Summary

RMgm-177
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_0933500; Gene model (P.falciparum): PF3D7_1114100; Gene product: rhomboid protease ROM1 (ROM1)
Phenotype Liver stage;
Last modified: 31 December 2013, 12:32
  *RMgm-177
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) : 23490234
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/ResearcherJ. Lin, G.R. Mair, C.J. Janse, S.M. Khan
Name Group/DepartmentLeiden Malaria Research Group
Name InstituteLeiden University Medical Center (LUMC)
CityLeiden
CountryThe Netherlands
Name of the mutant parasite
RMgm numberRMgm-177
Principal name538cl1; 538cl2
Alternative name∆rom1-p
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
SporozoiteNot different from wild type
Liver stageLiver-stage development was reduced as shown by a 1-day extension of the prepatent period in mice following the inoculation with 10(4) purified sporozoites.
While gliding motility and the rate of cell traversal of sporozoites were similar to wild type parasites, in two out of three experiments a reduction in sporozoite in vitro invasion rates was observed that could explain (in part) the delay in the prepatent period. Immunofluorescence analyses of liver stage parasites stained with antibodies against markers for parasite development (HSP70), PVM (UIS4 and EXP1) and merozoite formation (MSP1), revealed no distinct differences in morphology and size between ∆rom1 and wild type liver stages at 24h or 48h after sporozoite invasion.
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of the rhomboid protease ROM1.

Protein (function)
Rhomboid proteins are intra-membrane proteases that play a role in multiple processes. They belong to a family of serine proteases that cleave cell-surface proteins within their transmembrane domains. The Plasmodium  genome encodes a total of 8 rhomboid proteases (ROM1, 3, 4, 6, 7, 8, 9 and 10).  ROM1 is expressed in both the blood stages and mosquito stages (sporozoites). ROM1 was localized to organelles of the apical complex of merozoites. In vitro  substrates of  P. falciparum ROM1 include the merozoite-specific proteins AMA1 (apical membrane antigen 1) and proteins of the EBL (erythrocyte binding ligands) and RBL (reticulocyte binding ligands) families as well as several proteins of the invasive ookinete- and sporozoite-stages, such as TRAP (thrombospondin-related anonymous protein), CTRP (circumsporozoite- and TRAP-related protein) and MAEBL (merozoite adhesive erythrocytic binding protein). 
In P. falciparum  only a small fraction of AMA1 is shed by ROM1 and the intramembrane cleavage can be reduced to undetectable levels by mutagenesis without discernible phenotypic consequences. The successful generation of P. berghei and P. yoelii  mutants that lack expression of ROM1 (see below) also demonstrates that ROM1 is not essential for blood stage multiplication

Phenotype
The phenotype analyses indicate that ROM1 is not essential throughout the complete life cycle in P. berghei.
Liver-stage development was reduced as shown by a 1-day extension of the prepatent period in mice following the inoculation with 10(4) purified sporozoites.
While gliding motility and the rate of cell traversal of sporozoites were similar to wild type parasites,  in two out of three experiments a reduction in sporozoite in vitro invasion rates was observed that could explain (in part) the delay in the prepatent period. Immunofluorescence analyses of liver stage parasites stained with antibodies against markers for parasite development (HSP70), PVM (UIS4 and EXP1) and merozoite formation (MSP1), revealed no distinct differences in morphology and size between ∆rom1 and wild type liver stages at 24h or 48h after sporozoite invasion.

Additional information
It has been reported that P. berghei  and P. yoelii mutants lacking ROM1 (see mutants RMgm-176, RMgm-659) exhibit a slight growth delay and appear less virulent in mice than wild type parasites. In contrast, the ∆rom1 mutant described here and in RMgm-761 neither a growth nor a virulence-attenuation phenotype have been detected. Mice infected with ∆rom1 parasites showed a normal course of infection and all C57BL/6 mice developed clear signs of experimental cerebral malaria (ECM). The cause for these discrepancies in blood stage phenotypes between the ∆rom1 P. berghei  mutants described here and in RMgm-761 and the mutant RMgm-176 is unknown. In RMgm-176 the mutant clone examined was derived from a single transfection experiment.
Although a significant reduction in expression of UIS4 has been reported for liver stages of P. yoelii  mutants lacking expression of ROM1 (RMgm-659), the ∆rom1 parasites showed only a slight, but not significant, reduction in liver-stages as determined by UIS4 staining and the liver stages of the ∆rom1 mutant had no unusual parasitophorous vacuole membrane as reported for the P. yoelii  mutants. Liver stages of ∆rom1 and wild-type parasites were also comparable in size at 48 hrs post infection.

