Summary

RMgm-346
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_1359600; Gene model (P.falciparum): PF3D7_1346700; Gene product: 6-cysteine protein | transmission blocking target antigen precursor (P48/45)
Transgene
Transgene not Plasmodium: GFP (gfp-mu3)
Promoter: Gene model: PBANKA_1133300; Gene model (P.falciparum): PF3D7_1357100; Gene product: elongation factor 1-alpha (eef1a)
3'UTR: Gene model: PBANKA_0719300; Gene product: bifunctional dihydrofolate reductase-thymidylate synthase, putative (dhfr-ts)
Replacement locus: Gene model: PBANKA_0306000; Gene product: 6-cysteine protein (230p)
Phenotype Gametocyte/Gamete; Fertilization and ookinete;
Last modified: 24 December 2009, 22:24
  *RMgm-346
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption, Introduction of a transgene
Reference (PubMed-PMID number) Not published (yet)
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 507cl1 (RMgm-7)
Other information parent lineP.berghei ANKA 507cl1 (RMgm-7) is a reference ANKA mutant line which expresses GFP under control of a constitutive promoter (PubMed: PMID: 16242190).
The mutant parasite was generated by
Name PI/ResearcherB. Franke-Fayard; C.J. Janse
Name Group/DepartmentLeiden Malaria Research Group
Name InstituteLeiden University Medical Center
CityLeiden
CountryThe Netherlands
Name of the mutant parasite
RMgm numberRMgm-346
Principal name764acl1
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteMale gametes are strongly affected in their fertility, resulting in nearly complete inhibition of ookinete formation in vitro (>0.99%); Motile males fail to attach to and penetrate female gametes.
Mutant female gametes are fertile as shown by cross-fertilization with wild type male gametes.
Mutant parasites produce low numbers of ookinetes in vivo (Anopheles stephensi). See also 'Additional remarks phenotype'.
Fertilization and ookineteMale gametes are strongly affected in their fertility, resulting in nearly complete inhibition of ookinete formation in vitro (>0.99%); Motile males fail to attach to and penetrate female gametes.
Mutant female gametes are fertile as shown by cross-fertilization with wild type male gametes.
Mutant parasites produce low numbers of ookinetes in vivo (Anopheles stephensi). See also 'Additional remarks phenotype'.
OocystNot different from wild type
SporozoiteNot different from wild type
Liver stageNot different from wild type
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of the P48/45 protein and expresses GFP under the control of the constitutive eef1a promoter.

Protein (function)
The P48/45 protein (PF13_0247) is a member of a small family of proteins, the 6-cysteine (cys) family of (surface) proteins (Thompson J. et al., Mol. Biochem. Parasitol. (2001)118, 147-54). The proteins are characterized by domains of roughly 120 amino acids in size that contain six positionally conserved cysteines (6-cys). Although some species of Plasmodium (may) contain unique members of the 6-cys family, ten members have been identified that are conserved both in structure as well as in genome organization throughout the genus (Thompson et al., 2001). Some of the conserved 6-cys proteins are encoded by genes that form paralogous gene-pairs which are closely linked in the genome separated by less then 2 kb of intergenic region. Most members have a GPI anchor and are predicted membrane surface proteins whereas others appear to be secreted and most members are expressed in a discrete stage-specific manner in gametocytes, sporozoites or merozoites.
P48/45 is a surface protein of both male and female gametes and is considered as a major candidate antigen for development of a transmission blocking vaccine.

Phenotype
Phenotype analyses demonstrate that P48/45 plays an important role in fertilization (male fertility factor). Motile males fail to attach to and penetrate female gametes (see phenotype description). Mutant female gametes are fertile as has been shown by cross-fertilisation with wild type male gametes.

This mutant shows the same phenotype as mutant RMgm-15 which has been characterized in more detail. This mutant has been generated to be able to distinguish the female gametocytes and ookinetes based on their high GFP expression in cross-fertilisation experiments with fertile males of other mutants (see below).

