Summary

RMgm-133
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PBANKA_1217700; Gene model (P.falciparum): PF3D7_0320800; Gene product: ATP-dependent RNA helicase DDX6 (DOZI, Protein development of zygote inhibited)
Name tag: EGFP
Phenotype Gametocyte/Gamete; Fertilization and ookinete;
Last modified: 21 February 2010, 13:40
  *RMgm-133
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene tagging
Reference (PubMed-PMID number) Reference 1 (PMID number) : 16888139
Reference 2 (PMID number) : 20169188
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/ResearcherG.R. Mair, C.J. Janse, A.P. Waters
Name Group/DepartmentLeiden Malaria Research Group
Name InstituteLeiden University Medical Center (LUMC)
CityLeiden
CountryThe Netherlands
Name of the mutant parasite
RMgm numberRMgm-133
Principal name683cl1; 683cl2
Alternative namePbDOZI::GFP
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNormal production of gametocytes and gametes. A punctate GFP-fluorescence pattern that appeared to be restricted to the cytoplasm of female gametocytes was observed in live and fixed cells after immunofluorescence assay (IFA) analysis with antibodies to GFP. The mutant showed wild-type fertilization rates and zygote/ookinete production.
Fertilization and ookineteThe mutant showed wild-type fertilization rates and zygote/ookinete production.
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses a c-terminal GFP-tagged DOZI (protein development of zygote inhibited; ATP-dependent RNA helicase, putative).

Protein (function)
DOZI is an RNA helicase that is highly up-regulated in female gametocytes and shows high sequence homology to the DDX6 family of RNA helicases.  It contains domains involved in RNA-binding and RNA-unwinding activity.  In other eukaryotes the DDX6 family of DEAD-box RNA helicases is tightly linked both to storage of mRNAs encoding proteins associated with progression through meiosis into translationally silent mRNPs and with the transport of mRNA to degradation centers in the cell (P-bodies). These helicases are found in organisms as diverse as yeast (e.g., Dhh1p) and humans (e.g., RCK/p54). The phenotype analysis of a mutant lacking expression of DOZI (RMgm-100)  indicate that DOZI has a central role in the silencing and maintenance of steady-state levels of a population of gametocyte-specific transcripts. The loss of DOZI severely affected the capacity of the parasite to store and stabilize a discrete subset of mRNAs in the female gametocyte, resulting in a failure to synthesize specific proteins and to complete normal zygote development.  Translational repression of transcripts encoding these proteins in Plasmodium may function to specifically regulate gene expression during meiosis in the zygote.

Phenotype
The phenotype analyses demonstrate normal fertilisation and zygote/ookinete development indicating that the function of DOZI is not affected by the GFP-tag (see RMgm-100 for the phenotype of a mutant lacking expression of DOZI).
A punctate GFP-fluorescence pattern that appeared to be restricted to the cytoplasm of female gametocytes was observed. Fluorescence in situ hybridization (FISH) analysis of the localization of translationally repressed transcripts of genes encoding ookinete surface proteins P25 and P28, showed also a punctate localization pattern comparable to that of DOZI-GFP, indicating that the repressed transcripts and DOZI are distributed similarly in the cytoplasm.
Immunoprecipitations of the DOZI::GFP fusion protein from gametocyte lysates have been made with monoclonal anti-GFP antibodies. The eluates have been analyzed for DOZI protein and transcript content, showing the presence of (known) translational repressed transcripts.

Additional information:
This mutant has also been used for analysis of the protein content by LC-MS/MS mass-spectrometry of imuunoprecipitations of the DOZI::GFP fusion protein from gametocyte lysates (see PubMed, PMID number 20169188). This analysis identified a number of proteins that show homology to proteins found in metazoan mRNP granules involved in translational repression of mRNAs indication the presence of messenger ribonucleoprotein (mRNP) bodies in gametocytes with homology to metazoan mRNP granules/P-bodies (see also mutant RMgm-358 for characterization of another protein, CITH; CAR-I/Trailer Hitch Homolog,  that is present in the mRNPs of gametocytes).

Other mutants
RMgm-100: A mutant lacking expression of DOZI.
RMgm-361: A mutant which lacks expression of DOZI  and expresses GFP under control of a male and RFP under control of a female gametocyte specific promoter.