Other mutants
RMgm-176: A P. berghei mutant lacking expression of rhomboid protease ROM1 (partial disruption)
RMgm-761: A P. berghei mutant lacking expression of rhomboid protease ROM1 (complete disruption)
RMgm-178: A P. berghei mutant lacking expression of rhomboid protease ROM3
RMgm-762: A P. berghei mutant lacking expression of rhomboid protease ROM9
RMgm-179: A P. berghei mutant lacking expression of rhomboid protease ROM10


RMgm-187: Unsuccessful attempts to disrupt P. berghei rhomboid protease rom4
RMgm-758: Unsuccessful attempts to disrupt P. berghei rhomboid protease rom6
RMgm-759: Unsuccessful attempts to disrupt P. berghei rhomboid protease rom7
RMgm-760: Unsuccessful attempts to disrupt P. berghei rhomboid protease rom8

RMgm-763: A P. berghei mutant expressing  a (C-terminal) GFP-tagged version of rhomboid protease ROM3
RMgm-764: A P. berghei mutant expressing  a (C-terminal) mCherry-tagged version of rhomboid protease ROM4

RMgm-659: A P. y. yoelii 17XNL mutant lacking expression of rhomboid protease ROM1
RMgm-660: A P. y. yoelii 17XNL mutant expressing  a (N-terminal) HA-tagged version of rhomboid protease ROM1


