RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-583
Malaria parasiteP. yoelii
Genotype
Transgene
Transgene Plasmodium: Gene model: PY17X_1446500; Gene model (P.falciparum): PF3D7_1229400; Gene product: macrophage migration inhibitory factor (MIF)
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)
Replacement locus: Gene model: PY04370; Gene product: bifunctional dihydrofolate reductase-thymidylate synthase (dhfr/ts)
Phenotype Asexual bloodstage;
Last modified: 10 April 2012, 19:33
  *RMgm-583
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) : 20837716
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. yoelii
Parent strain/lineP. yoelii 17XL
Name parent line/clone Not applicable
Other information parent line17XL is a lethal strain of P. yoelii
The mutant parasite was generated by
Name PI/ResearcherS. Thorat; J.M. Burns
Name Group/DepartmentDepartment of Microbiology and Immunology
Name InstituteDrexel University, College of Medicine
CityPhiladelphia
CountryUSA
Name of the mutant parasite
RMgm numberRMgm-583
Principal namePy17XL-MIF(+)
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageBALB/cByJ mice infected with Py17XL-MIF(+) parasites showed a significant delay in time to death and as well as higher survival rates than those infected with control parasites (see 'Additional information phenotype')
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant contains one additional copy of the macrophage migration inhibitory factor gene (mif) of P. yoelii integrated into the dhfr/ts gene locus (the construct was integrated via a single crossover event instead of the expected double crossover).

Protein (function)
Macrophage migration inhibitory factor (MIF) is a mammalian cytokine that participates in innate and adaptive immune responses. Homologues of mammalian MIF have been discovered in parasite species (nematodes and malaria parasites). Like human MIF, histidine-tagged purified recombinant PMIF shows tautomerase and oxidoreductase activities (although the activities are reduced compared to those of histidine-tagged human MIF) and efficiently inhibits AP-1 activity in human embryonic kidney cells (Augustijn et al., Infect Immun. (2007),75:1116-28). P. berghei MIF is expressed in both the mammalian host and mosquito vector. In blood stages, MIF is secreted into the infected erythrocytes and released upon schizont rupture.
See also RMgm-26 for a P. berghei mutant lacking expression of MIF. This mutant was able to complete the entire life cycle and exhibited no significant changes in growth characteristics or virulence features during blood stage infection. However, rodent hosts infected with this mutant had significantly higher numbers of circulating reticulocytes.

Phenotype
BALB/cByJ mice infected with Py17XL-MIF(+) parasites showed a significant delay in time to death and as well as higher survival rates than those infected with control parasites (see 'additional Information'). These data indicate that that increased expression of Plasmodium MIF by blood-stage  parasites can alter the course of infection and reduce overall mortality.  

See also the phenotype of mutant RMgm-582. This mutant contains 2 additional copies of the macrophage migration inhibitory factor gene (mif) of P. yoelii integrated into the c-ssu-rrna gene locus.

Additional information
Increased expression of pymif Py17XL-MIF(+) parasites was assessed at both RNA and protein levels. Quantitative RT-PCR analysis indicated an increase in expression of approximately 3.6 fold which was comparable to the increase of 3.7 observed at the protein level as measured by immunoblot analysis.

The median survival for Py17XLMIF(+) infected mice in the two experiments was 19 and 20 days respectively, in comparison to 9 and 8.5 days for wt infected mice. In the first experiment, parasitemia in Py17XL-408 MIF(+) infected mice was slightly lower than in wt infected mice during the first week of infection. However, this was not the case in the second experiments where parasitemia levels in the two groups were comparable through day 8 of infection. The surviving mice, in both experiments cleared their infection completely by day 24 post-infection.

Other mutants
RMgm-582: A P.yoelii (Py17X) mutant containing  2 additional copies of the macrophage migration inhibitory factor gene (mif) of P. yoelii integrated into the c-ssu-rrna gene locus. These mif copies are under control of the strong consecutive eef1a promoter.
RMgm-665: A P. yoelii (17XNL) mutant lacking expression of MIF
RMgm-26 is a P. berghei mutant lacking expression of MIF.
RMgm-27 is a transgenic P. berghei mutant expressing GFP-tagged MIF.
RMgm-31 is a P. berghei transgenic mutant expressing c-myc-tagged MIF.


  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: Plasmodium
Gene Model of Parasite PY17X_1446500
Gene Model P. falciparum ortholog PF3D7_1229400
Gene productmacrophage migration inhibitory factor
Gene product: Alternative nameMIF
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 KpnI, SacII
Selectable marker used to select the mutant parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe complete coding region of pymif was PCR amplified from the cDNA clone MS36 obtained from a sequenced P. yoelii 17XL blood-stage cDNA library using forward 5'-GATATCATATGATGCCTTGCTGCGAATTAATAACAAAC-3´ and reverse 5´-GAATTCTCGAGTTAGCCAAATAGTGAACCACTAAAA–3´ primers.
A BamHI pymif cDNA fragment was cloned into gel-purified pPbGFPcon after excision of the PY00068 gene to generate pPyMIF(dhfr). The pPY00068-OE plasmid contains a gfp expression cassette under the control of a constitutive P. berghei eef1a promoter, designed to replace the pydhfr locus by a double crossover event.
Genetic analysis of Py17XL-MIF(+) parasites showed the presence of an intact, endogenous copy of the pymif gene and the integration of the Pb-eef1a-pymif construct at the 5’ end of the pydhfr gene locus via a single crossover event instead of the expected double crossover.
Additional remarks selection procedure
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 / InsertionReplacement locus
Gene Model of Parasite PY04370
Gene productbifunctional dihydrofolate reductase-thymidylate synthase
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