Summary

RMgm-665
Malaria parasiteP. yoelii
Genotype
DisruptedGene model (rodent): PY17X_1446500; Gene model (P.falciparum): PF3D7_1229400; Gene product: macrophage migration inhibitory factor (MIF)
Transgene
Transgene not Plasmodium: RFP
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: PY05452; Gene product: macrophage migration inhibitory factor (MIF)
Phenotype Liver stage;
Last modified: 31 July 2012, 17:49
  *RMgm-665
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption, Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 22252874
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. yoelii
Parent strain/lineP. y. yoelii 17XNL
Name parent line/clone Not applicable
Other information parent line17XNL is a non-lethal strain of P. yoelii
The mutant parasite was generated by
Name PI/ResearcherJ.L. Miller; S.H.I. Kappe; S.A. Mikolajczak
Name Group/DepartmentSeattle Biomedical Research Institute
Name InstituteSeattle Biomedical Research Institute
CitySeattle
CountryUSA
Name of the mutant parasite
RMgm numberRMgm-665
Principal nameP.yoeliiΔmif
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNot different from wild type
Fertilization and ookineteNot different from wild type
OocystNot different from wild type
SporozoiteNot different from wild type
Liver stageLess than 50% of mice infected with 1x10(3) P.yoeliiΔmif sporozoites became patent. For mice that became patent, the onset of blood-stage parasitemia was delayed 2 days compared to that with the control. Patency was more frequent in mice after injecting with 1x10(3) P.yoeliiΔmif sporozoites, and the onset of patency was delayed 1 day in mice that did develop blood-stage infection.

Fewer P.yoeliiΔmif liverstage parasites than control parasites were observed in mouse livers; however, these differences were not statistically significant Thus, the initial steps of hepatocyte invasion and dedifferentiation into a trophozoite appeared not to be affected significantly by the deletion of the mif gene. Microscopic valuation of infected livers revealed that the P.yoeliiΔmif parasites were significantly smaller than control P. yoelii parasites at 44 hpi, with the average diameter of a control parasite being 40.1 ± 10.3 µm and the diameter of a mif-deficient parasite being 24.9 ± 7.6 µm
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of macrophage migration inhibitory factor (MIF).

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 Plasmodium MIF 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 (Augustijn et al., Infect Immun. (2007),75:1116-28).

Phenotype
Phenotype analyses indicate that mutants lacking expression of MIF have a normal blood stage and mosquito development that is not different from wild type parasites. Liver stages of the mutants are affected in development as shown by a delay of the prepatent period in mice infected with mutant sporozoites and a reduced size of developing liver stages.

Additional information
See RMgm-26 for a P. berghei mutant lacking expression of MIF. This mutant showed normal development throughout the complete life cycle. However, no detailed analyses of liver stage development have been published for this mutant

By RT-PCR Py-mif transcripts were detected in mixed blood-stage parasites and 24-h and 44-h liver-stage schizonts but were not observed in infectious salivary gland sporozoites. By IFA using a polyclonal antibody raised against Py-MIF, Py-MIF was detected at low levels in salivary gland sporozoites, but its expression was strong in liver-stage schizonts. The small amounts of MIF observed in sporozoites were not likely to be unspecific staining, since a similar pattern was observed in parasites expressing a myc-tagged version of Py-MIF by use of an anti-myc antibody. At 24 h postinfection (hpi), Py-MIF was distributed uniformly throughout the  cytoplasm of liver stages. At 48 hpi, at which point nuclear centers had formed in the late schizonts, Py-MIF appeared to be restricted to the interior of the parasites and was concentrated in globular patterns. In 16-h and 30-h liver-stage schizonts, MIF exhibited a uniform distribution throughout the parasite, while at a later time point (44 hpi) Py-MIF became more compartmentalized and the distribution was focused in the nuclear centers. Throughout the exoerythrocytic stages, Py-MIF staining was always observed within the confines of the parasite and never within the host cell, as shown by staining with antibodies against the parasitophorous vacuole membrane (PVM) marker UIS4. However, the parasite plasma membrane is in close proximity to the PVM. Thus, it was difficult to determine whether some MIF was also found in the vacuolar lumen. No evidence was found for secretion of Py-MIF by liver stages

