Summary

RMgm-26
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_1444000; Gene model (P.falciparum): PF3D7_1229400; Gene product: macrophage migration inhibitory factor (MIF)
Phenotype Asexual bloodstage;
Last modified: 10 April 2012, 19:30
  *RMgm-26
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption
Reference (PubMed-PMID number) Reference 1 (PMID number) : 17158894
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone P. berghei ANKA cl15cy1
Other information parent lineA reference wild type clone from the ANKA strain of P. berghei (PubMed: PMID: 17406255).
The mutant parasite was generated by
Name PI/ResearcherK.D. Augustijn, C.J. Janse, A.P. Waters
Name Group/DepartmentLeiden Malaria Research Group
Name InstituteLeiden University Medical Center
CityLeiden
CountryThe Netherlands
Name of the mutant parasite
RMgm numberRMgm-26
Principal name425cl1; 573cl1
Alternative namepbmif-ko1; pbmif-ko2
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNormal growth and multiplication rate. Infections in C57BL/6 and BALB/c mice result in increased numbers of reticulocytes
Gametocyte/GameteNot different from wild type
Fertilization and ookineteNot different from wild type
OocystNot different from wild type
SporozoiteNot different from wild type
Liver stageNot different from wild type
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 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.

Phenotype
Mutants were 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 mutants had significantly higher numbers of circulating reticulocytes. 

Other mutants 
RMgm-27 is a transgenic mutant expressing GFP-tagged MIF.
RMgm-31 is a transgenic mutant expressing c-myc-tagged MIF.
RMgm-665: A P. yoelii (17XNL) mutant lacking expression of 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 PBANKA_1444000
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
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Plasmid/construct sequence
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AATTCTATGTAAATTATTTTTCCTATGCCCTTAACATTTATATAAATTATAAATATATTT
TTAAACATATATAATGTCATAATAGGGTATACCATTTTTGTGATCAAAATATAAATTGTA
CTTATGTTTCAATTTTTTATTTATTTAAGAATAAATAAGTTTTCTTTATATGTTGTAAAA
ATATGTACATATGAAATTAAAAAAAAAAAACATATAATTTTGTATAGTATAATTAATAGT
ACCATAAATAAAAGGTGGGAATGAATATTGGAAAATTTGGACATAATATTGCAAAAGCTA
TTTTTATATGTTTATATTTAGCATATAGACATTTATATACGACTAAATATTGTCCTTATT
TGTTATTAGCATATTTTTTTTATAAATTCATATGTAAATATTTTATGAACTTTTTAAAAA
TTCAGTTTATATATATTTCATTTGGTGTGTAATATTATTTATTTTTTTTTATTAAGTACA
ACCTTGTCAAAAAAATTGTTTATATAAATATTTATGAATAAAAAGTGAGAAAGGAAAAAA
ATAAAATTTGTCCTTGTATATAGGATATATAAGGAAAATATAAATAAATTAAAAATACTG
AAATACATTAAATATTTTGATAAATATTAAAAACCAACAAAATATAGAATACCCCATTAA
AAAAAAATGTATGCACAAAATTATTAATTTGGTATACTAAAATATTAAAAAAAGGTAGTG
AAATATTAAAAAAAAAGGCGGAGAAATATTTAAAAAAAACTAGGTAAACACATAACATGT
AGAAAAGTATAAATATTTTTTTCTCCTTTGAGAATTATAAAATTAATTAAAAATTGTATT
TATTTATATTTGATTTATTTTTTCACCATTTTATTAATTAAAACAATTTTTCACCATGCT
TCAATATATATACGCATAAGGATCCACTAGTTCTAGAGCGGCCGCCACCGCGGTGGAGCT
