Summary

RMgm-247
Malaria parasiteP. berghei
Genotype
Genetic modification not successful
DisruptedGene model (rodent): PBANKA_1443000; Gene model (P.falciparum): PF3D7_1228300; Gene product: NIMA related kinase 1 (nek-1)
PhenotypeNo phenotype has been described
Last modified: 21 December 2011, 16:31
  *RMgm-247
Successful modificationThe gene/parasite could not be changed/generated by the genetic modification.
The following genetic modifications were attempted Gene disruption
Number of attempts to introduce the genetic modification 4
Reference (PubMed-PMID number) Not published (yet)
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).
Attempts to generate the mutant parasite were performed by
Name PI/ResearcherG.R. Mair, S.M. Khan, C.J. Janse
Name Group/DepartmentLeiden Malaria Research Group
Name InstituteLeiden University Medical Center
CityLeiden
CountryThe Netherlands

  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1443000
Gene Model P. falciparum ortholog PF3D7_1228300
Gene productNIMA related kinase 1
Gene product: Alternative namenek-1
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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AATTCTTAATTTACTCTCATTTTATCGTTTTATACATTTTTAATAAAAGCCATTGAATTA
TTATACATATTTTTATATAGTTTTTTAAATTTAATTAATATATTCCATTTATTCTTAAAA
AGCGAGATAGAGAAAAAAAAATCTAGCTATATTTTAATCAAACAACTATAATACACTTTT
TTTTTAACTCATTTTAGCTTTTCTTAATAATGCATATCATTAAAGTGGCGTAAAACATTT
TTAGAGTTTTAAAATATTTATTTTTGTATATAACGAATTGTTTTTTACACAAAGTATTTT
ATTTTAAGGGAAAATAATATTACAAATATATATTTTTTTATTCAGTTTAAAAAAAAATAA
ACAAGTTTCCCCAAAAGATGATGAAAATTAACATAAAAATTACAGTATAAGGCATAAACA
ACAAAGTTATTATTTAATCAATCTCCATTACTATATGGGACATAACACATGCTTGATACA
CAATTATGACCATGTGCTTGGAATAATTCGTAAAAAACAAATAATAGTGAAAAATTAAAA
AAAAATATGAGTAAAACTTAATAAAGCATAATATTCCAAAAAATAACCAAAAATTTTCGA
AAGATAAAAAAATGATAAAGAACAAATTTGAGAGAACATAAAAGTAATAATATTAAAGCA
TAAAACCCCTTATTTAAACATATGTCTCAAATGTGTACATAATATTATGTATTTTAATTG
AGTCAAGTTATACCATCTTTCACAAGTTATATGCTTTTGACTATATTTGTTAAACATTTA
TATATATAGCTTTATTTCATTTTAATTAATATAATATTAAAAAAAAATTGTCTTTTAAAA
AATAAAACGTATATTTTTTTGTTGTTTTTTTAATGTATGTATGCATACTTGTAATGGCAT
ATTAATATTTAATTTATTCCTACAATTTACGGTACATATAAAGCTTGCATGCCTGCAGGT
CAACAATAAATAATAAATAAATATTGTGGAAATAAAATAACATATAATTATTTTTAATAC
ATTGATTTCCCTTTTATTTTTTTAAATTTCATTGATATAAAAATATATAATAATAACATA
TATGATTTCAAATTAATCTTTTCAAAAATGGTGTTTATTTTTGTATGTTTGTGTATGAAT
TAATCACATAACACATCTATTAAATTGAGTTGGTAATATAGACACAAATAAATATATATA
