Summary

RMgm-62
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_0933700; Gene model (P.falciparum): PF3D7_1113900; Gene product: mitogen-activated protein kinase 2 (MAP2; MAP-2; MAPK2)
Phenotype Fertilization and ookinete;
Last modified: 29 March 2013, 18:28
  *RMgm-62
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) : 15935755
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/ResearcherS.M. Khan, 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-62
Principal name546cl1
Alternative nameMAP2−
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 ookineteMale and female gametocyte production is not affected. Genome replication occurs in the male but no nuclear division/ segmentation. No exflagellation of male gametocytes (no male gamete production).
No fertilization since male gametocytes do not produce gametes.
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of the MAP2 protein.

Protein (function)
MAP kinases are serine/threonine protein kinases involved in a variety of functions including cell proliferation.

Phenotype
The mutant produces wild-type levels of gametocytes but complete male gamete formation was inhibited. Male gametogenesis progressed to genome replication but further development was arrested; neither nuclear division nor gamete formation (cytokensis) occurred, resulting in the presence of rounded male gametes with a single, enlarged nucleus. Female gametes could develop to completion and were fertile, as was shown by the formation of ookinetes in cross-fertilization experiments with fertile males of the mutant line P47− (RMgm-348; which produces infertile females). This observation indicates that MAP2 is essential for male gamete formation and is only active after CDPK4 activation (RMgm-12; a mutant lacking expression of CDPK4).

See also the paper by Dorin-Semblat D. et al. (2007, Mol Microbiol 65, 1170-1180) for unsuccessful attempts to knock-out map2 in P. falciparum, indicating that in P. falciparum map2 has an essential function during asexual blood stage development.

Additional information
Disruption of the P. falciparum ortholog has been attempted (Solyakov et al., 2011, Nat Commun, 2:565).
The gene is likely essential for asexual proliferation as integration of the KO vector was not achieved. Accesibility of the locus to recombination was verified by C-terminal tagging.
 

Other mutants
Independent P. berghei map2- mutants (RMgm-63RMgm-66) have been generated that also lacks MAP2 expression and have essentially the same phenotype.