Mutants lacking expression of P48/45 are frequently used in cross-fertilization studies in which the fertile females are used to test the fertility of males of other mutants (Khan et al., Cell (2005) 121, 675-87; Raine, J.D. et al., PLoS Pathog (2007) 3,e30).
These mutants  show very low levels of fertilization with higher fertilization rates in the mosquito than in in vitro ookinete cultures. Mutant parasites produce low numbers of ookinetes and oocysts in Anopheles stephensi mosquitoes (see also the phenotype description 'Fertilization and ookinete'). Both the ookinetes and oocysts show a normal morphology and oocysts produce normal numbers of sporozoites that are infectious to mice.

Additional information

Table: P. falciparum gene members of the 6-cys family

Gene P. falciparum Gene P. falciparum
p48/45  PF13_0247 p12p  PFF0620c
p47  PF13_0248 p230p  PFB0400w
p36  PFD0210c p230  PFB0405w
p52  PFD0215c p38  PFE0395c
p12  PFF0615c p41  PFD0240c

 

Other mutants
RMgm-15: An independent mutant lacking expression of P48/45
RMgm-349: A mutant lacking expression of both P47 and P48/45
RMgm-345: The mutant lacks expression of the P48/45 protein and expresses GFP under the control of a male specific promoter and RFP under the control of a female specific promoter. This mutant has been generated to be able to distinguish the female gametocytes/gametes and ookinetes based on their RFP expression in cross-fertilisation experiments with fertile males of other mutants and for determination of meiosis in cross-fertilisation studies. Meiosis in zygotes is determined by FACS analysis of the DNA content of Hoechst-stained gametes/zygotes selected based on their RFP-fluorescence.


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1359600
Gene Model P. falciparum ortholog PF3D7_1346700
Gene product6-cysteine protein | transmission blocking target antigen precursor
Gene product: Alternative nameP48/45
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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GGGCCCCCCCTCGAGGTCGACGGTATCGATAAGCTTGGATCCGAAGGCATATATAAGCAT
ACGTCAATACCCACGCACACTTCCCCGCACATCAAAGTTATCAAGTGCAAATAAATCAGT
GAATATATGTAGTGAAGTAAAAGAAGAGTAATTTTTTTCTGCATTTTTGCCGTGTGTGTC
CCTTTTCTCTCTTTAATAATGCTCTACTTTTTTGGGAACAGCCGTTTTTTTCTTTTTTTT
TTTTACTTTTTTTTTTATTTTGTATTAGTTATAAAATCGTCAGTTGGTAAAAATGAGTAT
GTTTCTCCAGATGAATTGAATATAAAAACATCCGGATTTTTAGGATACAAATGCGACTTT
TCCACCGAAGGAATTCATAATTTAGAACCTGATATTGTTGAAAGAAGATCAGTAATATGT
AGTATAAATTCATATTTTATTTATGATAAAATAAAACTAATTATACCTAAACAGGATGAC
CCAAAATCTAAATTTAAATTGCTACCAGAAAATTGTTTTGCAAAAGTATATTCAGATATT
GAAGGAAAAACAGAAATACCAATAGAACAAACAGGATTAATAGAATATACACTTGAAGAA
AATGATACTAATCAAGATTATAGTGAAAGGATTATTCAAATATCGCCATTTAATAATAAA
GATATAGAATTTTATTGCATTTGTGATAATACTGAACAAGTTATATCTCATATTGATGGA
AGAAGTGCATTAGTGCATGTAACTGTAAAGCTTGCATGCCTGCAGGTCAACAATAAATAA
TAAATAAATATTGTGGAAATAAAATAACATATAATTATTTTTAATACATTGATTTCCCTT
TTATTTTTTTAAATTTCATTGATATAAAAATATATAATAATAACATATATGATTTCAAAT
TAATCTTTTCAAAAATGGTGTTTATTTTTGTATGTTTGTGTATGAATTAATCACATAACA
CATCTATTAAATTGAGTTGGTAATATAGACACAAATAAATATATATATTTTTATAGCTTA
