Summary

RMgm-582
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)
Insertion locus: Gene model: Not available; Gene product: Not available (small subunit ribosomal rna gene (c-type unit))
Phenotype Asexual bloodstage;
Last modified: 10 April 2012, 19:32
  *RMgm-582
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. y. yoelii 17X
Name parent line/clone Not applicable
Other information parent line17X is a non-lethal strain of P. yoelii (17XNL)
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-582
Principal namePy17X-MIF(+)
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageBALB/cByJ and C57BL/6 mice challenged with Py17X-MIF(+) parasites showed a modest decrease in the severity of 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 contains 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 constitutive eef1a promoter.

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
Analyses of the course of infection of the mutant parasites in two mice strains (see 'Additional information') indicate that increased expression of Plasmodium MIF during blood-stage malaria does not increase the severity of disease but may in fact attenuate parasite virulence.

See also the phenotype of mutant RMgm-583. This mutant contains one additional copy of the macrophage migration inhibitory factor gene (mif) of P. yoelii integrated into the dhfr/ts gene locus.

Additional information
By immunoblot analysis and quantitation by densitometry, the PyMIF protein level in Py17X-MIF(+) transgenic parasites was increased approximately 5.5 fold over wt P. yoelii 17X parasites

BALB/cByJ and C57BL/6 mice challenged with Py17X-MIF(+) parasites showed a modest decrease in the severity of infection. The mean peak parasitemia in BALB/cByJ mice infected with Py17X-MIF(+) parasites was 21.7% ± 11.7 compared to 30.9% ± 6.8 in wt infected control mice. Similarly, the mean peak parasitemia in Py17X-MIF(+)  infected C57BL/6 mice was 15.5% ± 7.4, significantly lower than the mean peak parasitemia of 25.0% ± 11.5% observed in wt infected control mice. In both strains of mice  no difference in the pre-patent period of infection, the slope of the ascending parasitemia curve, day 8 parasitemia or day 10 parasitemia was noted between mice infected with Py17X-MIF(+) and wt infected mice. As such, the in vivo growth rate of the two transgenic parasites during the first 8-10 days of infection was comparable. These data indicate that increased expression of Plasmodium MIF during blood-stage malaria does not increase the severity of disease but may in fact attenuate parasite virulence.

Other mutants
RMgm-583: A P. yoelii (17XL) mutant containing one additional copy of the macrophage migration inhibitory factor gene (mif) of P. yoelii integrated into the dhfr/ts gene locus
RMgm-665: A P. yoelii (17XNL) mutant lacking expression of MIF
RMgm-26: a P. berghei mutant lacking expression of MIF.
RMgm-27: a transgenic P. berghei mutant expressing GFP-tagged MIF.
RMgm-31: 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 single cross-over
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid ApaI, 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 gfp gene fragment to generate pPyMIF(rrna). The pPbGFPcon plasmid contains a gfp expression cassette under the control of a constitutive P. berghei ef-1aα promoter, flanked by sequences to target insertion into the c- and/or d– ssu-rna subunit loci by a single crossover event. P. berghei ssu-rrna genes are 96% identical to P. yoelii ssu-rrna genes. The gfp gene was replaced with a pymif cDNA fragment to generate pPyMIF(rrna).
Genetic analysis showed that in addition to the endogenous pymif gene, Py17X-MIF(+) parasites contained two copies of the pymif transgene construct integrated in tandem into the c-ssu rrna gene.
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 / InsertionInsertion locus
Gene Model of Parasite Not available
Gene productNot available
Gene product: Alternative namesmall subunit ribosomal rna gene (c-type unit)
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