Summary

RMgm-125
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_1300700; Gene model (P.falciparum): PF3D7_1475500; Gene product: LCCL domain-containing protein (LAP2; LCCL/lectin adhesive-like protein 2; CCp1)
DisruptedGene model (rodent): PBANKA_0417600; Gene model (P.falciparum): PF3D7_0903800; Gene product: LCCL domain-containing protein (LAP6; LCCL/lectin adhesive-like protein 6; CCP4)
Phenotype Oocyst; Sporozoite;
Last modified: 19 February 2009, 21:18
  *RMgm-125
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption, Gene disruption
Reference (PubMed-PMID number) Reference 1 (PMID number) : 18848846
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. Lavazec, G.R. Mair, C.J. Janse, J. Templeton
Name Group/DepartmentDepartment of Microbiology and Immunology
Name InstituteWeill Cornell Medical College
CityNew York
CountryUSA
Name of the mutant parasite
RMgm numberRMgm-125
Principal name∆PbCCp1/∆PbCCp4
Alternative name953 m0-m1cl1
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
OocystNormal numbers of oocysts are produced. Sporozoite formation within the oocysts is blocked. Analysis of the morphology of oocysts by light microscopy revealed that the majority of 17–22 day oocysts were either vacuolated or non-sporulated, and did not contain sporozoites.
SporozoiteNo hemocoel and salivary gland sporozoites were detected.
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of two proteins CCp1 (LAP2, LCCL/lectin adhesive-like protein 2; LCCL domain containing protein CCp1) and CCp4 (LAP6).

Protein (function)
The ccp1 and ccp4 genes are members of a small conserved gene family, encoding proteins with multiple adhesive domains, for example a Lgl1 (LCCL)-lectin adhesive domain. In P. falciparum the LCCL domain-containing proteins are termed PfCCp's and in P. berghei PbLAP's.

Phenotype
Phenotype analyses indicate a role during oocyst development and sporozoite formation/production (see also additional information).

Additional information
In another study (Raine, J.D. et al., 2007, PloS Pathogens 3, e30) it has been shown that crossings of ∆lap2 (ccp1) and ∆lap6  (ccp4) mutant females with wild type males did not rescue the formation of sporozoites. In contrast, crossing of the mutant males with wild type females resulted in wild type production of sporozoites that were infectious to C57BL/6 mice.
The lack of rescue of the mutant phenotypes by crossing of mutant females with wild type males is suggestive of a role of these proteins within a few hours after fertilisation.

Other members of the LAP/CCp family of proteins have been analysed by targeted gene disruption. A number of mutants lacking expression of these proteins show a comparable defect in development of oocysts and formation of sporozoites (RMgm-113, RMgm-114, RMgm-115: LAP1/PbSLAP/PbSR/CCp3; RMgm-119: LAP4/CCp2; RMgm-98: LAP5/FNPA; RMgm-120: LAP6, CCp4).

Other mutants
RMgm-118: A mutant lacking expression of LAP2/CCp1
RMgm-121: A mutant lacking expression of LAP2/CCp1
RMgm-120: A mutant lacking expression of LAP6/CCP4
RMgm-124: A mutant lacking expression of both LAP2/CCP1  and PbSR/PSLAP/LAP1/CCp3