CCAATTCGCCCTATAGTGAGTCGTATTACGCGCGCTCACTGGCCGTCGTTTTACAACGTC
GTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCG
CCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCC
TGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTA
CGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCC
CTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTT
TAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATG
GTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCA
CGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCT
ATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGA
TTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTAGGTGGCAC
TTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATAT
GTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAG
TATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCC
TGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGC
ACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCC
CGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATC
CCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTT
GGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATT
ATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGAT
CGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCT
TGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGAT
GCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGC
TTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCG
CTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTC
TCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTA
CACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGC
CTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGA
TTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCAT
GACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGAT
CAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAA
ACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAA
GGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTT
AGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTT
ACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATA
GTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTT
GGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCAC
GCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGA
GCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCG
CCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAA
AAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACAT
GTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGC
TGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGA
AGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTG
GCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTA
GCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGG
AATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCT
CGAAATTAACCCTCACTAAAGGGAACAAAAGCTGGTACCGGCGAATCTTATCAACCATTT
ATACCAAAAGACTTTAATCAAAACTATAGCGAATCAGCTTCTTATGATAGTGCATATAAT
CCACTTCAAAATACATATGAATTTCATAATTAATTTTTTATAAATAATATATAAGTAAAT
TACGGATTATGAATTGAAATAATTATACATTTTTTTTCTAATTTTCTGAGGCTAATAAAA
TGTATGTAAATATATGCATACACCTCCTTTTCCTATGCTTTTTTTCAGTATAGTTTTTTA