TTTTTATAGCTTAAAAGTGTGTTATGAATATTTTAAGCATATTTTCTTTTTCTTTGGATT
GTGTAAAATGAACTCATATAATGCGTTTTTTTGTTTTTGTTATTTTGTCATTTTGTTATT
TTGCTATTTTATGGATTAATTTTTGTTTATAAAATGGGAAATAATTTTAACATATTTAAA
TAAATGGAGAAAAAATATAAAATAATTATAAAAAAAAGTTAATACACATTTTTTCCTGTT
ATAGACCTTATATTTATTTATCCATATATATATATATATATATATATATATATATATACA
TACCAAGTGAATTAAGAGGAAAGCTAATTTATTATTCAGAATAATATATGAACTATATAT
AATTTTTATTATTTTGGTGTATATTAATCTGTCTATATGCATACATGCAATAATTTATCG
ACTTATATATCAAATAACATAAAATAGAAGTGTTTTAAATTATGGATATATGCTCAATAT
TCATTTTTTTTAATAAGTTAGCTATATTTAAATTATACATTTTATATATGGTCTCTTTTT
TTTTTAAATATTATTTAAGTGATCATGAAAATATAAATAATTTTTTTTTATTTAATATCC
TTTTGCTTGCATGTGGTAAATGGAAATTTGGATGTGTTTTGAARGTTCGGATATAGTTGT
ATGGACATATAATATATTTTGTGAAAAATTGGTTTTATGTTTATACTTATGCCAATACTT
TTTGAGTAAAACAAAGCAAGTGCTTATAAATAATTAAAGCCAATTTTATAATATATATTT
TTTTATTTAATTTGAATTTAGTAGTATAATTTTTTATGGTAAGTGCTCAAAGAGAGTTGC
TTATAAAGTATGGTTTGTTTCTTTTTCGCCATTTTGAATTACACATTAAAAATATATAGA
TACATATATTATAATATGAAATCATTAATAATTTAGGGAAATTCTACAAATTTAAAAACG
AATAAAATAATTGTTTTTCATCATGCCATAACACAATATTGATATATACATGTACAAACA
TTTTTTTTATTTGGAAAATATAAATTATATAAAAAAAAATGTATAGTATACAAAATGAGC
ATATTCACACGGGGTGGACGTTCATTTTTTCATTTTTCCCCTGTTTTTTATGAGTATATG
ATAAAATTTTATGAACATTTACACAAAATGAAAATGGATATATAGGAAAAATGGAGCGGT
ATTTCATTTATCTTTGATTGTCATTTGGATATTATATTACCYTGGGTAGGCAATTAAAAA
TGTTAAATAACAATTTAAGGAAATTATATTTTATATATTAAAATTAACACTGTATTATAT
GATTCGCTTATAAAAGCCACTCTTTCCCCATGCAAAGCTGTTTAATATCAATTTTAACAA
ATTACACACATGTTAATATATTTATATATATAATTTATATATTTATAATTTATATATTTA
TATTTTTATTATTTATATATTTATTATTTATTGTGTGTGTCAATTCGGGTAGGATATACC
TCTTTTTTATTGTTTAAAGCGATTTGTATTCTAAAATATAAAGRATTTGAAAAAGAGAAA
GATAGAATATGATCCCATCATATATAGCCCTATAATTTTTATTTAGCAGCGAATTAATTT
TTCTATTAAGTTTATGTGTAATTAAAATAACGGAATATATATAATACAATAAAAAAGTGC
ATAAATTAAAATTTTTTCAATTAAATTTTTTTTTTTAAGGGGTTATATAATATTAAATAT
ATAAAATACGATTATATATTTTTGCTACAATTTTTTATATTAAGATATAAATAGTAAATA
AATGGTATTATATGGCATGTAATATATAAATTTTTTCCAATTTTTATTTTATATACACTT
TTCCTTTTTTTGTCATAAAACTTAAACAATTTACACATTCATTTTAAAAATTGACTATTT
GTTTCAACATTTTTTGAGTTTCCGTTTTATAATAGTATTTTCATTTGTATATTGCTTATA
TATATAAATACACACCTAAATGTTACAAAGGATCAATGCATAAACCGGTGTGTCTGGTCG
TCGCGATGACCCCCAAGAGGGGCATCGGCATCAACAACGGCCTCCCGTGGCCCCACTTGA
CCACAGATTTCAAACACTTTTCTCGTGTGACAAAAACGACGCCCGAAGAAGCCAGTCGCC
TGAACGGGTGGCTTCCCAGGAAATTTGCAAAGACGGGCGACTCTGGACTTCCCTCTCCAT
CAGTCGGCAAGAGATTCAACGCCGTTGTCATGGGACGGAAAACCTGGGAAAGCATGCCTC
GAAAGTTTAGACCCCTCGTGGACAGATTGAACATCGTCGTTTCCTCTTCCCTCAAAGAAG
AAGACATTGCGGCGGAGAAGCCTCAAGCTGAAGGCCAGCAGCGCGTCCGAGTCTGTGCTT
