc

Evidence is presented indicating that bone marrow-derived DC were critical for the induction of T cell responses during blood-stage infection and data is presented that suggests that  CD8α DC are the major antigen-presenting cells for CD8+ T cells during blood-stage infection, whereas CD4 DC play a more extensive role in CD4+ T cell stimulation.

Other mutants
RMgm-5: mutant expressing GFP under the control of the constitutive promoter of the elongation factor 1 alpha (eef1a) gene of P. berghei.

 

 


  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameCD4+ and CD8+ T cell epitopes and B cell epitopes (OVA, HA, gB) fused to GFP
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/constructCircular plasmid
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 markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationSequences of overlapping oligonucleotides designed for PCR amplification of the selected CD4+ and CD8+ T cell epitopes (OVA257–264 [SIINFEKL], H-2Kb restricted; OVA323–339 [KISQAVHAAHAEINEAG], I-Ab and IAd restricted; gB498–505 [SSIEFARL], H-2Kb restricted; HA518–526 [IYSTVASSL], H-2Kd restricted; and HA126–138 [HNTNGVTAACSHE], I-Ad restricted), B cell epitopes FLAG (DYKDDDK) and c-myc (EQKLISEEDL), and restriction sites to facilitate cloning are illustrated in Fig. S1.

To construct the polytope, oligonucleotides (F1–F6 and R1–R6) were denatured at 94°C for 2 min and then annealed at 37°C for 10 min before the addition of Klenow enzyme for 30 min at 30°C. After a 10-min incubation at 75°C, a PCR was performed on the annealed oligonucleotides using PLATINUM TaqDNA polymerase High Fidelity Enzyme and oligonucleotides F1 (5′-AGGATCCATGGATTACAAGGATGACGAACGATAAGTTAG-3′) and R1 (5′-TGGATCCTCAAGATCTTCTAGACAGATCCTCTTCAGAGATTAG-3′). PCR conditions were 94°C denaturation, 50°C annealing, and 68°C for nucleotide extension, incubating for 30 sec at each step for a total of 30 cycles. BamHI restriction sites (bold) were introduced in F1 and R1 to facilitate cloning into the expression vector PbGFPCON. Unique BglII (italicized) and XbaI (italicized in bold) sites were introduced in R1 to enable fusion to GFP and orientation screening, respectively.

To fuse the polytope to GFP, the polytope PCR product was first subcloned into the multicloning site of vector pGEM-Teasy (Promega). Digestion of this vector with BglII then created a compatible restriction site for the in-frame fusion of GFP (see Figure), released as a BamHI fragment from vector PbGFPCON. The final polytope-GFP fusion product was introduced as a BamHI fragment into the expression cassette of the expression plasmid PbGFPcon (see RMgm-5), to create vector PbTGFPcon.
Additional remarks selection procedureTransgenes were maintained as episomal plasmids under pyrimethamine selection in vivo.
Other details transgene
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
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 / InsertionNot available
Gene Model of Parasite Not available
Gene productNot available
Gene product: Alternative name
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