AATATTAAAAAAATACAAATCCAAGCTTTTAGTTGCACACATCATATATATAAGGTGAAA
ATGAGGAGATAGACATACTTTGGAATTATTTATATTGTATTTTTTTTTAAGTAGATTTTT
ACAAATACAATGGCATTATGGCTCTCTCCTTTTTAAATTATATCAAATAAATGCATGCCC
CAAATATTTATATTTTCATATATAAAAATATACTATATAAAATAAGACCCAAATGTAATA
AAAGATAAACTACAAAAAACACGTTGCTTGTGTTGCGTATGCTATTCGTTCACAAATGAT
TTAAAATTGCTCACTCGTTATTTGTTGTTTTATTATTTTTTATTTTCATTTTCTTTTTCA
TTTTTCATGTTTATATAGCTATATTATAATAAACTTTAATTTTTTTGCTTCATTATATTG
AACTTCAAATTTTTTTTTTTTATTTCATTCCACTTTTAATTATCACCATTTGAATAATAC
AAATAATATTATAGTACGTATAAAAGGCATTGTTTGCTTCGATGTTTTATTCACAATAAT
ATATATAATATGTGCTGATATTTAAAATATATTTTTATCCCCCGAGGTGTTTTGACATTG
TGGGAATAATGCATTATGGCGTGTATGGATTAATATCACAAGAGATCGATAAGCTTGCAT
GCCTGCAGGTCAACAATAAATAATAAATAAATATTGTGGAAATAAAATAACATATAATTA
TTTTTAATACATTGATTTCCCTTTTATTTTTTTAAATTTCATTGATATAAAAATATATAA
TAATAACATATATGATTTCAAATTAATCTTTTCAAAAATGGTGTTTATTTTTGTATGTTT
GTGTATGAATTAATCACATAACACATCTATTAAATTGAGTTGGTAATATAGACACAAATA
AATATATATATTTTTATAGCTTAAAAGTGTGTTATGAATATTTTAAGCATATTTTCTTTT
TCTTTGGATTGTGTAAAATGAACTCATATAATGCGTTTTTTTGTTTTTGTTATTTTGTCA
TTTTGTTATTTTGCTATTTTATGGATTAATTTTTGTTTATAAAATGGGAAATAATTTTAA
CATATTTAAATAAATGGAGAAAAAATATAAAATAATTATAAAAAAAAGTTAATACACATT
TTTTCCTGTTATAGACCTTATATTTATTTATCCATATATATATATATATATATATATATA
TATATATACATACCAAGTGAATTAAGAGGAAAGCTAATTTATTATTCAGAATAATATATG
AACTATATATAATTTTTATTATTTTGGTGTATATTAATCTGTCTATATGCATACATGCAA
TAATTTATCGACTTATATATCAAATAACATAAAATAGAAGTGTTTTAAATTATGGATATA
TGCTCAATATTCATTTTTTTTAATAAGTTAGCTATATTTAAATTATACATTTTATATATG
GTCTCTTTTTTTTTTAAATATTATTTAAGTGATCATGAAAATATAAATAATTTTTTTTTA
TTTAATATCCTTTTGCTTGCATGTGGTAAATGGAAATTTGGATGTGTTTTGAARGTTCGG
ATATAGTTGTATGGACATATAATATATTTTGTGAAAAATTGGTTTTATGTTTATACTTAT
GCCAATACTTTTTGAGTAAAACAAAGCAAGTGCTTATAAATAATTAAAGCCAATTTTATA
ATATATATTTTTTTATTTAATTTGAATTTAGTAGTATAATTTTTTATGGTAAGTGCTCAA
AGAGAGTTGCTTATAAAGTATGGTTTGTTTCTTTTTCGCCATTTTGAATTACACATTAAA
AATATATAGATACATATATTATAATATGAAATCATTAATAATTTAGGGAAATTCTACAAA
TTTAAAAACGAATAAAATAATTGTTTTTCATCATGCCATAACACAATATTGATATATACA
TGTACAAACATTTTTTTTATTTGGAAAATATAAATTATATAAAAAAAAATGTATAGTATA
CAAAATGAGCATATTCACACGGGGTGGACGTTCATTTTTTCATTTTTCCCCTGTTTTTTA
TGAGTATATGATAAAATTTTATGAACATTTACACAAAATGAAAATGGATATATAGGAAAA
ATGGAGCGGTATTTCATTTATCTTTGATTGTCATTTGGATATTATATTACCYTGGGTAGG
CAATTAAAAATGTTAAATAACAATTTAAGGAAATTATATTTTATATATTAAAATTAACAC
TGTATTATATGATTCGCTTATAAAAGCCACTCTTTCCCCATGCAAAGCTGTTTAATATCA
ATTTTAACAAATTACACACATGTTAATATATTTATATATATAATTTATATATTTATAATT
TATATATTTATATTTTTATTATTTATATATTTATTATTTATTGTGTGTGTCAATTCGGGT
AGGATATACCTCTTTTTTATTGTTTAAAGCGATTTGTATTCTAAAATATAAAGRATTTGA
AAAAGAGAAAGATAGAATATGATCCCATCATATATAGCCCTATAATTTTTATTTAGCAGC
GAATTAATTTTTCTATTAAGTTTATGTGTAATTAAAATAACGGAATATATATAATACAAT
AAAAAAGTGCATAAATTAAAATTTTTTCAATTAAATTTTTTTTTTTAAGGGGTTATATAA
TATTAAATATATAAAATACGATTATATATTTTTGCTACAATTTTTTATATTAAGATATAA
ATAGTAAATAAATGGTATTATATGGCATGTAATATATAAATTTTTTCCAATTTTTATTTT
ATATACACTTTTCCTTTTTTTGTCATAAAACTTAAACAATTTACACATTCATTTTAAAAA
TTGACTATTTGTTTCAACATTTTTTGAGTTTCCGTTTTATAATAGTATTTTCATTTGTAT
ATTGCTTATATATATAAATACACACCTAAATGTTACAAAGGATCAATGCATAAACCGGTG