CACTCCCAGCAGCTCTCAGCCTTCTGGAGGAAGAGTACAAGGATTCTGTCGACCAGATTT
TTGTCGTGGGAGGAGCGGGACTGTACGAGGCAGCGCTGTCTCTGGGCGTTGCCTCTCACC
TGTACATCACGCGTGTAGCCCGCGAGTTTCCGTGCGACGTTTTCTTCCCTGCGTTCCCCG
GAGATGACATTCTTTCAAACAAATCAACTGCTGCGCAGGCTGCAGCTCCTGCCGAGTCTG
TGTTCGTTCCCTTTTGTCCGGAGCTCGGAAGAGAGAAGGACAATGAAGCGACGTATCGAC
CCATCTTCATTTCCAAGACCTTCTCAGACAACGGGGTTCCCTACGACTTTGTGGTTCTCG
AGAAGAGAAGGAAGACTGACGACGCAGCCACTGCGGAACCGAGCAACGCAATGAGCTCCT
TGACGTCCACGAGGGAGACAACTCCCGTGCACGGGTTGCAGGCTCCTTCTTCGGCCGCAG
CCATTGCCCCGGTGTTGGCGTGGATGGACGAAGAAGACCGGAAAAAACGCGAGCAAAAGG
AACTGATTCGGGCCGTTCCGCATGTTCACTTTAGAGGCCATGAAGAGTTCCAGTACCTTG
ATCTCATTGCCGACATTATTAACAATGGAAGGACAATGGATGACCGAACGGGCGTTGGTG
TCATCTCCAAATTCGGCTGCACTATGCGCTACTCGCTGGATCAGGCCTTTCCACTTCTCA
CCACAAAGCGTGTGTTCTGGAAAGGGGTCCTCGAAGAGTTGCTGTGGTTCATTCGCGGCG
ACACGAACGCAAACCATCTTTCTGAGAAGGGCGTGAAGATCTGGGACAAGAATGTGACAC
GCGAGTTCCTCGATTCGCGCAATCTCCCCCACCGAGAGGTCGGAGACATCGGCCCGGGCT
ACGGCTTCCAGTGGAGACACTTCGGCGCGGCATACAAAGACATGCACACAGACTACACAG
GGCAGGGCGTCGACCAGCTGAAGAATGTGATCCAGATGCTGAGAACGAATCCAACAGATC
GTCGCATGCTCATGACTGCCTGGAATCCTGCAGCGCTGGACGAAATGGCGCTGCCGCCTT
GTCACTTGTTGTGCCAGTTCTACGTGAACGACCAGAAGGAGCTGTCGTGCATCATGTATC
AGCGGTCGTGCGATGTCGGCCTCGGCGTCCCCTTCAACATCGCTTCCTATTCGCTTTTGA
CGCTCATGGTTGCACACGTCTGCAACCTAAAACCTAAGGAGTTCATTCACTTCATGGGGA
ACACGCATGTCTACACGAACCATGTCGAGGCTTTAAAAGAGCAGCTGCGGAGAGAACCGA
GACCGTTCCCCATTGTGAACATCCTCAACAAGGAACGCATCAAGGAAATCGACGATTTCA
CCGCCGAGGATTTTGAGGTCGTGGGCTACGTCCCGCACGGACGAATCCAGATGGAGATGG
CTGTCTAGCGGAAATACAGAAGCTAGCTTTGATCCCGTTTTTCTTACTTATATATTTATA
CCAATTGATTGTATTTATAACTGTAAAAATGTGTATGTTGTGTGCATATTTTTTTTTGTG
CATGCACATGCATGTAAATAGCTAAAATTATGAACATTTTATTTTTTGTTCAGAAAAAAA
AAACTTTACACACATAAAATGGCTAGTATGAATAGCCATATTTTATATAAATTAAATCCT
ATGAATTTATGACCATATTAAAAATTTAGATATTTATGGAACATAATATGTTTGAAACAA
TAAGACAAAATTATTATTATTATTATTATTTTTACTGTTATAATTATGTTGTCTCTTCAA
TGATTCATAAATAGTTGGACTTGATTTTTAAAATGTTTATAATATGATTAGCATAGTTAA
ATAAAAAAAGTTGAAAAATTAAAAAAAAACATATAAACACAAATGATGTTTTTTCCTTCA
ATTTCGATGGGTGAGAAAATATGGGGAATACAGAAAATTATTTTGATTCTTTCCCCTTTA
TTTCCCTTTTTGCCATTTAATTGTTATTATTATATATATTTTTTTTTCTGTTCATTAAAT
GGCTTAATTATGACATAAAATATTTCATTATGTGGCATTTATTATACTCTATCATTATTA
TATATATTTAGATATCGCTACCCACGATATTATATGAAATAGTTTTGCTTCATTCTCTAT
TTTTTCTTCTTGTTATTACACATTTTATCATATATATATTAATATTGTTATTTACATATT
ATGGAAATATAAATGCACTTAATTTTTGGAACGAATGCGAAAAATATCTATGATATATAT
ATGCATATCGTTCCATATATACACACATACACATGTATGTATGTATGTATACTTGCCCGT
GGTACATAATATTTACATATTTGTGCATGCTAATATAAATTCTTCTAGTTTAATTTGTTT
