Summary

RMgm-318
Malaria parasiteP. berghei
Genotype
Genetic modification not successful
DisruptedGene model (rodent): PBANKA_1426200; Gene model (P.falciparum): PF3D7_0811300; Gene product: CCR4-associated factor 1|poly(A) ribonuclease POP2
PhenotypeNo phenotype has been described
Last modified: 28 August 2011, 18:05
  *RMgm-318
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 2
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 RMgm-7
Other information parent lineRMgm-7 is a P.berghei ANKA line (507cl1) which constitutively expresses GFP under control of the eukaryotic elongation factor-1α (eef1a) promoter of P. berghei
Attempts to generate the mutant parasite were performed by
Name PI/ResearcherG.R. Mair, C.J. Janse, A.P. Waters
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_1426200
Gene Model P. falciparum ortholog PF3D7_0811300
Gene productCCR4-associated factor 1|poly(A) ribonuclease POP2
Gene product: Alternative name
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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GCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTCGAAATTA
ACCCTCACTAAAGGGAACAAAAGCTGGTACCCCCTAAATCGAAATGCTGTTTTTAAATAA
AATATTGGATATTGAAAAAACAATTGCTAAGTGAAAAGCATATACATAATACACATTCAT
GCATTCATAATATATATACATATATCGTGCTAGTACTATTATTATTTTTTTGTGTGTTGT
GTTGTCAATTTTTATGAAAATGCTGGATACATTTCATCAATACAATTTTAAAATATACGA
CTAGAAAGAGTAAAACATTAAAAACGCGCATATATATACATATATGAGTGTACAATATGT
ATATCAGCAAATACGAATAAATGCCCTGAATTTTTTGTTTTTTAGTAAGTTGGAAAAAAC
ACATTCAAGCGTTTTTCGGTATAAAGAAACAATTTTTAATAAGTTTATATTTTTTCTAAA
TTTGGTGCAAAAAGCGTATATGCATTTATATATATATAAGCCGACGTAAAAGGAAATAAA
TATAGCATTGTTGAAGTAATGCCAATTACAGGAATTATGATTTTTTTTGTGTGTTAGAAA
ATAAGATAGATGATTGATGGATTAAAAAAAAAAATACAAAAGATAAAAGTGATAAAATAC
GAGGCTTGTTTATATACATATATGCACATGTACAATTTGATATTCCCGTTTTATATATGT
GTAGTATGCAAAAAGTGAAAATATTTTGAATGTAAATGCATAAGAACTAAAATAAGCTTG
CATGCCTGCAGGTCAACAATAAATAATAAATAAATATTGTGGAAATAAAATAACATATAA
TTATTTTTAATACATTGATTTCCCTTTTATTTTTTTAAATTTCATTGATATAAAAATATA
TAATAATAACATATATGATTTCAAATTAATCTTTTCAAAAATGGTGTTTATTTTTGTATG
TTTGTGTATGAATTAATCACATAACACATCTATTAAATTGAGTTGGTAATATAGACACAA
ATAAATATATATATTTTTATAGCTTAAAAGTGTGTTATGAATATTTTAAGCATATTTTCT
TTTTCTTTGGATTGTGTAAAATGAACTCATATAATGCGTTTTTTTGTTTTTGTTATTTTG
TCATTTTGTTATTTTGCTATTTTATGGATTAATTTTTGTTTATAAAATGGGAAATAATTT
TAACATATTTAAATAAATGGAGAAAAAATATAAAATAATTATAAAAAAAAGTTAATACAC
ATTTTTTCCTGTTATAGACCTTATATTTATTTATCCATATATATATATATATATATATAT
ATATATATATACATACCAAGTGAATTAAGAGGAAAGCTAATTTATTATTCAGAATAATAT
ATGAACTATATATAATTTTTATTATTTTGGTGTATATTAATCTGTCTATATGCATACATG
CAATAATTTATCGACTTATATATCAAATAACATAAAATAGAAGTGTTTTAAATTATGGAT
ATATGCTCAATATTCATTTTTTTTAATAAGTTAGCTATATTTAAATTATACATTTTATAT
ATGGTCTCTTTTTTTTTTAAATATTATTTAAGTGATCATGAAAATATAAATAATTTTTTT
TTATTTAATATCCTTTTGCTTGCATGTGGTAAATGGAAATTTGGATGTGTTTTGAARGTT
CGGATATAGTTGTATGGACATATAATATATTTTGTGAAAAATTGGTTTTATGTTTATACT
TATGCCAATACTTTTTGAGTAAAACAAAGCAAGTGCTTATAAATAATTAAAGCCAATTTT
ATAATATATATTTTTTTATTTAATTTGAATTTAGTAGTATAATTTTTTATGGTAAGTGCT
CAAAGAGAGTTGCTTATAAAGTATGGTTTGTTTCTTTTTCGCCATTTTGAATTACACATT