AAAGTGTGTTATGAATATTTTAAGCATATTTTCTTTTTCTTTGGATTGTGTAAAATGAAC
TCATATAATGCGTTTTTTTGTTTTTGTTATTTTGTCATTTTGTTATTTTGCTATTTTATG
GATTAATTTTTGTTTATAAAATGGGAAATAATTTTAACATATTTAAATAAATGGAGAAAA
AATATAAAATAATTATAAAAAAAAGTTAATACACATTTTTTCCTGTTATAGACCTTATAT
TTATTTATCCATATATATATATATATATATATATATATATATATACATACCAAGTGAATT
AAGAGGAAAGCTAATTTATTATTCAGAATAATATATGAACTATATATAATTTTTATTATT
TTGGTGTATATTAATCTGTCTATATGCATACATGCAATAATTTATCGACTTATATATCAA
ATAACATAAAATAGAAGTGTTTTAAATTATGGATATATGCTCAATATTCATTTTTTTTAA
TAAGTTAGCTATATTTAAATTATACATTTTATATATGGTCTCTTTTTTTTTTAAATATTA
TTTAAGTGATCATGAAAATATAAATAATTTTTTTTTATTTAATATCCTTTTGCTTGCATG
TGGTAAATGGAAATTTGGATGTGTTTTGAARGTTCGGATATAGTTGTATGGACATATAAT
ATATTTTGTGAAAAATTGGTTTTATGTTTATACTTATGCCAATACTTTTTGAGTAAAACA
AAGCAAGTGCTTATAAATAATTAAAGCCAATTTTATAATATATATTTTTTTATTTAATTT
GAATTTAGTAGTATAATTTTTTATGGTAAGTGCTCAAAGAGAGTTGCTTATAAAGTATGG
TTTGTTTCTTTTTCGCCATTTTGAATTACACATTAAAAATATATAGATACATATATTATA
ATATGAAATCATTAATAATTTAGGGAAATTCTACAAATTTAAAAACGAATAAAATAATTG
TTTTTCATCATGCCATAACACAATATTGATATATACATGTACAAACATTTTTTTTATTTG
GAAAATATAAATTATATAAAAAAAAATGTATAGTATACAAAATGAGCATATTCACACGGG
GTGGACGTTCATTTTTTCATTTTTCCCCTGTTTTTTATGAGTATATGATAAAATTTTATG
AACATTTACACAAAATGAAAATGGATATATAGGAAAAATGGAGCGGTATTTCATTTATCT
TTGATTGTCATTTGGATATTATATTACCYTGGGTAGGCAATTAAAAATGTTAAATAACAA
TTTAAGGAAATTATATTTTATATATTAAAATTAACACTGTATTATATGATTCGCTTATAA
AAGCCACTCTTTCCCCATGCAAAGCTGTTTAATATCAATTTTAACAAATTACACACATGT
TAATATATTTATATATATAATTTATATATTTATAATTTATATATTTATATTTTTATTATT
TATATATTTATTATTTATTGTGTGTGTCAATTCGGGTAGGATATACCTCTTTTTTATTGT
TTAAAGCGATTTGTATTCTAAAATATAAAGRATTTGAAAAAGAGAAAGATAGAATATGAT
CCCATCATATATAGCCCTATAATTTTTATTTAGCAGCGAATTAATTTTTCTATTAAGTTT
ATGTGTAATTAAAATAACGGAATATATATAATACAATAAAAAAGTGCATAAATTAAAATT
TTTTCAATTAAATTTTTTTTTTTAAGGGGTTATATAATATTAAATATATAAAATACGATT
ATATATTTTTGCTACAATTTTTTATATTAAGATATAAATAGTAAATAAATGGTATTATAT
GGCATGTAATATATAAATTTTTTCCAATTTTTATTTTATATACACTTTTCCTTTTTTTGT
CATAAAACTTAAACAATTTACACATTCATTTTAAAAATTGACTATTTGTTTCAACATTTT
TTGAGTTTCCGTTTTATAATAGTATTTTCATTTGTATATTGCTTATATATATAAATACAC
ACCTAAATGTTACAAAGGATCAATGCATAAACCGGTGTGTCTGGTCGTCGCGATGACCCC
CAAGAGGGGCATCGGCATCAACAACGGCCTCCCGTGGCCCCACTTGACCACAGATTTCAA
ACACTTTTCTCGTGTGACAAAAACGACGCCCGAAGAAGCCAGTCGCCTGAACGGGTGGCT
TCCCAGGAAATTTGCAAAGACGGGCGACTCTGGACTTCCCTCTCCATCAGTCGGCAAGAG
ATTCAACGCCGTTGTCATGGGACGGAAAACCTGGGAAAGCATGCCTCGAAAGTTTAGACC
CCTCGTGGACAGATTGAACATCGTCGTTTCCTCTTCCCTCAAAGAAGAAGACATTGCGGC
GGAGAAGCCTCAAGCTGAAGGCCAGCAGCGCGTCCGAGTCTGTGCTTCACTCCCAGCAGC
TCTCAGCCTTCTGGAGGAAGAGTACAAGGATTCTGTCGACCAGATTTTTGTCGTGGGAGG
AGCGGGACTGTACGAGGCAGCGCTGTCTCTGGGCGTTGCCTCTCACCTGTACATCACGCG
TGTAGCCCGCGAGTTTCCGTGCGACGTTTTCTTCCCTGCGTTCCCCGGAGATGACATTCT
TTCAAACAAATCAACTGCTGCGCAGGCTGCAGCTCCTGCCGAGTCTGTGTTCGTTCCCTT
TTGTCCGGAGCTCGGAAGAGAGAAGGACAATGAAGCGACGTATCGACCCATCTTCATTTC
CAAGACCTTCTCAGACAACGGGGTTCCCTACGACTTTGTGGTTCTCGAGAAGAGAAGGAA
GACTGACGACGCAGCCACTGCGGAACCGAGCAACGCAATGAGCTCCTTGACGTCCACGAG
GGAGACAACTCCCGTGCACGGGTTGCAGGCTCCTTCTTCGGCCGCAGCCATTGCCCCGGT
GTTGGCGTGGATGGACGAAGAAGACCGGAAAAAACGCGAGCAAAAGGAACTGATTCGGGC
CGTTCCGCATGTTCACTTTAGAGGCCATGAAGAGTTCCAGTACCTTGATCTCATTGCCGA