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0933700
Gene Model P. falciparum ortholog PF3D7_1113900
Gene productmitogen-activated protein kinase 2
Gene product: Alternative nameMAP2; MAP-2; MAPK2
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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ATCCAGTTAATATATATATATTTCGTCGAAAGGGGTGCCCATAAACTGAGCTTGCTATCA
ATAAAACATCGAACTAACCCTTTATACAACTGCTTGTAAATGGGTGTAAAACAATTCTAA
CTTTAATACTAAATTAGTATATATTGCCAAATCGTATATTTTTTTGCGCATTTTTTGAAT
TTCAACATTGTGTTTCTGTAATTTATGACTTTTCTATATGTAATTCAACTTATATATATG
CTAAAAGAATGAACTATTTTAAAAGTTTCCCGCATTTTCTAGACCAAGTTATTCTACATT
AAATTATTCTTAATTTTTAATTAACTTTTCTCTTATAATGTTAAATTTGTACAGAAACTC
AAACAAATGAAAGGGGGTATTTTTTATGCCTCATTAGTTTCACAAACACGCATTTTATGA
AATGGGAGAAACACTGTTTTTTCCACGTCATATGCATGTCTAAATGCATATAATTTTTTT
ATTATTTTCCACCTTTTTCTTTCATTAAAATTTTTTTTAATTATTGAGAAATGCTTTTTC
ATTTATATAATTTTTTAATATTCTACGAATGCTCATGTTCCCAATTTACTATGAATTCGA
TCAATCTTATTATTAACCCATTTTATTTTACACATTATTTTTATTCCGGTAATTTTGTCA
ATGTATAAATACATATATTTATTTGTGTTGATTTATTCATAATGTGAATATGTCTCACTA
AAAGTTCAATTTCAATAATTTTATTTTCACCTAATGAAATTGGGATATATAACAGAATAA
AATAATTCAATAAGGGGGCAAAAAGGATACAATGATTTGTGTGTCAAATCGAAGAGTAAA
TATGCACTCATGGATCCACTAGTTCTAGAGCGGCCGCCACCGCGGTGGAGCTCCAATTCG
CCCTATAGTGAGTCGTATTACGCGCGCTCACTGGCCGTCGTTTTACAACGTCGTGACTGG
GAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGG
CGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGC
GAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGC
GTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTT
CTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTC
CGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGT
AGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTT
AATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTT
GATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAA
AAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTAGGTGGCACTTTTCGGG
GAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGC
TCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTA
TTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTG
CTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGG
GTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAAC
GTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTG
ACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGT
ACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTG
CTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGAC
CGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTT
GGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAG
CAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGC
AACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCC
TTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTA
TCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGG
GGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGA
TTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAAC
TTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAA
TCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGAT
CTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGC
TACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTG
GCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACC
ACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGG
CTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGG
ATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAA
CGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCG
AAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGA
GGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCT
GACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCA
GCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTC
CTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCG
CTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCC
CAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACA
GGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTC
ATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGA
GCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTCGAAATTA
ACCCTCACTAAAGGGAACAAAAGCTGGTACCGGGCCCCCCCTCGAGGTCGACGGTATCGA
TGAATTCCATATATGGAAATGCTAAATCGATCCATAAATAATAGTAATAACATGTATTCA
AAAAGAGAATATATAATTTTAATAGCTAAAATAACGATTTTGTCAATCCATATATTTTTT
GCCCGTTTATTGTTTTTAGCGTTTTCCCATACGATCATATAATATATATTTACCTGAATA
ATATTATATACATTGAGGAAATAAATATATACGCACATTCTCCTTTTTTACGTATAAATG
TAAATTTTTTTTTCAAAATGTTGAAAAAAAAAAAAGAATACTCTGGAATTGAAAAAGAGG
AAAAAAATACAAAAAAAAAAAACAATGCTTCAGAATGTTTAAGCATGCAAAATGATGATA
GCACATATATAGATAGCAATGAAGAAGATAAAAGTTGGGAATACATAAATGAGAACACAA
ATGAAAATATAAAAAATAACGACGATATTAACAAAAAATCTGGCGAAAAAGAATACAATG
CATCCGAGACAAAAAAAACCAATAATAACGAGAAAAACAAAAATAATAGTAACAATAAAA
CTAGCAAAATAAATGATGAAAAAATAAATATAAAAGAAGCTATTATAAAAAATGTGCATG
TGCCAGATAATTACATAATTAAGCACTTAATAGGAAGAGGGTCGTATGGGTATGTATATT
AAGCTTGCATGCCTGCAGGTCAACAATAAATAATAAATAAATATTGTGGAAATAAAATAA
CATATAATTATTTTTAATACATTGATTTCCCTTTTATTTTTTTAAATTTCATTGATATAA
AAATATATAATAATAACATATATGATTTCAAATTAATCTTTTCAAAAATGGTGTTTATTT
TTGTATGTTTGTGTATGAATTAATCACATAACACATCTATTAAATTGAGTTGGTAATATA
GACACAAATAAATATATATATTTTTATAGCTTAAAAGTGTGTTATGAATATTTTAAGCAT
ATTTTCTTTTTCTTTGGATTGTGTAAAATGAACTCATATAATGCGTTTTTTTGTTTTTGT
TATTTTGTCATTTTGTTATTTTGCTATTTTATGGATTAATTTTTGTTTATAAAATGGGAA
ATAATTTTAACATATTTAAATAAATGGAGAAAAAATATAAAATAATTATAAAAAAAAGTT
AATACACATTTTTTCCTGTTATAGACCTTATATTTATTTATCCATATATATATATATATA
TATATATATATATATATACATACCAAGTGAATTAAGAGGAAAGCTAATTTATTATTCAGA
ATAATATATGAACTATATATAATTTTTATTATTTTGGTGTATATTAATCTGTCTATATGC
ATACATGCAATAATTTATCGACTTATATATCAAATAACATAAAATAGAAGTGTTTTAAAT
TATGGATATATGCTCAATATTCATTTTTTTTAATAAGTTAGCTATATTTAAATTATACAT
TTTATATATGGTCTCTTTTTTTTTTAAATATTATTTAAGTGATCATGAAAATATAAATAA
TTTTTTTTTATTTAATATCCTTTTGCTTGCATGTGGTAAATGGAAATTTGGATGTGTTTT
GAARGTTCGGATATAGTTGTATGGACATATAATATATTTTGTGAAAAATTGGTTTTATGT
TTATACTTATGCCAATACTTTTTGAGTAAAACAAAGCAAGTGCTTATAAATAATTAAAGC
CAATTTTATAATATATATTTTTTTATTTAATTTGAATTTAGTAGTATAATTTTTTATGGT
AAGTGCTCAAAGAGAGTTGCTTATAAAGTATGGTTTGTTTCTTTTTCGCCATTTTGAATT
ACACATTAAAAATATATAGATACATATATTATAATATGAAATCATTAATAATTTAGGGAA
ATTCTACAAATTTAAAAACGAATAAAATAATTGTTTTTCATCATGCCATAACACAATATT
GATATATACATGTACAAACATTTTTTTTATTTGGAAAATATAAATTATATAAAAAAAAAT
GTATAGTATACAAAATGAGCATATTCACACGGGGTGGACGTTCATTTTTTCATTTTTCCC
CTGTTTTTTATGAGTATATGATAAAATTTTATGAACATTTACACAAAATGAAAATGGATA
TATAGGAAAAATGGAGCGGTATTTCATTTATCTTTGATTGTCATTTGGATATTATATTAC
CYTGGGTAGGCAATTAAAAATGTTAAATAACAATTTAAGGAAATTATATTTTATATATTA
AAATTAACACTGTATTATATGATTCGCTTATAAAAGCCACTCTTTCCCCATGCAAAGCTG
TTTAATATCAATTTTAACAAATTACACACATGTTAATATATTTATATATATAATTTATAT
ATTTATAATTTATATATTTATATTTTTATTATTTATATATTTATTATTTATTGTGTGTGT
CAATTCGGGTAGGATATACCTCTTTTTTATTGTTTAAAGCGATTTGTATTCTAAAATATA
AAGRATTTGAAAAAGAGAAAGATAGAATATGATCCCATCATATATAGCCCTATAATTTTT
ATTTAGCAGCGAATTAATTTTTCTATTAAGTTTATGTGTAATTAAAATAACGGAATATAT
ATAATACAATAAAAAAGTGCATAAATTAAAATTTTTTCAATTAAATTTTTTTTTTTAAGG