AAAAATATATAGATACATATATTATAATATGAAATCATTAATAATTTAGGGAAATTCTAC
AAATTTAAAAACGAATAAAATAATTGTTTTTCATCATGCCATAACACAATATTGATATAT
ACATGTACAAACATTTTTTTTATTTGGAAAATATAAATTATATAAAAAAAAATGTATAGT
ATACAAAATGAGCATATTCACACGGGGTGGACGTTCATTTTTTCATTTTTCCCCTGTTTT
TTATGAGTATATGATAAAATTTTATGAACATTTACACAAAATGAAAATGGATATATAGGA
AAAATGGAGCGGTATTTCATTTATCTTTGATTGTCATTTGGATATTATATTACCYTGGGT
AGGCAATTAAAAATGTTAAATAACAATTTAAGGAAATTATATTTTATATATTAAAATTAA
CACTGTATTATATGATTCGCTTATAAAAGCCACTCTTTCCCCATGCAAAGCTGTTTAATA
TCAATTTTAACAAATTACACACATGTTAATATATTTATATATATAATTTATATATTTATA
ATTTATATATTTATATTTTTATTATTTATATATTTATTATTTATTGTGTGTGTCAATTCG
GGTAGGATATACCTCTTTTTTATTGTTTAAAGCGATTTGTATTCTAAAATATAAAGRATT
TGAAAAAGAGAAAGATAGAATATGATCCCATCATATATAGCCCTATAATTTTTATTTAGC
AGCGAATTAATTTTTCTATTAAGTTTATGTGTAATTAAAATAACGGAATATATATAATAC
AATAAAAAAGTGCATAAATTAAAATTTTTTCAATTAAATTTTTTTTTTTAAGGGGTTATA
TAATATTAAATATATAAAATACGATTATATATTTTTGCTACAATTTTTTATATTAAGATA
TAAATAGTAAATAAATGGTATTATATGGCATGTAATATATAAATTTTTTCCAATTTTTAT
TTTATATACACTTTTCCTTTTTTTGTCATAAAACTTAAACAATTTACACATTCATTTTAA
AAATTGACTATTTGTTTCAACATTTTTTGAGTTTCCGTTTTATAATAGTATTTTCATTTG
TATATTGCTTATATATATAAATACACACCTAAATGTTACAAAGGATCAATGCATAAACCG
GTGTGTCTGGTCGTCGCGATGACCCCCAAGAGGGGCATCGGCATCAACAACGGCCTCCCG
TGGCCCCACTTGACCACAGATTTCAAACACTTTTCTCGTGTGACAAAAACGACGCCCGAA
GAAGCCAGTCGCCTGAACGGGTGGCTTCCCAGGAAATTTGCAAAGACGGGCGACTCTGGA
CTTCCCTCTCCATCAGTCGGCAAGAGATTCAACGCCGTTGTCATGGGACGGAAAACCTGG
GAAAGCATGCCTCGAAAGTTTAGACCCCTCGTGGACAGATTGAACATCGTCGTTTCCTCT
TCCCTCAAAGAAGAAGACATTGCGGCGGAGAAGCCTCAAGCTGAAGGCCAGCAGCGCGTC
CGAGTCTGTGCTTCACTCCCAGCAGCTCTCAGCCTTCTGGAGGAAGAGTACAAGGATTCT
GTCGACCAGATTTTTGTCGTGGGAGGAGCGGGACTGTACGAGGCAGCGCTGTCTCTGGGC
GTTGCCTCTCACCTGTACATCACGCGTGTAGCCCGCGAGTTTCCGTGCGACGTTTTCTTC
CCTGCGTTCCCCGGAGATGACATTCTTTCAAACAAATCAACTGCTGCGCAGGCTGCAGCT
CCTGCCGAGTCTGTGTTCGTTCCCTTTTGTCCGGAGCTCGGAAGAGAGAAGGACAATGAA
GCGACGTATCGACCCATCTTCATTTCCAAGACCTTCTCAGACAACGGGGTTCCCTACGAC
TTTGTGGTTCTCGAGAAGAGAAGGAAGACTGACGACGCAGCCACTGCGGAACCGAGCAAC
GCAATGAGCTCCTTGACGTCCACGAGGGAGACAACTCCCGTGCACGGGTTGCAGGCTCCT
TCTTCGGCCGCAGCCATTGCCCCGGTGTTGGCGTGGATGGACGAAGAAGACCGGAAAAAA
CGCGAGCAAAAGGAACTGATTCGGGCCGTTCCGCATGTTCACTTTAGAGGCCATGAAGAG
TTCCAGTACCTTGATCTCATTGCCGACATTATTAACAATGGAAGGACAATGGATGACCGA
ACGGGCGTTGGTGTCATCTCCAAATTCGGCTGCACTATGCGCTACTCGCTGGATCAGGCC
TTTCCACTTCTCACCACAAAGCGTGTGTTCTGGAAAGGGGTCCTCGAAGAGTTGCTGTGG
TTCATTCGCGGCGACACGAACGCAAACCATCTTTCTGAGAAGGGCGTGAAGATCTGGGAC
AAGAATGTGACACGCGAGTTCCTCGATTCGCGCAATCTCCCCCACCGAGAGGTCGGAGAC
ATCGGCCCGGGCTACGGCTTCCAGTGGAGACACTTCGGCGCGGCATACAAAGACATGCAC
ACAGACTACACAGGGCAGGGCGTCGACCAGCTGAAGAATGTGATCCAGATGCTGAGAACG
AATCCAACAGATCGTCGCATGCTCATGACTGCCTGGAATCCTGCAGCGCTGGACGAAATG
GCGCTGCCGCCTTGTCACTTGTTGTGCCAGTTCTACGTGAACGACCAGAAGGAGCTGTCG
TGCATCATGTATCAGCGGTCGTGCGATGTCGGCCTCGGCGTCCCCTTCAACATCGCTTCC
TATTCGCTTTTGACGCTCATGGTTGCACACGTCTGCAACCTAAAACCTAAGGAGTTCATT
CACTTCATGGGGAACACGCATGTCTACACGAACCATGTCGAGGCTTTAAAAGAGCAGCTG
CGGAGAGAACCGAGACCGTTCCCCATTGTGAACATCCTCAACAAGGAACGCATCAAGGAA