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0933500
Gene Model P. falciparum ortholog PF3D7_1114100
Gene productrhomboid protease ROM1
Gene product: Alternative nameROM1
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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AATTGATTCGTTGTGAATATAATCAGGAAATAATATATACACACAATTATTACACATATA
TAGTATATATATGCCACATATATGCTTTGAGTTTGGACTTATTTTTTAAATTTCATACAT
TTTGAGGGTACTTTATATATATGCTAATGCATTTCATAATTATTTTTTCATAATATATAT
TTTTTTGTGTATATTATATATGATTAATTTATGAATTCTTAATTAATTTACATTTTTTTG
ATTTTAATTTATAATTATTACGGAGTGTTTCTTCCTTTTTCATTATTATTTAAATAAAAA
AGAAGCAATTATTGATAAAAAAAAATAATAATAGAAAATGAGTAACATCCATACATTAGC
AGAGTATAGGGATGGGTATGGTGAAAATCTTCCATTCAATAGAAGTATAAAAAATAATTA
TAAAAATAATAAAATGCATAAAAAATATACAATATAAATTTCTTTATTGTCTTTCAATAT
AATTAATTTTGTCCATAAAATTTAACGTTTCTGTCTCATACTTTTTAATTCGAATATGCA
CCAGCACATGCATAAGTACATGTTTATATATATATATGTCGATTTATTTATGTTCACCTT
TTATTTTTATTTTGTAAGCACCAGGGTATTATGCGTCACAGAGTAGTTTTATTCAAAGAT
CAAAACCTATTGACGTAGTTAACTTGATTTTCCCCAATTTTACATGGAAAAGTTTTGTCA
TGGTTGTTTCCATAATACAGATTATCGTTTTTATAATATCTCTTAGTATAAGTCCTGGAG
AATTTTTAACTCCTTCTGGTATTAATTGTGTCCGAATGCAAGCTTGCATGCCTGCAGGTC
AACAATAAATAATAAATAAATATTGTGGAAATAAAATAACATATAATTATTTTTAATACA
TTGATTTCCCTTTTATTTTTTTAAATTTCATTGATATAAAAATATATAATAATAACATAT
ATGATTTCAAATTAATCTTTTCAAAAATGGTGTTTATTTTTGTATGTTTGTGTATGAATT
AATCACATAACACATCTATTAAATTGAGTTGGTAATATAGACACAAATAAATATATATAT
TTTTATAGCTTAAAAGTGTGTTATGAATATTTTAAGCATATTTTCTTTTTCTTTGGATTG
TGTAAAATGAACTCATATAATGCGTTTTTTTGTTTTTGTTATTTTGTCATTTTGTTATTT
TGCTATTTTATGGATTAATTTTTGTTTATAAAATGGGAAATAATTTTAACATATTTAAAT
AAATGGAGAAAAAATATAAAATAATTATAAAAAAAAGTTAATACACATTTTTTCCTGTTA
TAGACCTTATATTTATTTATCCATATATATATATATATATATATATATATATATATACAT
ACCAAGTGAATTAAGAGGAAAGCTAATTTATTATTCAGAATAATATATGAACTATATATA
ATTTTTATTATTTTGGTGTATATTAATCTGTCTATATGCATACATGCAATAATTTATCGA
CTTATATATCAAATAACATAAAATAGAAGTGTTTTAAATTATGGATATATGCTCAATATT
CATTTTTTTTAATAAGTTAGCTATATTTAAATTATACATTTTATATATGGTCTCTTTTTT
TTTTAAATATTATTTAAGTGATCATGAAAATATAAATAATTTTTTTTTATTTAATATCCT
TTTGCTTGCATGTGGTAAATGGAAATTTGGATGTGTTTTGAARGTTCGGATATAGTTGTA
TGGACATATAATATATTTTGTGAAAAATTGGTTTTATGTTTATACTTATGCCAATACTTT
TTGAGTAAAACAAAGCAAGTGCTTATAAATAATTAAAGCCAATTTTATAATATATATTTT
TTTATTTAATTTGAATTTAGTAGTATAATTTTTTATGGTAAGTGCTCAAAGAGAGTTGCT
TATAAAGTATGGTTTGTTTCTTTTTCGCCATTTTGAATTACACATTAAAAATATATAGAT
ACATATATTATAATATGAAATCATTAATAATTTAGGGAAATTCTACAAATTTAAAAACGA
ATAAAATAATTGTTTTTCATCATGCCATAACACAATATTGATATATACATGTACAAACAT
TTTTTTTATTTGGAAAATATAAATTATATAAAAAAAAATGTATAGTATACAAAATGAGCA
TATTCACACGGGGTGGACGTTCATTTTTTCATTTTTCCCCTGTTTTTTATGAGTATATGA
TAAAATTTTATGAACATTTACACAAAATGAAAATGGATATATAGGAAAAATGGAGCGGTA
TTTCATTTATCTTTGATTGTCATTTGGATATTATATTACCYTGGGTAGGCAATTAAAAAT
GTTAAATAACAATTTAAGGAAATTATATTTTATATATTAAAATTAACACTGTATTATATG
ATTCGCTTATAAAAGCCACTCTTTCCCCATGCAAAGCTGTTTAATATCAATTTTAACAAA
TTACACACATGTTAATATATTTATATATATAATTTATATATTTATAATTTATATATTTAT
ATTTTTATTATTTATATATTTATTATTTATTGTGTGTGTCAATTCGGGTAGGATATACCT
CTTTTTTATTGTTTAAAGCGATTTGTATTCTAAAATATAAAGRATTTGAAAAAGAGAAAG
ATAGAATATGATCCCATCATATATAGCCCTATAATTTTTATTTAGCAGCGAATTAATTTT
TCTATTAAGTTTATGTGTAATTAAAATAACGGAATATATATAATACAATAAAAAAGTGCA
TAAATTAAAATTTTTTCAATTAAATTTTTTTTTTTAAGGGGTTATATAATATTAAATATA
TAAAATACGATTATATATTTTTGCTACAATTTTTTATATTAAGATATAAATAGTAAATAA
ATGGTATTATATGGCATGTAATATATAAATTTTTTCCAATTTTTATTTTATATACACTTT
TCCTTTTTTTGTCATAAAACTTAAACAATTTACACATTCATTTTAAAAATTGACTATTTG
TTTCAACATTTTTTGAGTTTCCGTTTTATAATAGTATTTTCATTTGTATATTGCTTATAT
ATATAAATACACACCTAAATGTTACAAAGGATCAATGCATAAACCGGTGTGTCTGGTCGT