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1217700
Gene Model P. falciparum ortholog PF3D7_0320800
Gene productATP-dependent RNA helicase DDX6
Gene product: Alternative nameDOZI, Protein development of zygote inhibited
Details of the genetic modification
Name of the tagEGFP
Details of taggingC-terminal
Additional remarks: tagging
Commercial source of tag-antibodiesRoche
Type of plasmid/constructPlasmid single 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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ATCTTTATTTATTATATATATTTTTATTTATTCTATTTTGTTCCTTTTTTTTTATTTGTA
TGTAGTTTATTATTATACTATTATTATTTTTTTTTTTGCTTTCATTCTTTAATTCATATC
GAGTTATTTTTCTTTCTTATAACATTTATATTCCTTTCCATATTCTATTCATATATTTCT
TTTATTACGCTGTTAATTTTATAATATTCTTTTTTACGGCATAATTCTTTCATCTCAATA
CGTTGCATATATATAATCCATATATGTATATAACATATCCTCTCCATGTTTTTCATATTT
TTTTCTACTATTCCCTTTTTCAACAAATGATATAAACAGCTATTCTCTCTCTCTCTATAT
ATATATATATTACTATTCACGCGTTTCATTTTAAAATTCTTATATAAATATAAAATTAAA
CTGTAATATATATAACACTTGTGTGTGTATTTGCATGTATATATAATATATATATACAGC
AAGATGAGTTATAAAACTAATTGTGTCGCTTCAAATGCTAATACCAATGCTTTGAATAAT
TCAAATAATTTAAATAAAATAGAAGACAATGTCATATTTGATGAAGCTTGGAAAAAAAAA
ATATTGGAGCCATTAAAAGATCCTCGATATAAAACAGAAGATGTGACAAAAACCAAAGGA
AATGAATTTGAGGATTACTGTTTGAAAGAAGAGCTTTGATGGGTATTTTTGAAAAAGGTT
ATGAAAAGCCATCGCCAATACAAGAGGAAAGTATTCCTGTGGCTTTGGCTGGAAAAAATA
TTTTAGCTCGAGCAAAAAATGGGACAGGAAAAACCGCCGCATTTGCTATTCCGTTACTAG
AAAAATGCAATACTCATAAAAATTTCATTCAAGGTAAAGCTATTCAAAAAATCAGTAAAG
AAATACATATAATATACATTTACATATGTGTATGGATACTAGTAATTTGTAGAAATTTTT
TACATATAGAAAATCCCAACCCAATGTTTATTTTCTTTGCCTTATATATATGTATACGTA
TATATTCATCTCTTTTCGCTCAAAAATGTTTATTGATATTTCTTTTCGTTCATAGGTTTA
ATACTAGTTCCCACAAGGGAATTGGCGCTACAAACATCTGCTATGATAAAAGAATTAGGA
AAACATATGAAAATTCAGTGTATGGTAACAACCGGTGGTACTTCACTAAGAGAAGATATT
ATGAGACTTTATAATGCCGTTCATATTTTATGTGGAACCCCTGGAAGAATCTTAGATTTA
GCAAATAAAGATGTTGCGAATTTATCAGGATGCCATATAATGGTAATGGATGAAGCAGAC
AAATTATTGTCCCCTGAATTTCAGCCTATAGTAGAAGAATTAATGAAATTTTTACCCAAA
GAAAAACAGATATTAATGTATTCAGCTACATTTCCAGTCACTGTTAAAGAATTTAGACAA
ATTTACTTATCAGATGCTCATGAAATAAATTTAATGGATGAATTGACATTAAAAGGTATT
ACCCAATATTATGCGTTTGTTAAAGAGAGGCAAAAAGTACATTGTTTAAATACTTTATTT
GCTAAATTACAAATTAATCAAGCTATAATATTTTGTAACAGTATAACAAGAGTTGAACTG
TTAGCAAAAAAAATTACAGAATTAGGTTATAGCTCTTTTTATATACATGCAAGAATGTCG
CAAACACATCGAAATCGTGTTTTTCATGATTTTAGAAATGGTGCTTGTCGATGTTTAGTT
TCCTCAGATCTTTTTACAAGAGGTATAGACATACAATCTGTTAATGTAGTAATTAATTTT
GATTTTCCTAAAAATTCAGAAACTTATCTACATAGAATTGGCAGATCAGGAAGATATGGT
CACTTAGGGTTAGCCATTAATTTAATTACATATGAAGACCGTTTTAATTTATATAAAATA
GAATTAGAACTAGGAACTGAAATTCAACCGATCCCTAACGAAATAGATCCATCCATCTAT
ACTGGATCCATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAA
TTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCA
ACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGG
CCAACACTTGTCACTACTTTCGGTTATGGTGTTCAATGCTTTGCGAGATACCCAGATCAT
ATGAAACAGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACT
ATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGAT
ACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTT
GGACACAAATTGGAATACAACTATAACTCACACAATGTATACATCATGGCAGACAAACAA
AAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAA
CTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGAC
AACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCAC
ATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATAC
AAATAAATTCTAGAAGATCCCGTTTTTCTTACTTATATATTTATACCAATTGATTGTATT
TATAACTGTAAAAATGTGTATGTTGTGTGCATATTTTTTTTTGTGCATGCACATGCATGT
AAATAGCTAAAATTATGAACATTTTATTTTTTGTTCAGAAAAAAAAAACTTTACACACAT
AAAATGGCTAGTATGAATAGCCATATTTTATATAAATTAAATCCTATGAATTTATGACCA
TATTAAAAATTTAGATATTTATGGAACATAATATGTTTGAAACAATAAGACAAAATTATT
ATTATTATTATTATTTTTACTGTTATAATTATGTTGTCTCTTCAATGATTCATAAATAGT
TGGACTTGATTTTTAAAATGTTTATAATATGATTAGCATAGTTAAATAAAAAAAGTTGAA
AAATTAAAAAAAAACATATAAACACAAATGATGTTTTTTCCTTCAATTTCGGGTACCGAG
CTCGAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCA
ACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCG
CACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCCTGATGCGGTA
TTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAAT
CTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCC
CTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAG
CTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGT
GATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGG
CACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAA
TATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAA
GAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCT
TCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGG
TGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCG
CCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATT
ATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGA
CTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGA
ATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAAC
GATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCG
CCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCAC
GATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCT
AGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCT
GCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGG
GTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTAT
CTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGG
TGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGAT
TGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCT
CATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAA
GATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAA
AAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCC
GAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTA
GTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCT
GTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACG
ATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAG
CTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGC
CACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGG
AGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTT
TCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATG
GAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCA
CATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTG
AGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGC
GGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAG
CTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAG
TTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTG
TGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAA
GCTTGCATGCCTGCAGGTCAACAATAAATAATAAATAAATATTGTGGAAATAAAATAACA
TATAATTATTTTTAATACATTGATTTCCCTTTTATTTTTTTAAATTTCATTGATATAAAA
ATATATAATAATAACATATATGATTTCAAATTAATCTTTTCAAAAATGGTGTTTATTTTT
GTATGTTTGTGTATGAATTAATCACATAACACATCTATTAAATTGAGTTGGTAATATAGA
CACAAATAAATATATATATTTTTATAGCTTAAAAGTGTGTTATGAATATTTTAAGCATAT
TTTCTTTTTCTTTGGATTGTGTAAAATGAACTCATATAATGCGTTTTTTTGTTTTTGTTA
TTTTGTCATTTTGTTATTTTGCTATTTTATGGATTAATTTTTGTTTATAAAATGGGAAAT
AATTTTAACATATTTAAATAAATGGAGAAAAAATATAAAATAATTATAAAAAAAAGTTAA
TACACATTTTTTCCTGTTATAGACCTTATATTTATTTATCCATATATATATATATATATA
TATATATATATATATACATACCAAGTGAATTAAGAGGAAAGCTAATTTATTATTCAGAAT
AATATATGAACTATATATAATTTTTATTATTTTGGTGTATATTAATCTGTCTATATGCAT
ACATGCAATAATTTATCGACTTATATATCAAATAACATAAAATAGAAGTGTTTTAAATTA
TGGATATATGCTCAATATTCATTTTTTTTAATAAGTTAGCTATATTTAAATTATACATTT
TATATATGGTCTCTTTTTTTTTTAAATATTATTTAAGTGATCATGAAAATATAAATAATT
TTTTTTTATTTAATATCCTTTTGCTTGCATGTGGTAAATGGAAATTTGGATGTGTTTTGA
ARGTTCGGATATAGTTGTATGGACATATAATATATTTTGTGAAAAATTGGTTTTATGTTT
ATACTTATGCCAATACTTTTTGAGTAAAACAAAGCAAGTGCTTATAAATAATTAAAGCCA
ATTTTATAATATATATTTTTTTATTTAATTTGAATTTAGTAGTATAATTTTTTATGGTAA
GTGCTCAAAGAGAGTTGCTTATAAAGTATGGTTTGTTTCTTTTTCGCCATTTTGAATTAC
ACATTAAAAATATATAGATACATATATTATAATATGAAATCATTAATAATTTAGGGAAAT
TCTACAAATTTAAAAACGAATAAAATAATTGTTTTTCATCATGCCATAACACAATATTGA
TATATACATGTACAAACATTTTTTTTATTTGGAAAATATAAATTATATAAAAAAAAATGT
ATAGTATACAAAATGAGCATATTCACACGGGGTGGACGTTCATTTTTTCATTTTTCCCCT
GTTTTTTATGAGTATATGATAAAATTTTATGAACATTTACACAAAATGAAAATGGATATA
TAGGAAAAATGGAGCGGTATTTCATTTATCTTTGATTGTCATTTGGATATTATATTACCY
TGGGTAGGCAATTAAAAATGTTAAATAACAATTTAAGGAAATTATATTTTATATATTAAA
ATTAACACTGTATTATATGATTCGCTTATAAAAGCCACTCTTTCCCCATGCAAAGCTGTT
TAATATCAATTTTAACAAATTACACACATGTTAATATATTTATATATATAATTTATATAT
TTATAATTTATATATTTATATTTTTATTATTTATATATTTATTATTTATTGTGTGTGTCA
ATTCGGGTAGGATATACCTCTTTTTTATTGTTTAAAGCGATTTGTATTCTAAAATATAAA
GRATTTGAAAAAGAGAAAGATAGAATATGATCCCATCATATATAGCCCTATAATTTTTAT
TTAGCAGCGAATTAATTTTTCTATTAAGTTTATGTGTAATTAAAATAACGGAATATATAT
AATACAATAAAAAAGTGCATAAATTAAAATTTTTTCAATTAAATTTTTTTTTTTAAGGGG
TTATATAATATTAAATATATAAAATACGATTATATATTTTTGCTACAATTTTTTATATTA
AGATATAAATAGTAAATAAATGGTATTATATGGCATGTAATATATAAATTTTTTCCAATT
TTTATTTTATATACACTTTTCCTTTTTTTGTCATAAAACTTAAACAATTTACACATTCAT
TTTAAAAATTGACTATTTGTTTCAACATTTTTTGAGTTTCCGTTTTATAATAGTATTTTC
ATTTGTATATTGCTTATATATATAAATACACACCTAAATGTTACAAAGGATCAATGCATA
AACCGGTGTGTCTGGTCGTCGCGATGACCCCCAAGAGGGGCATCGGCATCAACAACGGCC
TCCCGTGGCCCCACTTGACCACAGATTTCAAACACTTTTCTCGTGTGACAAAAACGACGC
CCGAAGAAGCCAGTCGCCTGAACGGGTGGCTTCCCAGGAAATTTGCAAAGACGGGCGACT
CTGGACTTCCCTCTCCATCAGTCGGCAAGAGATTCAACGCCGTTGTCATGGGACGGAAAA
CCTGGGAAAGCATGCCTCGAAAGTTTAGACCCCTCGTGGACAGATTGAACATCGTCGTTT