ATCGACGATTTCACCGCCGAGGATTTTGAGGTCGTGGGCTACGTCCCGCACGGACGAATC
CAGATGGAGATGGCTGTCTAGCGGAAATACAGAAGCTAGCTTTGATCCCGTTTTTCTTAC
TTATATATTTATACCAATTGATTGTATTTATAACTGTAAAAATGTGTATGTTGTGTGCAT
ATTTTTTTTTGTGCATGCACATGCATGTAAATAGCTAAAATTATGAACATTTTATTTTTT
GTTCAGAAAAAAAAAACTTTACACACATAAAATGGCTAGTATGAATAGCCATATTTTATA
TAAATTAAATCCTATGAATTTATGACCATATTAAAAATTTAGATATTTATGGAACATAAT
ATGTTTGAAACAATAAGACAAAATTATTATTATTATTATTATTTTTACTGTTATAATTAT
GTTGTCTCTTCAATGATTCATAAATAGTTGGACTTGATTTTTAAAATGTTTATAATATGA
TTAGCATAGTTAAATAAAAAAAGTTGAAAAATTAAAAAAAAACATATAAACACAAATGAT
GTTTTTTCCTTCAATTTCGATATCGAATTCTAATGGTGTTTACTACGGAAATAATAATTA
TCTGAAGAAAAACAATAATAATGGTAATAATACGTCAATTTACGGAAATCATTATTGCAA
CGATAAAAATAATGACAATAATAATAACAATAGCCAATATTGGGACGGAAAAAATGATAT
AATGTCAGCAAATAATACTAACAACCATTATATACATAACAACATTAATAGTAACAATAA
TAGTGATTTATATACTTATGTGGATAGTACAAACAATAGGGAATATATGATTGCTTTTAG
TAAAGATGCAGTAAAAGGAAATATAAAAAGTGATGTACTATATAACTTATATTCAGATAT
TAATCAAAACAATTATAATATGATACCAGGTATGAATAATATGGGAGCAAATAATAATTT
TAACACAAATATATGCAATAATAACAATAATAACAATAATAACAACGGAAACATCAACAA
TACTAATAGCAGTATGAACAATTTTTCTATTAATAAATATAATACATCACAAATAATTAA
TTCTATAAATAAAAATGGAAGAATGAGTTTAGTTAATAATGCATACATTAACAATAACAA
TAAAATTGGAGATAATATTAATGAAAATAACCATAGTAGAATGAACACCAATGATGTTAG
TAGTAATAGTAATGTTGTTAGTATTGGCACACATAATATGGGAATGCTGGATACTATTGG
AAAGCGGCCGCCACCGCGGTGGAGCTCCAATTCGCCCTATAGTGAGTCGTATTACGCGCG
CTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAA
TCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGA
TCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGGACGCGCCCTGTAGCGGCGC
ATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCT
AGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCG
TCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGA
CCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGT
TTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGG
AACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTC
GGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAAT
ATTAACGCTTACAATTTAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGT
TTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATG
CTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATT
CCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTA
AAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGC
GGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAA
GTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGC
CGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTT
ACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACT
GCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCAC
AACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATA
CCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTA
TTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCG
GATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGAT
AAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGT
AAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGA
AATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAA
GTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAG
GTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCAC
TGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGC
GTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGAT
CAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAAT
ACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCT
ACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGT
CTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACG
GGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTA
CAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCG
GTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGG
TATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGC
TCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTG
GCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGAT
AACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGC
AGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCG
CGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGT
GAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTT
ATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGA
Restriction sites to linearize plasmid
Partial or complete disruption of the genePartial
Additional remarks partial/complete disruption The carbon catabolite repressor protein 4 (CCR4)-Not complex is a well-conserved, eukaryotic gene regulatory complex, broadly divided into two modules: CCR4 and CCR4-associated factor 1 (CAF1), regulating mRNA decay, and the Not proteins, regulating transcription and protein degradation. In yeast and other eukaryotes, CAF1 has a major role in determining transcript levels through regulated deadenylation of mRNA. Members of the CCR4-Not complex are well conserved in all Plasmodium species, suggestive of their significance in parasite gene regulation.

The unsuccessful attempts to disrupt caf1 indicates an essential role during asexual blood stage development of P. berghei.

In P. falciparum a 'CAF1-knock out mutant' has been generated and selected by piggyBac-mediated mutagenesis. Asexaul blood stages of this mutant show a severely attenuated growth rate. Phenotype analyses of the P. falciparum mutant indicates that the average number of merozoites/schizont and the length of the cell cycle is similar to that of wild type parasites. However, merozoites show a defect in egress from the host erythrocyte.

The construct used here for disruption of P. berghei caf1 was aimed to disrupt the first exon and a large part of the second exon of the 3 exons.

See RMgm-639 for independent attempts to disrupt P. berghei caf1
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 1L2660 (KpnI); 5'F
Additional information primer 1aaaggtaccCCCTAAATCGAAATGCTG
Sequence Primer 2L2661 (HindIII); 5'R
Additional information primer 2aaaaagcTTATTTTAGTTCTTATGC
Sequence Primer 3L2662 (EcoRI); 3'F
Additional information primer 3aaagaattcTAATGGTGTTTACTACGG
Sequence Primer 4L2663 (NotI); 3'R
Additional information primer 4aaagcggccgcTTTCCAATAGTATCCAGC
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6