Summary

RMgm-515
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_0719200; Gene model (P.falciparum): PF3D7_0417100; Gene product: mRNA-binding protein PUF2 (Pumilio2)
Phenotype Sporozoite; Liver stage;
Last modified: 1 June 2011, 16:33
  *RMgm-515
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) : 21625527
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/ResearcherC.S.S Gomes-Santos; J.A.M. Braks: G.R. Mair
Name Group/DepartmentMalaria Unit
Name InstituteInstituto de Medicina Molecular
CityLisbon
CountryPortugal
Name of the mutant parasite
RMgm numberRMgm-515
Principal name375cl1
Alternative namePbpuf2-KOa
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
SporozoiteNormal numbers of oocysts and salivary gland sporozoites are formed. Sporozoites are defective in gliding motility and hepatocyte cell traversal in vitro (see also 'Additional remarks phenotype' for further description of the sporozoites). Sporozoites are less infective to hepatocytes in vitro and to mice after intravenous inoculation of sporozoites. Sporozoites could not be transmitted to naive mice by mosquito bite.
Liver stageNormal numbers of oocysts and salivary gland sporozoites are formed. Sporozoites are defective in gliding motility and hepatocyte cell traversal in vitro. Sporozoites are less infective to hepatocytes in vitro and to mice after intravenous inoculation of sporozoites. Sporozoites could not be transmitted to naive mice by mosquito bite.
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of the RNA binding protein Puf2.

Protein (function)
The roles of Puf (Pumilio and fem-3 mRNA binding factor) proteins are diverse yet intimately involved in the translational regulation of developmental and differentiation factors in organisms as diverse as yeast, C. elegans, Drosophila and humans. Two such proteins, Puf1 (PFE0935c) and Puf2 (PFD0825c), are known in the human malaria parasite, P. falciparum, with orthologs in all Plasmodium spp. characterized. The Plasmodium Puf proteins have the typical highly conserved organization that includes the eight tandem copies of the PUM RNA binding domain (or Pumilio Homology Domain, PHD) at the carboxyterminus of the protein and expectedly PfPuf2 was shown to bind RNA in vitro. In P. falciparum evidence has been reported for a role for Puf2 in gametocyte development although Pfpuf2 is most highly transcribed in sporozoites (see also 'Additional Information').

Phenotype
The phenotype analyses indicate that Puf2 is not essential for blood stage development, production of gametocytes, ookinetes, oocysts and sporozoites. However, sporozoite gliding motility, cell traversal capacity and infectivity is affected. Evidence is presented that sporozoites in the absence of Puf2 'prematurely' initiate development into the early livers stage (EEF's) inside salivary glands (see below). The phenotype analyses indicate that the RNA binding protein Puf2 regulates the transition of sporozoites into liver stage forms.

Additional information
Evidence is presented that sporozoites in the absence of Puf2 'prematurely' initiate development into the early livers stage (EEF's) inside salivary glands. This is based on morphological changes of sporozoites in salivary glands, at day 18-27after mosquito infection, resembling transformation of sporozoites into the early liver stage after hepatocyte invasion and on genome-wide transcriptional changes in sporozoites that accompany the morphological changes.

In P. falciparum Puf2 expression has been demonstrated in gametocytes and disruption of Pfpuf2 (PFD0825c) resulted in increased gametocyte production and increased differentiation of male gametocytes (Miao J. et al., 2010, 123-1039-1049). The phenotype of the P. falciparum mutants lacking expression of Puf2 has not been analysed during mosquito development.

See mutants RMgm-513 and RMgm-514 lacking expression of Puf1 ((PBANKA_123350) . Phenotype analyses of these mutants indicate  that Puf1 is not essential for blood stage development, production of gametocytes, ookinetes, oocysts and infective sporozoites and for mosquito transmission and liver stage development .

Mutant RMgm-591 that lacks expression of both Puf1 and Puf2 shows the same phenotype as mutants lacking only expression of Puf2.