CATTATTAACAATGGAAGGACAATGGATGACCGAACGGGCGTTGGTGTCATCTCCAAATT
CGGCTGCACTATGCGCTACTCGCTGGATCAGGCCTTTCCACTTCTCACCACAAAGCGTGT
GTTCTGGAAAGGGGTCCTCGAAGAGTTGCTGTGGTTCATTCGCGGCGACACGAACGCAAA
CCATCTTTCTGAGAAGGGCGTGAAGATCTGGGACAAGAATGTGACACGCGAGTTCCTCGA
TTCGCGCAATCTCCCCCACCGAGAGGTCGGAGACATCGGCCCGGGCTACGGCTTCCAGTG
GAGACACTTCGGCGCGGCATACAAAGACATGCACACAGACTACACAGGGCAGGGCGTCGA
CCAGCTGAAGAATGTGATCCAGATGCTGAGAACGAATCCAACAGATCGTCGCATGCTCAT
GACTGCCTGGAATCCTGCAGCGCTGGACGAAATGGCGCTGCCGCCTTGTCACTTGTTGTG
CCAGTTCTACGTGAACGACCAGAAGGAGCTGTCGTGCATCATGTATCAGCGGTCGTGCGA
TGTCGGCCTCGGCGTCCCCTTCAACATCGCTTCCTATTCGCTTTTGACGCTCATGGTTGC
ACACGTCTGCAACCTAAAACCTAAGGAGTTCATTCACTTCATGGGGAACACGCATGTCTA
CACGAACCATGTCGAGGCTTTAAAAGAGCAGCTGCGGAGAGAACCGAGACCGTTCCCCAT
TGTGAACATCCTCAACAAGGAACGCATCAAGGAAATCGACGATTTCACCGCCGAGGATTT
TGAGGTCGTGGGCTACGTCCCGCACGGACGAATCCAGATGGAGATGGCTGTCTAGCGGAA
ATACAGAAGCTAGCTTTGATCCCGTTTTTCTTACTTATATATTTATACCAATTGATTGTA
TTTATAACTGTAAAAATGTGTATGTTGTGTGCATATTTTTTTTTGTGCATGCACATGCAT
GTAAATAGCTAAAATTATGAACATTTTATTTTTTGTTCAGAAAAAAAAAACTTTACACAC
ATAAAATGGCTAGTATGAATAGCCATATTTTATATAAATTAAATCCTATGAATTTATGAC
CATATTAAAAATTTAGATATTTATGGAACATAATATGTTTGAAACAATAAGACAAAATTA
TTATTATTATTATTATTTTTACTGTTATAATTATGTTGTCTCTTCAATGATTCATAAATA
GTTGGACTTGATTTTTAAAATGTTTATAATATGATTAGCATAGTTAAATAAAAAAAGTTG
AAAAATTAAAAAAAAACATATAAACACAAATGATGTTTTTTCCTTCAATTTCGATATCAG
CAATACCTCAATCAGCATCTTTTACATGTATGTGTAAAATGGATAAAATAACTGGTTTTA
TGAATATTAAAATTGGTTCCGCATATTATGCTTTTCTCTCAAAATTGTTTATTATATTTA
TTCCCTTATTTTTTATGTGGCTATAAAGTAGTGTGTAGCGTATTCTTTTATTTTACAGAA
AATGTATATTATCATTTTTCGCATTTCATCGTTTAAACAAGTACATGAACGTGTGCATGC
AATTATTTCTGTGTTTGGATTTGTATTCAAATCATCATGAACTTTTTTTGAATATTCGTT
ACAAGCCATAATAGAATATACACCATCTACAATATAATTTTTTTTTCACTATTTTATATT
TCATCCTATTGTGTATGTATATTCCTTTAATAAAAAATCCATATGCTTAACATTCACATA
TATTAATAATTTTAATTAATTTTTTTTTTTGCACAGATAAAACTTTTCTTTTTAAAAAAA
AGGTATATACACTCTTTTTTACCCCACATTTAACTGATTTTCACTCACCATTACATATAG
CTATATATTGCATATTACTATTATGTAAGGCAAATGTTTATTATTATTGCTGTTATTTTT
TATTTATATATTTTTTTCCATATATCAATTTAAGGAGAAATAAAATAATTTGCTATTTGA
AAAAATATAAAACAAATATCTAGTCGTGCAATACTGAATTCGAATACTTAAAACAAAAAA
TAAATTAATTAAATACCCTTTTAATATTGTAGTTAAAATCGATGTGTTTAGAGAATATTT
TTTATTGCTATTTATCTTCTTTTTTTTATTTTTCTTCCATGTTTTCTCATTCAGCTTTCT
ATGTATTTTAGTAATATTCTCCACACACAAACTATAAATTTTCTTATGAAAAATCACAAA
ATTTAGCAAATAGCGCTTAGGCATGTACTCGTAAATAAGCATGTGCATAGTATAATACAC
TTCTCATCAATTTTGAGAGGATTTTATTTAATTTCGGCACGCCCCTCTCAACTTGTGTTT
ATCACATATTCAATACTCCATAAAAAATGATAGCATCATTTCGATATGCGCATCTCAAGA
GAGTAAAAAATACACCTTATAGTAAGGGACATAAATATATTCAAGAAAATAGGGGATACT
GTTACTTAATAGACAAAATTAATAATTGTGGCAACATTAATAGTCATCAAATTAATACGA
AGAAAGTTTTTTTTCCTGGCAATAAAAGAAACTACTTTACATATTTATACAACAATGTGA
AAGGCAAAGAAGAGTTAAAGGGAGATATGGAAAATTTGTACAACAATATCATAACAAGCA
ATTGGAACGACTCAGTAAATTTGGTTTTAAATGTTCCAATTTGGGAAGGACGATTAAATG
ATATAGAAGAAAAAATCAAAGGGTATGAAAATGATAAGTCTATAATCGATCAAAAAAAAA
AAATTAATGAATTATTTGATGTTTTATTTATATTGGAAGATTTACGTGATCATATTAATC
TCGAGGGGGGGCCCGGTACCGAATTCCTGCAGCCCGGGACTAGTTCTAGAGCGGCCGCCA
CCGCGGTGGAGCTCCAATTCGCCCTATAGTGAGTCGTATTACGCGCGCTCACTGGCCGTC
GTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCA
CATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAA
CAGTTGCGCAGCCTGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCG
GGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCT
TTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAAT
CGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTT
GATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTG
ACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAAC
CCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTA
AAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACA
ATTTAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAA
TACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATT
GAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGG
CATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAG
ATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTG
AGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTG
GCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATT
CTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGA
CAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTAC
TTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATC
ATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGC
GTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAAC
TACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAG
GACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCG
GTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTA
TCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCG
CTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATA
TACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTT
TTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACC
CCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCT
TGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAA
CTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAG
TGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTC
TGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGG
ACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCA
CACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTAT
GAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGG
TCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTC
CTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGC
GGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGC
CTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCG
CCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGA
GCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTC
ATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAA
TTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTC
GTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATG
ATTACGCCAAGCTCGAAATTAACCCTCACTAAAGGGAACAAAAGCT
Restriction sites to linearize plasmid
Partial or complete disruption of the genePartial
Additional remarks partial/complete disruption 5'UTR and 550bp of coding region intact
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 1CCCAAGCTTGGATCCGAAGGCATATATAAGCATACG
Additional information primer 1L406 (HindIII,BamHI); Pbs48/45 F 5'KO target region
Sequence Primer 2CCCAAGCTTTACAGTTACATGCACTAATGC
Additional information primer 2L407 (HindIII); Pbs48/45 R 5'KO target region
Sequence Primer 3GGATATCAGCAATACCTCAATCAGCATC
Additional information primer 3L408 (EcoRV); Pbs48/45 F 3'KO target region
Sequence Primer 4?
Additional information primer 4?
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6

  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameGFP (gfp-mu3)
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/constructPlasmid 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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AATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTT
AATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACC
GATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCCTGATGCGGTATTTT
CTCCTTACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGC
TCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGA
CGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGC
ATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATA
CGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACT
TTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATG
TATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGT
ATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCT
GTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCA
CGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCC
GAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCC
CGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTG
GTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTA
TGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATC
GGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTT
GATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATG
CCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCT
TCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGC
TCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCT
CGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTAC
ACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCC
TCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGAT
TTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATG
ACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATC
AAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAA
CCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAG
GTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTA
GGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTA
CCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAG
TTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTG
GAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCACG
CTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAG
CGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGC
CACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAA
AACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATG
TTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCT
GATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAA
GAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGG
CACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAG
CTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGA
ATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTT
CCGCGGGTATATGGTAAAGAACCTACTAACACAATAAAATATTTAAATAATGTATTTCCT
ATAAATAAATTTACAGATTTATTTTTTAATACAAAAGATATAGATATACCAGAAATAAAT
GATCAGTTTAAAGGTTTTAAATTCTTTATGACATCATTTATAAATCATGGATCATATCCA
CTAACAATAGAATGTGGTGTAACAAATGGTGGAACTAGTTATAAAAGAGCAATTATTTTA
TTGCATGTTCGAACTGATTTAAAAGATAGACCAGTTTCATTTTGTGATTTTCGAAAAGGA
GAATTATATAATTATTTGAATGCTTATACTGAAGGGGATGTATGCATAATAATTTCCAAA
TCAAATACAAGTTTTGGTTTTAGATGCCCAGTAAATACAAAAAAAATGCCAAAAAATTGT
TTTACGCAAGTATATGAAAAAGGGTATCTAAATGACGCCAATAAAATTAATACTAAAAAT
GTTATTAACTATTCATTTGAAAATCCAGAATATGCGCTAGCTGGTTYTAATTATACATTA
ACAAAATCGTATCAATTTGAATGTCATTGTGTAGATAAAGAAACAGAACAAATTGTAAAA
ACGGTTTTAGTCAAATATGTAAATGAAGATGAAATATATGATTATAATGATTTTCCAATG
GTGAATCACAAACCTATTATTGCACATCCAAATAAAACACATCAAGCTTGCATGCCTGCA
GCCCAGCTTAATTCTTTTCGAGCTCTTTATGCTTAAGTTTACAATTTAATATTCATACTT
TAAGTATTTTTTGTAGTATCCTAGATATTGTGCTTTAAATGCTCACCCCTCAAAGCACCA
GTAATATTTTCATCCACTGAAATACCATTAAATTTTCAAAAAAATACTATGCATATAATG
TTATACATATAAACATAAAACGCCATGTAAATCAAAAAATATATAAAAATATGTATAAAA
ATAAATATGCACTAAATATAAGCTAATTATGCATAAAAATTAAAGTGCCCTTTATTAACT
AGTCGTAATTATTTATATTTCTATGTTATAAAAAAATCCTCATATAATAATATAATTAAT
ATATGTAATGTTTTTTTTATTTTATAATTTTAATATAAAATAATATGTAAATTAATTCAA
AAAATAAATATAATTGTTGTGAAACAAAAAACGTAATTTTTTCATTTGCCTTCAAAATTT
AAATTTATTTTAATATTTCCTAAAATATATATACTTTGTGTATAAATATATAAAAATATA
TATTTGCTTATAAATAAATAAAAAATTTTATAAAACATAGGGGGATCCATGAGTAAAGGA
GAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGG
CACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTT
AAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTC
GGTTATGGTGTTCAATGCTTTGCGAGATACCCAGATCATATGAAACAGCATGACTTTTTC
AAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGG
AACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAG
TTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAAC
TATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAAC
TTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAA
AATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCACACAA
TCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTA
ACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAATAAATGGATCCCGTTTTT
CTTACTTATATATTTATACCAATTGATTGTATTTATAACTGTAAAAATGTGTATGTTGTG
TGCATATTTTTTTTTGTGCATGCACATGCATGTAAATAGCTAAAATTATGAACATTTTAT
TTTTTGTTCAGAAAAAAAAAACTTTACACACATAAAATGGCTAGTATGAATAGCCATATT
TTATATAAATTAAATCCTATGAATTTATGACCATATTAAAAATTTAGATATTTATGGAAC
ATAATATGTTTGAAACAATAAGACAAAATTATTATTATTATTATTATTTTTACTGTTATA
ATTATGTTGTCTCTTCAATGATTCATAAATAGTTGGACTTGATTTTTAAAATGTTTATAA
TATGATTAGCATAGTTAAATAAAAAAAGTTGAAAAATTAAAAAAAAACATATAAACACAA
ATGATGTTTTTTCCTTCAATTTCGGGTACCGAGCTCGAATTCTCTTGAGCCCGTTAATGA
AATAGATACAATTCATTCATGTTATATACATCTAGAACATAATCTGAATATGGTTCAAGT
TAAATGTCCAAAAATTATAAAAAGTGATGATATTTTTGATGGTAATACCATAATAGACAC
CAAGGTAACATCACGAAGTAGTCAACAAAATAATTTTTATTTAGAAAATACAGATGTTGA
ACCAGAAGAAATAGAGAAATATAAAAATATAGAATACATACCAGAAAACGATGAAGTAAT
GCATCTAGACAAAAAAGAAAAGCTAGATGATATATTACCAGGTGTTATCATATTAGATAA