TGGTTTCATAATAAAATGTTGTTTATTTTATTAAACATAATTTTTCCCATTTTATGCTTA
ACATTTTTATTTTATACTATTTATTTTATTTTTAATTTGGCGATGTGGCAACAGAGATTC
TTAAGTTAAACACGTATTATAATGGTATGTGATTTTCAAATTTCATTTTTCATAGTTCAA
TTCCTTTTTGCGGTTTTTTAAGCTTAGTTTTTCTGACTGATTAATTTTAGCGTGCATATA
TAATGTATGCAGGATCCACTAGTTCTAGAGCGGCCGCCACCGCGGTGGAGCTCCAATTCG
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
TG
Restriction sites to linearize plasmid
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 modificationDisruption experiments were performed in the Leiden Malaria Research Group (exp. 583, 607, 628, 660; pl1024).

The NIMA-related protein kinases (Neks) constitute an extended family of eukaryotic mitotic serine/threonine kinases. The best characterized members of the Nek family include NIMA (never in mitosis/Aspergillus), the founding member from the fungus Aspergillus nidulans, and its closest homologue in mammals, Nek2. Initially identified as a kinase essential for mitotic entry in Aspergillus, NIMA has been also shown to participate in nuclear membrane fission. Eleven members of the NIMA kinase family (Nek1-11) have now been identified in various human tissues, and together fulfil a number of cell cycle-related functions in centrosome separation, mitosis, meiosis and checkpoint control. The P. falciparum kinome includes four NIMA-related serine/threonine kinases. Pfnek-1 (PlasmoDB identifier PFL1370w) clusters within the Aspergillus NIMA/human Nek2 branch in phylogenetic trees, while clear orthology to mammalian or yeast Neks could not be assigned for the three other P. falciparum sequences (Pfnek-2, -3 and -4, PlasmoDB identifiers PFE1290w, PFL0080c and MAL7P1.100,respectively).

The unsuccessful attempts to disrupt this gene indicates an essential function during blood stage development.

See also RMgm-564 for unsuccessful attempts to disrupt PBANKA_144300

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.
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 1L2218 (EcoRI); 5'
Additional information primer 1aaagaattcTTAATTTACTCTCATTTTATCG
Sequence Primer 2L2219 (HindIII); 5'
Additional information primer 2aaaaagcttTATATGTACCGTAAATTGTAGG
Sequence Primer 3L2220 (XmaI); 3'
Additional information primer 3aaacccgggTGAGAAAATATGGGGAATACAG
Sequence Primer 4L2221 (KpnI); 3'
Additional information primer 4aaaggatccTGCATACATTATATATGCACGC
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6