Other mutants
RMgm-513: A mutant lacking expression of Puf1 (PBANKA_123350)
RMgm-514: A mutant lacking expression of Puf1 (PBANKA_123350) which expresses GFP under the constitutive eef1α promoter
RMgm-516: An independent mutant lacking expression of Puf2 which expresses GFP under the constitutive eef1α promoter
RMgm-591: A mutant lacking expression of both Puf1 and Puf2 which expresses GFP under the constitutive eef1α promote


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0719200
Gene Model P. falciparum ortholog PF3D7_0417100
Gene productmRNA-binding protein PUF2
Gene product: Alternative namePumilio2
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
Click to view information
Click to hide information
Plasmid/construct sequence
Click to view information
Click to hide information
GTCAACAATAAATAATAAATAAATATTGTGGAAATAAAATAACATATAATTATTTTTAAT
ACATTGATTTCCCTTTTATTTTTTTAAATTTCATTGATATAAAAATATATAATAATAACA
TATATGATTTCAAATTAATCTTTTCAAAAATGGTGTTTATTTTTGTATGTTTGTGTATGA
ATTAATCACATAACACATCTATTAAATTGAGTTGGTAATATAGACACAAATAAATATATA
TATTTTTATAGCTTAAAAGTGTGTTATGAATATTTTAAGCATATTTTCTTTTTCTTTGGA
TTGTGTAAAATGAACTCATATAATGCGTTTTTTTGTTTTTGTTATTTTGTCATTTTGTTA
TTTTGCTATTTTATGGATTAATTTTTGTTTATAAAATGGGAAATAATTTTAACATATTTA
AATAAATGGAGAAAAAATATAAAATAATTATAAAAAAAAGTTAATACACATTTTTTCCTG
TTATAGACCTTATATTTATTTATCCATATATATATATATATATATATATATATATATATA
CATACCAAGTGAATTAAGAGGAAAGCTAATTTATTATTCAGAATAATATATGAACTATAT
ATAATTTTTATTATTTTGGTGTATATTAATCTGTCTATATGCATACATGCAATAATTTAT
CGACTTATATATCAAATAACATAAAATAGAAGTGTTTTAAATTATGGATATATGCTCAAT
ATTCATTTTTTTTAATAAGTTAGCTATATTTAAATTATACATTTTATATATGGTCTCTTT
TTTTTTTAAATATTATTTAAGTGATCATGAAAATATAAATAATTTTTTTTTATTTAATAT
CCTTTTGCTTGCATGTGGTAAATGGAAATTTGGATGTGTTTTGAARGTTCGGATATAGTT
GTATGGACATATAATATATTTTGTGAAAAATTGGTTTTATGTTTATACTTATGCCAATAC
TTTTTGAGTAAAACAAAGCAAGTGCTTATAAATAATTAAAGCCAATTTTATAATATATAT
TTTTTTATTTAATTTGAATTTAGTAGTATAATTTTTTATGGTAAGTGCTCAAAGAGAGTT
GCTTATAAAGTATGGTTTGTTTCTTTTTCGCCATTTTGAATTACACATTAAAAATATATA
GATACATATATTATAATATGAAATCATTAATAATTTAGGGAAATTCTACAAATTTAAAAA
CGAATAAAATAATTGTTTTTCATCATGCCATAACACAATATTGATATATACATGTACAAA
CATTTTTTTTATTTGGAAAATATAAATTATATAAAAAAAAATGTATAGTATACAAAATGA
GCATATTCACACGGGGTGGACGTTCATTTTTTCATTTTTCCCCTGTTTTTTATGAGTATA
TGATAAAATTTTATGAACATTTACACAAAATGAAAATGGATATATAGGAAAAATGGAGCG
GTATTTCATTTATCTTTGATTGTCATTTGGATATTATATTACCYTGGGTAGGCAATTAAA
AATGTTAAATAACAATTTAAGGAAATTATATTTTATATATTAAAATTAACACTGTATTAT
ATGATTCGCTTATAAAAGCCACTCTTTCCCCATGCAAAGCTGTTTAATATCAATTTTAAC
AAATTACACACATGTTAATATATTTATATATATAATTTATATATTTATAATTTATATATT
TATATTTTTATTATTTATATATTTATTATTTATTGTGTGTGTCAATTCGGGTAGGATATA
CCTCTTTTTTATTGTTTAAAGCGATTTGTATTCTAAAATATAAAGRATTTGAAAAAGAGA
AAGATAGAATATGATCCCATCATATATAGCCCTATAATTTTTATTTAGCAGCGAATTAAT
TTTTCTATTAAGTTTATGTGTAATTAAAATAACGGAATATATATAATACAATAAAAAAGT