Other mutants
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 transgenic mutant P. berghei expressing c-myc-tagged MIF.
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-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


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent 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/construct usedPlasmid double cross-over
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid ApaI
Partial or complete disruption of the geneComplete
Additional remarks partial/complete disruption
Selectable marker used to select the mutant parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationUsing a double-crossover recombination strategy, the mif locus was replaced with a selectable marker (dihydrofolate reductase [DHFR]) and an RFP cassette under the control of a constitutive promoter.

P. yoelii 17XNL genomic DNA was used as a template to amplify a 0.5-kb fragment of the 5′-untranslated region (5′UTR) and a 0.7-kb fragment of the 3′UTR of Py-mif, using oligonucleotide primers PyMIF-1 SacII forward (F), PyMIF-2 ApaI reverse (R), PyMIF-3 ApaI F, and PyMIF-4 NotI R. PyMIF-2 F and PyMIF-3 R contained a unique ApaI site and overlapping sequences. The 5′UTR and 3′UTR fragments were then combined by sequence overlap extension PCR, and the resulting fragment was inserted into the transfection plasmid between SacII and NotI restriction enzyme sites. The resulting plasmid was digested with ApaI between the 5′UTR and the 3′UTR to linearize the plasmid for transfection.
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 1ccgcggTATCCAAAATATTTATTCCTATAGC
Additional information primer 1PyMIF-1 (SacII); 5'UTR
Sequence Primer 2ccataatgtctcagggcccTCTCCATTTTCTATCTCTTCATTTT
Additional information primer 2PyMIF-2 (ApaI); 5'UTR
Sequence Primer 3gatagaaaatggagagggcccTGAGACATTATGGTTCTTTCCTTTT
Additional information primer 3PyMIF-3 (ApaI); 3'UTR
Sequence Primer 4gcggccgcGTTAAAACAATTCGAGAGATGAAAA
Additional information primer 4PyMIF-4 (NotI); 3'UTR
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 nameRFP
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 ApaI
Selectable marker used to select the mutant parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationUsing a double-crossover recombination strategy, the mif locus was replaced with a selectable marker (dihydrofolate reductase [DHFR]) and an RFP cassette under the control of a constitutive promoter.

P. yoelii 17XNL genomic DNA was used as a template to amplify a 0.5-kb fragment of the 5′-untranslated region (5′UTR) and a 0.7-kb fragment of the 3′UTR of Py-mif, using oligonucleotide primers PyMIF-1 SacII forward (F), PyMIF-2 ApaI reverse (R), PyMIF-3 ApaI F, and PyMIF-4 NotI R. PyMIF-2 F and PyMIF-3 R contained a unique ApaI site and overlapping sequences. The 5′UTR and 3′UTR fragments were then combined by sequence overlap extension PCR, and the resulting fragment was inserted into the transfection plasmid between SacII and NotI restriction enzyme sites. The resulting plasmid was digested with ApaI between the 5′UTR and the 3′UTR to linearize the plasmid for transfection.
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 PY05452
Gene productmacrophage migration inhibitory factor
Gene product: Alternative nameMIF
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 1ccgcggTATCCAAAATATTTATTCCTATAGC
Additional information primer 1PyMIF-1 (SacII); 5'UTR
Sequence Primer 2ccataatgtctcagggcccTCTCCATTTTCTATCTCTTCATTTT
Additional information primer 2PyMIF-2 (ApaI); 5'UTR
Sequence Primer 3gatagaaaatggagagggcccTGAGACATTATGGTTCTTTCCTTTT
Additional information primer 3PyMIF-3 (ApaI); 3'UTR
Sequence Primer 4gcggccgcGTTAAAACAATTCGAGAGATGAAAA
Additional information primer 4PyMIF-4 (NotI); 3'UTR