GGTTATATAATATTAAATATATAAAATACGATTATATATTTTTGCTACAATTTTTTATAT
TAAGATATAAATAGTAAATAAATGGTATTATATGGCATGTAATATATAAATTTTTTCCAA
TTTTTATTTTATATACACTTTTCCTTTTTTTGTCATAAAACTTAAACAATTTACACATTC
ATTTTAAAAATTGACTATTTGTTTCAACATTTTTTGAGTTTCCGTTTTATAATAGTATTT
TCATTTGTATATTGCTTATATATATAAATACACACCTAAATGTTACAAAGGATCAATGCA
TAAACCGGTGTGTCTGGTCGTCGCGATGACCCCCAAGAGGGGCATCGGCATCAACAACGG
CCTCCCGTGGCCCCACTTGACCACAGATTTCAAACACTTTTCTCGTGTGACAAAAACGAC
GCCCGAAGAAGCCAGTCGCCTGAACGGGTGGCTTCCCAGGAAATTTGCAAAGACGGGCGA
CTCTGGACTTCCCTCTCCATCAGTCGGCAAGAGATTCAACGCCGTTGTCATGGGACGGAA
AACCTGGGAAAGCATGCCTCGAAAGTTTAGACCCCTCGTGGACAGATTGAACATCGTCGT
TTCCTCTTCCCTCAAAGAAGAAGACATTGCGGCGGAGAAGCCTCAAGCTGAAGGCCAGCA
GCGCGTCCGAGTCTGTGCTTCACTCCCAGCAGCTCTCAGCCTTCTGGAGGAAGAGTACAA
GGATTCTGTCGACCAGATTTTTGTCGTGGGAGGAGCGGGACTGTACGAGGCAGCGCTGTC
TCTGGGCGTTGCCTCTCACCTGTACATCACGCGTGTAGCCCGCGAGTTTCCGTGCGACGT
TTTCTTCCCTGCGTTCCCCGGAGATGACATTCTTTCAAACAAATCAACTGCTGCGCAGGC
TGCAGCTCCTGCCGAGTCTGTGTTCGTTCCCTTTTGTCCGGAGCTCGGAAGAGAGAAGGA
CAATGAAGCGACGTATCGACCCATCTTCATTTCCAAGACCTTCTCAGACAACGGGGTTCC
CTACGACTTTGTGGTTCTCGAGAAGAGAAGGAAGACTGACGACGCAGCCACTGCGGAACC
GAGCAACGCAATGAGCTCCTTGACGTCCACGAGGGAGACAACTCCCGTGCACGGGTTGCA
GGCTCCTTCTTCGGCCGCAGCCATTGCCCCGGTGTTGGCGTGGATGGACGAAGAAGACCG
GAAAAAACGCGAGCAAAAGGAACTGATTCGGGCCGTTCCGCATGTTCACTTTAGAGGCCA
TGAAGAGTTCCAGTACCTTGATCTCATTGCCGACATTATTAACAATGGAAGGACAATGGA
TGACCGAACGGGCGTTGGTGTCATCTCCAAATTCGGCTGCACTATGCGCTACTCGCTGGA
TCAGGCCTTTCCACTTCTCACCACAAAGCGTGTGTTCTGGAAAGGGGTCCTCGAAGAGTT
GCTGTGGTTCATTCGCGGCGACACGAACGCAAACCATCTTTCTGAGAAGGGCGTGAAGAT
CTGGGACAAGAATGTGACACGCGAGTTCCTCGATTCGCGCAATCTCCCCCACCGAGAGGT
CGGAGACATCGGCCCGGGCTACGGCTTCCAGTGGAGACACTTCGGCGCGGCATACAAAGA
CATGCACACAGACTACACAGGGCAGGGCGTCGACCAGCTGAAGAATGTGATCCAGATGCT
GAGAACGAATCCAACAGATCGTCGCATGCTCATGACTGCCTGGAATCCTGCAGCGCTGGA
CGAAATGGCGCTGCCGCCTTGTCACTTGTTGTGCCAGTTCTACGTGAACGACCAGAAGGA
GCTGTCGTGCATCATGTATCAGCGGTCGTGCGATGTCGGCCTCGGCGTCCCCTTCAACAT
CGCTTCCTATTCGCTTTTGACGCTCATGGTTGCACACGTCTGCAACCTAAAACCTAAGGA
GTTCATTCACTTCATGGGGAACACGCATGTCTACACGAACCATGTCGAGGCTTTAAAAGA
GCAGCTGCGGAGAGAACCGAGACCGTTCCCCATTGTGAACATCCTCAACAAGGAACGCAT
CAAGGAAATCGACGATTTCACCGCCGAGGATTTTGAGGTCGTGGGCTACGTCCCGCACGG
ACGAATCCAGATGGAGATGGCTGTCTAGCGGAAATACAGAAGCTAGCTTTGATCCCGTTT
TTCTTACTTATATATTTATACCAATTGATTGTATTTATAACTGTAAAAATGTGTATGTTG
TGTGCATATTTTTTTTTGTGCATGCACATGCATGTAAATAGCTAAAATTATGAACATTTT
ATTTTTTGTTCAGAAAAAAAAAACTTTACACACATAAAATGGCTAGTATGAATAGCCATA
TTTTATATAAATTAAATCCTATGAATTTATGACCATATTAAAAATTTAGATATTTATGGA
ACATAATATGTTTGAAACAATAAGACAAAATTATTATTATTATTATTATTTTTACTGTTA
TAATTATGTTGTCTCTTCAATGATTCATAAATAGTTGGACTTGATTTTTAAAATGTTTAT
AATATGATTAGCATAGTTAAATAAAAAAAGTTGAAAAATTAAAAAAAAACATATAAACAC
AAATGATGTTTTTTCCTTCAATTTCGAT
Restriction sites to linearize plasmid BamHI, Asp718I
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 modificationIn the same study an independent uncloned map2- mutant was generated using the same sequences (line 610).
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 1aaagaattcCATATATGGAAATGCTAAATCG
Additional information primer 12214EcoRI; Primer F 5'
Sequence Primer 2aaaaagcttAATATACATACCCATACGACCC
Additional information primer 22215HindIII; Primer R 5'
Sequence Primer 3aaagatatcCAGTTAATATATATATATTTCG
Additional information primer 32216EcoRV; Primer F 3'
Sequence Primer 4aaaggatccATGAGTGCATATTTACTCTTCG
Additional information primer 42217BamHI; Primer R 3'
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6