GCATAAATTAAAATTTTTTCAATTAAATTTTTTTTTTTAAGGGGTTATATAATATTAAAT
ATATAAAATACGATTATATATTTTTGCTACAATTTTTTATATTAAGATATAAATAGTAAA
TAAATGGTATTATATGGCATGTAATATATAAATTTTTTCCAATTTTTATTTTATATACAC
TTTTCCTTTTTTTGTCATAAAACTTAAACAATTTACACATTCATTTTAAAAATTGACTAT
TTGTTTCAACATTTTTTGAGTTTCCGTTTTATAATAGTATTTTCATTTGTATATTGCTTA
TATATATAAATACACACCTAAATGTTACAAAGGATCAATGCATAAACCGGTGTGTCTGGT
CGTCGCGATGACCCCCAAGAGGGGCATCGGCATCAACAACGGCCTCCCGTGGCCCCACTT
GACCACAGATTTCAAACACTTTTCTCGTGTGACAAAAACGACGCCCGAAGAAGCCAGTCG
CCTGAACGGGTGGCTTCCCAGGAAATTTGCAAAGACGGGCGACTCTGGACTTCCCTCTCC
ATCAGTCGGCAAGAGATTCAACGCCGTTGTCATGGGACGGAAAACCTGGGAAAGCATGCC
TCGAAAGTTTAGACCCCTCGTGGACAGATTGAACATCGTCGTTTCCTCTTCCCTCAAAGA
AGAAGACATTGCGGCGGAGAAGCCTCAAGCTGAAGGCCAGCAGCGCGTCCGAGTCTGTGC
TTCACTCCCAGCAGCTCTCAGCCTTCTGGAGGAAGAGTACAAGGATTCTGTCGACCAGAT
TTTTGTCGTGGGAGGAGCGGGACTGTACGAGGCAGCGCTGTCTCTGGGCGTTGCCTCTCA
CCTGTACATCACGCGTGTAGCCCGCGAGTTTCCGTGCGACGTTTTCTTCCCTGCGTTCCC
CGGAGATGACATTCTTTCAAACAAATCAACTGCTGCGCAGGCTGCAGCTCCTGCCGAGTC
TGTGTTCGTTCCCTTTTGTCCGGAGCTCGGAAGAGAGAAGGACAATGAAGCGACGTATCG
ACCCATCTTCATTTCCAAGACCTTCTCAGACAACGGGGTTCCCTACGACTTTGTGGTTCT
CGAGAAGAGAAGGAAGACTGACGACGCAGCCACTGCGGAACCGAGCAACGCAATGAGCTC
CTTGACGTCCACGAGGGAGACAACTCCCGTGCACGGGTTGCAGGCTCCTTCTTCGGCCGC
AGCCATTGCCCCGGTGTTGGCGTGGATGGACGAAGAAGACCGGAAAAAACGCGAGCAAAA
GGAACTGATTCGGGCCGTTCCGCATGTTCACTTTAGAGGCCATGAAGAGTTCCAGTACCT
TGATCTCATTGCCGACATTATTAACAATGGAAGGACAATGGATGACCGAACGGGCGTTGG
TGTCATCTCCAAATTCGGCTGCACTATGCGCTACTCGCTGGATCAGGCCTTTCCACTTCT
CACCACAAAGCGTGTGTTCTGGAAAGGGGTCCTCGAAGAGTTGCTGTGGTTCATTCGCGG
CGACACGAACGCAAACCATCTTTCTGAGAAGGGCGTGAAGATCTGGGACAAGAATGTGAC
ACGCGAGTTCCTCGATTCGCGCAATCTCCCCCACCGAGAGGTCGGAGACATCGGCCCGGG
CTACGGCTTCCAGTGGAGACACTTCGGCGCGGCATACAAAGACATGCACACAGACTACAC
AGGGCAGGGCGTCGACCAGCTGAAGAATGTGATCCAGATGCTGAGAACGAATCCAACAGA
TCGTCGCATGCTCATGACTGCCTGGAATCCTGCAGCGCTGGACGAAATGGCGCTGCCGCC
TTGTCACTTGTTGTGCCAGTTCTACGTGAACGACCAGAAGGAGCTGTCGTGCATCATGTA
TCAGCGGTCGTGCGATGTCGGCCTCGGCGTCCCCTTCAACATCGCTTCCTATTCGCTTTT
GACGCTCATGGTTGCACACGTCTGCAACCTAAAACCTAAGGAGTTCATTCACTTCATGGG
GAACACGCATGTCTACACGAACCATGTCGAGGCTTTAAAAGAGCAGCTGCGGAGAGAACC
GAGACCGTTCCCCATTGTGAACATCCTCAACAAGGAACGCATCAAGGAAATCGACGATTT
CACCGCCGAGGATTTTGAGGTCGTGGGCTACGTCCCGCACGGACGAATCCAGATGGAGAT
GGCTGTCTAGCGGAAATACAGAAGCTGGCTTTGATCCCGTTTTTCTTACTTATATATTTA
TACCAATTGATTGTATTTATAACTGTAAAAATGTGTATGTTGTGTGCATATTTTTTTTTG
TGCATGCACATGCATGTAAATAGCTAAAATTATGAACATTTTATTTTTTGTTCAGAAAAA
AAAAACTTTACACACATAAAATGGCTAGTATGAATAGCCATATTTTATATAAATTAAATC
CTATGAATTTATGACCATATTAAAAATTTAGATATTTATGGAACATAATATGTTTGAAAC
AATAAGACAAAATTATTATTATTATTATTATTTTTACTGTTATAATTATGTTGTCTCTTC
AATGATTCATAAATAGTTGGACTTGATTTTTAAAATGTTTATAATATGATTAGCATAGTT
AAATAAAAAAAGTTGAAAAATTAAAAAAAAACATATAAACACAAATGATGTTTTTTCCTT
CAATTTCGATATCTTCTGTGTCATTTTCTTTAAGCTTTTTTATAACATACTCACATCCAT
TTTTATGAAAACATAAAAAAAATATATCATTCATTATTTTATTTAAATCCTCATCTTTTA