AAATAAAATGTTCAAAGAAAAAGGACATTTCACTTTTGTTACTCCATTAATTGTAGAAAA
GGTATTAATATTAAAAATATATTGTGATAATACTAAAACAATAATTAATAATATGAAAGG
GAAAAAAGGTATTACAGTAATAAGGATTTCTCAAAATACAACAAAAAATAAATTTTATGG
ATGTGACTTTTCAGGTAATTCTAAAAAAACATTTTACTATTCCAATGTTTATGATTTAGA
AAAAAAAAATGAGTTTTGTGAAATAGAATTAAAAGAAAATATAGTAGTTAGCTTAAATTG
TCCAACTGGTAAAATTAATCCAAAAAATTGTTTTAGAAATGTATATATAAAAAGTAATAT
GAATGAACAAACAACCGAAAATATAGAAAATATATTTAACGAAATAAAAGTTATAGATGC
AGATTATTTTATAAATAATTCATCAACCTTTTTGATGATTTCCAAAATTACAAAAAAAGA
GTTTGATTTTTATTGTACATGTGAAGATTATAAAACCAAAAATATAGGAACAATATATAT
TAAAAATTATGAATATCTAGATTCAAAACCTAAATATAAAAATAAACAAATTTCCTATAT
AGATGTAGTTCCATACCCGCGGGGAAAGGGCG
Restriction sites to linearize plasmid KspI (SacII)
Selectable marker used to select the mutant parasitegfp (FACS)
Promoter of the selectable markerNo
Selection (positive) procedureFACS (flowsorting)
Selection (negative) procedureNo
Additional remarks genetic modificationThe GFP gene (1 copy) has been inserted into the 230p locus (PB000214.00.0) by double cross-over integration.
Additional remarks selection procedureThis reporter mutant expressing GFP does not contain a drug-selectable marker. This mutant has been selected by FACS sorting after transfection based on GFP fluorescence.
Other details transgene
Promoter
Gene Model of Parasite PBANKA_1133300
Gene Model P. falciparum ortholog PF3D7_1357100
Gene productelongation factor 1-alpha
Gene product: Alternative nameeef1a
Primer information details of the primers used for amplification of the promoter sequence  Click to view information
Primer information details of the primers used for amplification of the promoter sequence  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
3'-UTR
Gene Model of Parasite PBANKA_0719300
Gene productbifunctional dihydrofolate reductase-thymidylate synthase, putative
Gene product: Alternative namedhfr-ts
Primer information details of the primers used for amplification the 3'-UTR sequences  Click to view information
Primer information details of the primers used for amplification the 3'-UTR sequences  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
Insertion/Replacement locus
Replacement / InsertionReplacement locus
Gene Model of Parasite PBANKA_0306000
Gene product6-cysteine protein
Gene product: Alternative name230p
Primer information details of the primers used for amplification of the target sequences  Click to view information
Primer information details of the primers used for amplification of the target sequences  Click to hide information
Sequence Primer 15'-cccaagcttccgcgggtatatggtaaagaacctactaacac
Additional information primer 1primer L1345: 0.7kb 5'region of PB000214.00.0 (230p gene)
Sequence Primer 25'-cccaagcttgatgtgttttatttggatgtgc
Additional information primer 2primer L1346: 0.7kb 5'region of PB000214.00.0 (230p gene)
Sequence Primer 35'-ccggaattctcttgagcccgttaatg
Additional information primer 3primer L1347: 1kb 3'region of PB000214.00.0 (230p gene)
Sequence Primer 45'-tccccgcgggtatggaactacatctatatagg
Additional information primer 4primer L1348: 1kb 3'region of PB000214.00.0 (230p gene)