TGTCTGGTCGTCGCGATGACCCCCAAGAGGGGCATCGGCATCAACAACGGCCTCCCGTGG
CCCCACTTGACCACAGATTTCAAACACTTTTCTCGTGTGACAAAAACGACGCCCGAAGAA
GCCAGTCGCCTGAACGGGTGGCTTCCCAGGAAATTTGCAAAGACGGGCGACTCTGGACTT
CCCTCTCCATCAGTCGGCAAGAGATTCAACGCCGTTGTCATGGGACGGAAAACCTGGGAA
AGCATGCCTCGAAAGTTTAGACCCCTCGTGGACAGATTGAACATCGTCGTTTCCTCTTCC
CTCAAAGAAGAAGACATTGCGGCGGAGAAGCCTCAAGCTGAAGGCCAGCAGCGCGTCCGA
GTCTGTGCTTCACTCCCAGCAGCTCTCAGCCTTCTGGAGGAAGAGTACAAGGATTCTGTC
GACCAGATTTTTGTCGTGGGAGGAGCGGGACTGTACGAGGCAGCGCTGTCTCTGGGCGTT
GCCTCTCACCTGTACATCACGCGTGTAGCCCGCGAGTTTCCGTGCGACGTTTTCTTCCCT
GCGTTCCCCGGAGATGACATTCTTTCAAACAAATCAACTGCTGCGCAGGCTGCAGCTCCT
GCCGAGTCTGTGTTCGTTCCCTTTTGTCCGGAGCTCGGAAGAGAGAAGGACAATGAAGCG
ACGTATCGACCCATCTTCATTTCCAAGACCTTCTCAGACAACGGGGTTCCCTACGACTTT
GTGGTTCTCGAGAAGAGAAGGAAGACTGACGACGCAGCCACTGCGGAACCGAGCAACGCA
ATGAGCTCCTTGACGTCCACGAGGGAGACAACTCCCGTGCACGGGTTGCAGGCTCCTTCT
TCGGCCGCAGCCATTGCCCCGGTGTTGGCGTGGATGGACGAAGAAGACCGGAAAAAACGC
GAGCAAAAGGAACTGATTCGGGCCGTTCCGCATGTTCACTTTAGAGGCCATGAAGAGTTC
CAGTACCTTGATCTCATTGCCGACATTATTAACAATGGAAGGACAATGGATGACCGAACG
GGCGTTGGTGTCATCTCCAAATTCGGCTGCACTATGCGCTACTCGCTGGATCAGGCCTTT
CCACTTCTCACCACAAAGCGTGTGTTCTGGAAAGGGGTCCTCGAAGAGTTGCTGTGGTTC
ATTCGCGGCGACACGAACGCAAACCATCTTTCTGAGAAGGGCGTGAAGATCTGGGACAAG
AATGTGACACGCGAGTTCCTCGATTCGCGCAATCTCCCCCACCGAGAGGTCGGAGACATC
GGCCCGGGCTACGGCTTCCAGTGGAGACACTTCGGCGCGGCATACAAAGACATGCACACA
GACTACACAGGGCAGGGCGTCGACCAGCTGAAGAATGTGATCCAGATGCTGAGAACGAAT
CCAACAGATCGTCGCATGCTCATGACTGCCTGGAATCCTGCAGCGCTGGACGAAATGGCG
CTGCCGCCTTGTCACTTGTTGTGCCAGTTCTACGTGAACGACCAGAAGGAGCTGTCGTGC
ATCATGTATCAGCGGTCGTGCGATGTCGGCCTCGGCGTCCCCTTCAACATCGCTTCCTAT
TCGCTTTTGACGCTCATGGTTGCACACGTCTGCAACCTAAAACCTAAGGAGTTCATTCAC
TTCATGGGGAACACGCATGTCTACACGAACCATGTCGAGGCTTTAAAAGAGCAGCTGCGG
AGAGAACCGAGACCGTTCCCCATTGTGAACATCCTCAACAAGGAACGCATCAAGGAAATC
GACGATTTCACCGCCGAGGATTTTGAGGTCGTGGGCTACGTCCCGCACGGACGAATCCAG
ATGGAGATGGCTGTCTAGCGGAAATACAGAAGCTAGCTTTGATCCCGTTTTTCTTACTTA
TATATTTATACCAATTGATTGTATTTATAACTGTAAAAATGTGTATGTTGTGTGCATATT
TTTTTTTGTGCATGCACATGCATGTAAATAGCTAAAATTATGAACATTTTATTTTTTGTT
CAGAAAAAAAAAACTTTACACACATAAAATGGCTAGTATGAATAGCCATATTTTATATAA
ATTAAATCCTATGAATTTATGACCATATTAAAAATTTAGATATTTATGGAACATAATATG
TTTGAAACAATAAGACAAAATTATTATTATTATTATTATTTTTACTGTTATAATTATGTT
GTCTCTTCAATGATTCATAAATAGTTGGACTTGATTTTTAAAATGTTTATAATATGATTA
GCATAGTTAAATAAAAAAAGTTGAAAAATTAAAAAAAAACATATAAACACAAATGATGTT
TTTTCCTTCAATTTCGATATCG
Restriction sites to linearize plasmid Asp718, BamHI
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 modification
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 1TTAAATCAAGGTACCGGCGAATCTTATCAACCATTTATACC
Additional information primer 15’KO MIF 5'UTR fwd asp718
Sequence Primer 2TTGTATTTCATCGATCTCTTGTGATATTAATCCATACACGCC
Additional information primer 25’KO MIF 5'UTR rev ClaI
Sequence Primer 3AAAGAGGCTGAATTCTATGTAAATTATTTTTCCTATGCCCTTAAC
Additional information primer 33’KO MIF 3'UTR fwd (EcoRI)
Sequence Primer 4ATCGGATCCTTATGCGTATATATATTGAAGCATGGTG
Additional information primer 43’KO MIF 3'UTR rev (BamHI)
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6