CGCGATGACCCCCAAGAGGGGCATCGGCATCAACAACGGCCTCCCGTGGCCCCACTTGAC
CACAGATTTCAAACACTTTTCTCGTGTGACAAAAACGACGCCCGAAGAAGCCAGTCGCCT
GAACGGGTGGCTTCCCAGGAAATTTGCAAAGACGGGCGACTCTGGACTTCCCTCTCCATC
AGTCGGCAAGAGATTCAACGCCGTTGTCATGGGACGGAAAACCTGGGAAAGCATGCCTCG
AAAGTTTAGACCCCTCGTGGACAGATTGAACATCGTCGTTTCCTCTTCCCTCAAAGAAGA
AGACATTGCGGCGGAGAAGCCTCAAGCTGAAGGCCAGCAGCGCGTCCGAGTCTGTGCTTC
ACTCCCAGCAGCTCTCAGCCTTCTGGAGGAAGAGTACAAGGATTCTGTCGACCAGATTTT
TGTCGTGGGAGGAGCGGGACTGTACGAGGCAGCGCTGTCTCTGGGCGTTGCCTCTCACCT
GTACATCACGCGTGTAGCCCGCGAGTTTCCGTGCGACGTTTTCTTCCCTGCGTTCCCCGG
AGATGACATTCTTTCAAACAAATCAACTGCTGCGCAGGCTGCAGCTCCTGCCGAGTCTGT
GTTCGTTCCCTTTTGTCCGGAGCTCGGAAGAGAGAAGGACAATGAAGCGACGTATCGACC
CATCTTCATTTCCAAGACCTTCTCAGACAACGGGGTTCCCTACGACTTTGTGGTTCTCGA
GAAGAGAAGGAAGACTGACGACGCAGCCACTGCGGAACCGAGCAACGCAATGAGCTCCTT
GACGTCCACGAGGGAGACAACTCCCGTGCACGGGTTGCAGGCTCCTTCTTCGGCCGCAGC
CATTGCCCCGGTGTTGGCGTGGATGGACGAAGAAGACCGGAAAAAACGCGAGCAAAAGGA
ACTGATTCGGGCCGTTCCGCATGTTCACTTTAGAGGCCATGAAGAGTTCCAGTACCTTGA
TCTCATTGCCGACATTATTAACAATGGAAGGACAATGGATGACCGAACGGGCGTTGGTGT
CATCTCCAAATTCGGCTGCACTATGCGCTACTCGCTGGATCAGGCCTTTCCACTTCTCAC
CACAAAGCGTGTGTTCTGGAAAGGGGTCCTCGAAGAGTTGCTGTGGTTCATTCGCGGCGA
CACGAACGCAAACCATCTTTCTGAGAAGGGCGTGAAGATCTGGGACAAGAATGTGACACG
CGAGTTCCTCGATTCGCGCAATCTCCCCCACCGAGAGGTCGGAGACATCGGCCCGGGCTA
CGGCTTCCAGTGGAGACACTTCGGCGCGGCATACAAAGACATGCACACAGACTACACAGG
GCAGGGCGTCGACCAGCTGAAGAATGTGATCCAGATGCTGAGAACGAATCCAACAGATCG
TCGCATGCTCATGACTGCCTGGAATCCTGCAGCGCTGGACGAAATGGCGCTGCCGCCTTG
TCACTTGTTGTGCCAGTTCTACGTGAACGACCAGAAGGAGCTGTCGTGCATCATGTATCA
GCGGTCGTGCGATGTCGGCCTCGGCGTCCCCTTCAACATCGCTTCCTATTCGCTTTTGAC
GCTCATGGTTGCACACGTCTGCAACCTAAAACCTAAGGAGTTCATTCACTTCATGGGGAA
CACGCATGTCTACACGAACCATGTCGAGGCTTTAAAAGAGCAGCTGCGGAGAGAACCGAG
ACCGTTCCCCATTGTGAACATCCTCAACAAGGAACGCATCAAGGAAATCGACGATTTCAC
CGCCGAGGATTTTGAGGTCGTGGGCTACGTCCCGCACGGACGAATCCAGATGGAGATGGC
TGTCTAGCGGAAATACAGAAGCTAGCTTTGATCCCGTTTTTCTTACTTATATATTTATAC
CAATTGATTGTATTTATAACTGTAAAAATGTGTATGTTGTGTGCATATTTTTTTTTGTGC
ATGCACATGCATGTAAATAGCTAAAATTATGAACATTTTATTTTTTGTTCAGAAAAAAAA
AACTTTACACACATAAAATGGCTAGTATGAATAGCCATATTTTATATAAATTAAATCCTA
TGAATTTATGACCATATTAAAAATTTAGATATTTATGGAACATAATATGTTTGAAACAAT
AAGACAAAATTATTATTATTATTATTATTTTTACTGTTATAATTATGTTGTCTCTTCAAT
GATTCATAAATAGTTGGACTTGATTTTTAAAATGTTTATAATATGATTAGCATAGTTAAA
TAAAAAAAGTTGAAAAATTAAAAAAAAACATATAAACACAAATGATGTTTTTTCCTTCAA
TTTCGATATCAAGTTATAGTAAATATGCTTTGCTCCAAATATATATATATATATGCCCAT
TTTGGCATATATATATAAAACATTTATTCCGCACATAGTAAGCGACCATTTACACACCTA
TATGATTAATAGATTCGAAATCCATACAATAAAAATTTAATGAATTATAATAATATTAAA
AACACATGCATATTCTTTAATTTGCTCCTTTAATTTTTTATAACCATTTTTTAATTTCTA
ATAATGATAACAATAGTAACAATTTTTTAAGTTATTATATTTATTTACCATAGTTTTTTC
ATTTATTCTCTTTTTCTTGTATTTTTGTATTGCCCTTTTTTGTTAAATTTTAAATATACA
GAAAAAAAAAACTTTTTTTTTAATTTAAATATATACATAACTATAAATATATTTGTGGAC
ACATAATTTAATTAATAAATTTGAAAAGTTAAAACCACATTTAAGCACAGCGTATATGTC
GTTATTTTTTTAACATGTTTTCTATGAGTTATGTTTTCTTTTGTGTTGTTTTAAAAATAT
GTATATCAAAATCAATAATAGACAAGCCAGTTTATTAAACTAGACTCAACGATATATTTA
AAAAATATTAAAGCTAAAAAATTATTTAAAAATATTAAATAATTTCATTTTTACAAAATA
AGTTGATTTTGTTAAGAATACTATTTAAAAATTAAATAATCATCGAATATTTGTTTTTTC
CATTGTTACATCGAGATATTTTTTCATTAAACAAACGAAAATATTTATAACTTAAAAAAT
TATAATATAAAAAAAATAAAACGACAAAAAAAACAAATAAAAATCGGTACAAAGATATAT
ACATAGGGGATCCACTAGTTCTAGAGCGGCCGCCACCGCGGTGGAGCTCCAATTCGCCCT
ATAGTGAGTCGTATTACGCGCGCTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAA
ACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTA
ATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAAT
GGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGA
CCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCG
CCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGAT
TTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTG
GGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATA
GTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATT
TATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAAT
TTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTAGGTGGCACTTTTCGGGGAAA
TGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCAT
GAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCA
ACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCA
CCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTA
CATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTT
TCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGC
CGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTC
ACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGC
CATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAA
GGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGA
ACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAAT
GGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACA
ATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCC
GGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCAT
TGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAG
TCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAA
GCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCA
TTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCC
TTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTC
TTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACC
AGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTT
CAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTT
CAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGC
TGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAA
GGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGAC
CTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGG
GAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGA
GCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACT
TGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAA
CGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGC
GTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCG
CCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAAT
ACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTT
TCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTA
GGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGG
ATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTCGAAATTAACCC
TCACTAAAGGGAACAAAAGCTGGTACCGGGCCCCCCCTCGAGGTCGACGGTATCGATG
Restriction sites to linearize plasmid
Partial or complete disruption of the genePartial
Additional remarks partial/complete disruption The construct replaces only the 3rd and 4th exons of the open reading frame (ORF) but retains 1st and 2nd exons in mutant
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 1aacaattgattcgttgtgaatataatcagg
Additional information primer 1L2082 (MunI); 5'rho1 sense
Sequence Primer 2ttaagcttgcattcggacacaattaatac
Additional information primer 2L2083 (HindIII); 5'rho1 antisense
Sequence Primer 3aagatatcaagttatagtaaatatgctttgc
Additional information primer 3L2086 (EcoRV); 3'rho1 sense
Sequence Primer 4ttggatcccctatgtatatatctttgtaccg
Additional information primer 4L2087 (BamHI); 3'rho1 antisense
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6