CCTCTTCCCTCAAAGAAGAAGACATTGCGGCGGAGAAGCCTCAAGCTGAAGGCCAGCAGC
GCGTCCGAGTCTGTGCTTCACTCCCAGCAGCTCTCAGCCTTCTGGAGGAAGAGTACAAGG
ATTCTGTCGACCAGATTTTTGTCGTGGGAGGAGCGGGACTGTACGAGGCAGCGCTGTCTC
TGGGCGTTGCCTCTCACCTGTACATCACGCGTGTAGCCCGCGAGTTTCCGTGCGACGTTT
TCTTCCCTGCGTTCCCCGGAGATGACATTCTTTCAAACAAATCAACTGCTGCGCAGGCTG
CAGCTCCTGCCGAGTCTGTGTTCGTTCCCTTTTGTCCGGAGCTCGGAAGAGAGAAGGACA
ATGAAGCGACGTATCGACCCATCTTCATTTCCAAGACCTTCTCAGACAACGGGGTTCCCT
ACGACTTTGTGGTTCTCGAGAAGAGAAGGAAGACTGACGACGCAGCCACTGCGGAACCGA
GCAACGCAATGAGCTCCTTGACGTCCACGAGGGAGACAACTCCCGTGCACGGGTTGCAGG
CTCCTTCTTCGGCCGCAGCCATTGCCCCGGTGTTGGCGTGGATGGACGAAGAAGACCGGA
AAAAACGCGAGCAAAAGGAACTGATTCGGGCCGTTCCGCATGTTCACTTTAGAGGCCATG
AAGAGTTCCAGTACCTTGATCTCATTGCCGACATTATTAACAATGGAAGGACAATGGATG
ACCGAACGGGCGTTGGTGTCATCTCCAAATTCGGCTGCACTATGCGCTACTCGCTGGATC
AGGCCTTTCCACTTCTCACCACAAAGCGTGTGTTCTGGAAAGGGGTCCTCGAAGAGTTGC
TGTGGTTCATTCGCGGCGACACGAACGCAAACCATCTTTCTGAGAAGGGCGTGAAGATCT
GGGACAAGAATGTGACACGCGAGTTCCTCGATTCGCGCAATCTCCCCCACCGAGAGGTCG
GAGACATCGGCCCGGGCTACGGCTTCCAGTGGAGACACTTCGGCGCGGCATACAAAGACA
TGCACACAGACTACACAGGGCAGGGCGTCGACCAGCTGAAGAATGTGATCCAGATGCTGA
GAACGAATCCAACAGATCGTCGCATGCTCATGACTGCCTGGAATCCTGCAGCGCTGGACG
AAATGGCGCTGCCGCCTTGTCACTTGTTGTGCCAGTTCTACGTGAACGACCAGAAGGAGC
TGTCGTGCATCATGTATCAGCGGTCGTGCGATGTCGGCCTCGGCGTCCCCTTCAACATCG
CTTCCTATTCGCTTTTGACGCTCATGGTTGCACACGTCTGCAACCTAAAACCTAAGGAGT
TCATTCACTTCATGGGGAACACGCATGTCTACACGAACCATGTCGAGGCTTTAAAAGAGC
AGCTGCGGAGAGAACCGAGACCGTTCCCCATTGTGAACATCCTCAACAAGGAACGCATCA
AGGAAATCGACGATTTCACCGCCGAGGATTTTGAGGTCGTGGGCTACGTCCCGCACGGAC
GAATCCAGATGGAGATGGCTGTCTAGCGGAAATACAGAAGCTAGCTTTGATCCCGTTTTT
CTTACTTATATATTTATACCAATTGATTGTATTTATAACTGTAAAAATGTGTATGTTGTG
TGCATATTTTTTTTTGTGCATGCACATGCATGTAAATAGCTAAAATTATGAACATTTTAT
TTTTTGTTCAGAAAAAAAAAACTTTACACACATAAAATGGCTAGTATGAATAGCCATATT
TTATATAAATTAAATCCTATGAATTTATGACCATATTAAAAATTTAGATATTTATGGAAC
ATAATATGTTTGAAACAATAAGACAAAATTATTATTATTATTATTATTTTTACTGTTATA
ATTATGTTGTCTCTTCAATGATTCATAAATAGTTGGACTTGATTTTTAAAATGTTTATAA
TATGATTAGCATAGTTAAATAAAAAAAGTTGAAAAATTAAAAAAAAACATATAAACACAA
ATGATGTTTTTTCCTTCAATTTCGAT
Restriction sites to linearize plasmid
Selectable marker used to select the mutant parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationC-terminal GFP-tagging of DOZI was performed using a construct that integrates through single cross-over homologous recombination into the pbdozi-locus and contains the tgdhfr/ts selectable marker. The disadvantage of using an insertion construct is that the construct can be removed from the genome, thereby restoring the wild type genotype. The primers as shown below were used to amplify the 2153 bps targeting region of pbdozi.
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 1ATAAAACTCGTTATGCATG
Additional information primer 12290BamHI
Sequence Primer 2CATGGATCCAGTATAGATGGATGGATC
Additional information primer 22381BamHI
Sequence Primer 3
Additional information primer 3
Sequence Primer 4
Additional information primer 4
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6