CAGTTTGTTTGATTTCCTTTGTATTTTCTAATATTTCATTGATTGGTTCATTTATTATGG
CATTATCATAATATTTTTCATTTTTTTTTTCACCTGACACTTTATTTATGACTTGTTCAG
GTAAATTTACATTGTGATTTGTGAACAAATTATTTTTGGCTAGCTCAGGGAATTTGTCAT
TATGATTGTCTTCGTATTTAATTTTTTCGAAGAATTTGCCATTTTGTTCGCAAGGGGAAT
TTTCAAAATCATTAATAAATGAAAAATTATCTTCGGTGAGAGTTTTGGGGGAAGATAAAA
ATTCGTCGCTTACTGAGTTTAAACTTTCATAATGAGTAGACCATGAGTTTAACTCTTTTA
AATTGTATAGATTATCTTTGAAGTTAAACTTTTTTTGAACATCCTTGAGGTTTTTTATGT
TTGTTTCGTCTAAAAACATGGATTGCATCGTTTTGGGTATACTTAAATGTATATATGCTG
AAATTTATATAGGTATATATAAAATAAATAATTTATGGTTGTACATGTATATATATTACA
TATGATTATAAAATATGAACAAGGAATATTTTTTTTAAATACGGTCTGAAAAAAAATAAA
TAGCACAATGATGAAATAATTAATGAATGGCTTTTTCAAAATATATGCTAAGTGTAAAAC
AAATATGTTAGAAGATTGCAATTTTTTATTATGTAGATATATAAAATGTGTATTTGAATT
GTTATACAAACTGTTTTCATATTATATTAATATGATGAAATGACAAAAAAAAAATTAAAA
GTATTATTATTTTTGTGTAGACAGCAATAAACTTAAACAATATTCTGTTTTCATGTTAAA
AATGTGTGTAGAATATTTAAGAATAGACAAATGAGAAATGATAAGAAGAAAATAAAAGTA
TACAATATATATCATGGGTATATGGAATGTGGGGATCCACTAGTTCTAGAGCGGCCGCCA
CCGCGGTGGAGCTCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTTCGAGCTTGGCGTAA
TCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATA
CGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTA
ATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAA
TGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCG
CTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAG
GCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAA
GGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTC
CGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACA
GGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCG
ACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCT
CATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGT
GTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAG
TCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGC
AGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTAC
ACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGA
GTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGC
AAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACG
GGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCA
AAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGT
ATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCA
GCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACG
ATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCA
CCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGT
CCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGT
AGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCA
CGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACA
TGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGA
AGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACT
GTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGA
GAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCG
CCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTC
TCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGA
TCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAAT
GCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTT
CAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGT
ATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTAAA
TTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTT
TTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAG
GGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACG
TCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAAT
CAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCC
GATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGA
AAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACAC
CCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCCCATTCGCCATTCAGGCTGCGCAACT
GTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGAT
GTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAA
CGACGGCCAGTGAATTGTAATACGACTCACTATAGGGCGAATTGGGTACCGGGCCCCCCC
TCGAGGTCGACGGTATCGATAAGCTTTCTAAATTTATGCAATTTAAAAACTAATTTATTT
TATTACTTGGGGTAGGCTTTTAGTCATATAAAAAAATAAGCTCCAATTTATAAATCAATA
AATATATGCACTAAAAATAGTTTAATTATAATAAATATATTGAAAGAAAAATTGTTAAGA
ATATATAAAAAACGAACTTATAAAGAATATTAAAATCAAAAAACATTCGTTTTAAATACC
TTTTTTATTAATCAGAAACTAAAATATAACATAAATGTGCAAAAAAAAATCAAAAATTAT
GAAATGTATGCAAACACATATCTAACGTAATTCTTAAATGTCTGTGTTGTCCTAAGTCGT
TTTGTTTTGTCCTCAAAAAATTTGAAGCCGCCAAAGGTTGGATTATATATATACATATGT
ATGCACTTTTTCACTAATAATAGTAAAAATTCATGCCTCTAAATTATTAATAGCCCTTAA
AATATATTTACCTATAAAATATGGAGAGGGGAAATATATATACTCCTATAACAATAATTT
TTCAAAAATATAATTAAAACATACAAGTGTGTCTATTTTTTGCATACACATTTTTATATA
TGAACAAATATACACGTGTACATGTTAATTTTGTTTATATTATATTTTTTAATACTTTTC
CTTTTTTGTTATTACCATAAGTTTCTTCCTTTAGCTTGTCCAAATTTTCAATAATAGTTT
TTACTATTAAATTTCTTTCTTTCCTTTTGCAAGTTGTTAATAATTTCTGCATCATAAAAT
TCCCGTAACAGTCTTTGCAAATGCTTATAATGTTGTCATTTCTGTGTGCAAAAAAAAAAA
AAAATATATAATAGCTAGCTAAGAATATTGAAAAGGGGATAAAAAATAACATATATTTAT
GACAAATAAATTTAATACCCCTCAGGAATGTCGTCAACAATTCTTTTGATAATTTTTTTT
TTGTATTTGTACTCCTTTTTAAGTAAAATTTTTTCTATGATATTGCTAAAAAAGTAGAAA
AGAAAGAAAAATAAATAACCTAAATTATGTTTGCATATAAAAACTCGGAAGCTTGCATGC
CTGCAG
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 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 1AGGATCCCCACATTCCATATACCC
Additional information primer 11544 (BamHI); 5’ targeting region
Sequence Primer 2TGATATCTTCTGTGTCATTTTCTTTAAG
Additional information primer 21545 (EcoRV); 5’ targeting region
Sequence Primer 3TAAGCTTCCGAGTTTTTATATGCAAACA
Additional information primer 31546 (HindIII); 3’ targeting region
Sequence Primer 4TAAGCTTTCTAAATTTATGCAATTTAAAAAC
Additional information primer 41547 (HindIII); 3’ targeting region
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6