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1300700
Gene Model P. falciparum ortholog PF3D7_1475500
Gene productLCCL domain-containing protein
Gene product: Alternative nameLAP2; LCCL/lectin adhesive-like protein 2; CCp1
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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AATTCGGAATTACTGAATAATGAAAAGCAAATAATAATATAAATAACAAATCATAATAAT
AGAATATATTATACAATGTGAAGTAAAATGATTATTTTTGAACGAAAATTATTTATTTGT
TCATTTGAACATATGCTTATGTGATGTTTTTTCTTATTTAGGTTGATTGGTTCACCAATC
ATGATAATTTTTTTATTGATGACACGTGGAAAGACAATAAATTAAATTTAAAACATATGC
AAAGCAATTTATTTCTATTACACATATACAGCATTATTATTATATGCTTTGTTTTCTCCT
AAATTAGCAAACTGTTTAGAAACAAATATAGGAAAAATCTATATATGCGCATATCCTTAA
TAAACAGTAGAATTATATAAATATAATAATATGCATACATTATTTGGTTAGTTCATTTTG
CGTATAAAAGCAATATATCGTGATCGTCTTTTGGGTTTTTTTTATCATGTAAAAAATACG
GGTTATTGTCCCCTAAATCAATTTTGCTAAATATAATAAAATCATATTAAGGGCGCACTT
TAGTTAATTATAACAAAATAAATGCATATATAGTGTAATGTTGATAGGATTATTTATAAT
TTTTTTAGATTCATGGTTCGTTTTGCTAATTTTATGAAATTTTCTTTGAGGTATTTATGT
ATGAACTTATTTAAATATTTGTATTATAGCAGTGAAATATATATTCCATCCATATAATAA
AATAAATAATTTATTGTATAAAATTGTAAATAGTCTTCTTATATATATAATAAATATAAT
TTTTATTAAATTTTATTATAAAAAGGGTTCAATTTCTTTATGAAAGTTTAAAATTTATAA
TAAAAAAGTACAAATAATTTTTATTTTGCAATAAGGAGCTGCATAAAGAAAAAACATAGA
TATCCAAAATGTGTAATAAAAGATATTTTTAGTGCGTATAATATACATACATTAACTAGT
TCTAGAGCGGCCGCCACCGCGGTGGAGCTCCAATTCGCCCTATAGTGAGTCGTATTACGC
GCGCTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACT
TAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCAC
CGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGGACGCGCCCTGTAGCGG
CGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGC
CCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCC
CCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCT
CGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGAC
GGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAAC
TGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGAT
TTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAA
AATATTAACGCTTACAATTTAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATT
TGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAA
ATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTT
ATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAA
GTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAAC
AGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTT
AAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGT
CGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCAT
CTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAAC
ACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTG
CACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCC
ATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAA
CTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAG
GCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCT
GATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGAT
GGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAA
CGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGAC
CAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATC
TAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTC
CACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTG
CGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCG
GATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCA
AATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCG
CCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCG
TGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGA
ACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATAC
CTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTAT
CCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCC
TGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGA
TGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTC
CTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTG
GATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAG
CGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCC
GCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGC
AGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACAC
TTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGA
AACAGCTATGACCATGATTACGCCAAGCTCGAAATTAACCCTCACTAAAGGGAACAAAAG
CTGGTACCGGGCCCCCCCTCGAGGTCGACGGTATCGATAAGCTATTAAAATTAATACGAA
AATAAAAATTAAAGGATGATGGCGATTATTAGTTCTTTTTAATTCCATCATCTATCCTTA
TATGTAGTATATTGGGGTTAGAGATAGTGGGTTATGAATACCAAAAATATAGAAAATTGC
AAAGGAGTTGATTTATTCTCTGTTGTATAAGAATGTACACAGTTAAAGTTAGTATATATG
TGTATATTTTTCAGTCTATATAAATATACTTTTGTTTTCTCATTGGTATGTTCAATTTTT
TATATTATTTTACGTGTCGATGGATACAATTTAGTATTTTTTTTAATTTTGTATTTTTGA
ATTATATTAATCTAGATTATTCATATATTTTTTCATATGCAGATAGAAAAAGGTTTCATA
CGTTAGTTTATCCTACAAATTGAATATTAACACATACATATATTTATATTTTAAGCTTGC
ATGCCTGCAGGTCAACAATAAATAATAAATAAATATTGTGGAAATAAAATAACATATAAT
TATTTTTAATACATTGATTTCCCTTTTATTTTTTTAAATTTCATTGATATAAAAATATAT
AATAATAACATATATGATTTCAAATTAATCTTTTCAAAAATGGTGTTTATTTTTGTATGT
TTGTGTATGAATTAATCACATAACACATCTATTAAATTGAGTTGGTAATATAGACACAAA
TAAATATATATATTTTTATAGCTTAAAAGTGTGTTATGAATATTTTAAGCATATTTTCTT
TTTCTTTGGATTGTGTAAAATGAACTCATATAATGCGTTTTTTTGTTTTTGTTATTTTGT
CATTTTGTTATTTTGCTATTTTATGGATTAATTTTTGTTTATAAAATGGGAAATAATTTT
AACATATTTAAATAAATGGAGAAAAAATATAAAATAATTATAAAAAAAAGTTAATACACA
TTTTTTCCTGTTATAGACCTTATATTTATTTATCCATATATATATATATATATATATATA
TATATATATACATACCAAGTGAATTAAGAGGAAAGCTAATTTATTATTCAGAATAATATA
TGAACTATATATAATTTTTATTATTTTGGTGTATATTAATCTGTCTATATGCATACATGC
AATAATTTATCGACTTATATATCAAATAACATAAAATAGAAGTGTTTTAAATTATGGATA
TATGCTCAATATTCATTTTTTTTAATAAGTTAGCTATATTTAAATTATACATTTTATATA
TGGTCTCTTTTTTTTTTAAATATTATTTAAGTGATCATGAAAATATAAATAATTTTTTTT
TATTTAATATCCTTTTGCTTGCATGTGGTAAATGGAAATTTGGATGTGTTTTGAARGTTC
GGATATAGTTGTATGGACATATAATATATTTTGTGAAAAATTGGTTTTATGTTTATACTT
ATGCCAATACTTTTTGAGTAAAACAAAGCAAGTGCTTATAAATAATTAAAGCCAATTTTA
TAATATATATTTTTTTATTTAATTTGAATTTAGTAGTATAATTTTTTATGGTAAGTGCTC
AAAGAGAGTTGCTTATAAAGTATGGTTTGTTTCTTTTTCGCCATTTTGAATTACACATTA
AAAATATATAGATACATATATTATAATATGAAATCATTAATAATTTAGGGAAATTCTACA
AATTTAAAAACGAATAAAATAATTGTTTTTCATCATGCCATAACACAATATTGATATATA
CATGTACAAACATTTTTTTTATTTGGAAAATATAAATTATATAAAAAAAAATGTATAGTA
TACAAAATGAGCATATTCACACGGGGTGGACGTTCATTTTTTCATTTTTCCCCTGTTTTT
TATGAGTATATGATAAAATTTTATGAACATTTACACAAAATGAAAATGGATATATAGGAA
AAATGGAGCGGTATTTCATTTATCTTTGATTGTCATTTGGATATTATATTACCYTGGGTA
GGCAATTAAAAATGTTAAATAACAATTTAAGGAAATTATATTTTATATATTAAAATTAAC
ACTGTATTATATGATTCGCTTATAAAAGCCACTCTTTCCCCATGCAAAGCTGTTTAATAT
CAATTTTAACAAATTACACACATGTTAATATATTTATATATATAATTTATATATTTATAA
TTTATATATTTATATTTTTATTATTTATATATTTATTATTTATTGTGTGTGTCAATTCGG
GTAGGATATACCTCTTTTTTATTGTTTAAAGCGATTTGTATTCTAAAATATAAAGRATTT
GAAAAAGAGAAAGATAGAATATGATCCCATCATATATAGCCCTATAATTTTTATTTAGCA
GCGAATTAATTTTTCTATTAAGTTTATGTGTAATTAAAATAACGGAATATATATAATACA
ATAAAAAAGTGCATAAATTAAAATTTTTTCAATTAAATTTTTTTTTTTAAGGGGTTATAT
AATATTAAATATATAAAATACGATTATATATTTTTGCTACAATTTTTTATATTAAGATAT
AAATAGTAAATAAATGGTATTATATGGCATGTAATATATAAATTTTTTCCAATTTTTATT
TTATATACACTTTTCCTTTTTTTGTCATAAAACTTAAACAATTTACACATTCATTTTAAA
AATTGACTATTTGTTTCAACATTTTTTGAGTTTCCGTTTTATAATAGTATTTTCATTTGT
ATATTGCTTATATATATAAATACACACCTAAATGTTACAAAGGATCAATGCATAAACCGG
TGTGTCTGGTCGTCGCGATGACCCCCAAGAGGGGCATCGGCATCAACAACGGCCTCCCGT
GGCCCCACTTGACCACAGATTTCAAACACTTTTCTCGTGTGACAAAAACGACGCCCGAAG
AAGCCAGTCGCCTGAACGGGTGGCTTCCCAGGAAATTTGCAAAGACGGGCGACTCTGGAC
TTCCCTCTCCATCAGTCGGCAAGAGATTCAACGCCGTTGTCATGGGACGGAAAACCTGGG
AAAGCATGCCTCGAAAGTTTAGACCCCTCGTGGACAGATTGAACATCGTCGTTTCCTCTT
CCCTCAAAGAAGAAGACATTGCGGCGGAGAAGCCTCAAGCTGAAGGCCAGCAGCGCGTCC
GAGTCTGTGCTTCACTCCCAGCAGCTCTCAGCCTTCTGGAGGAAGAGTACAAGGATTCTG
TCGACCAGATTTTTGTCGTGGGAGGAGCGGGACTGTACGAGGCAGCGCTGTCTCTGGGCG
TTGCCTCTCACCTGTACATCACGCGTGTAGCCCGCGAGTTTCCGTGCGACGTTTTCTTCC
CTGCGTTCCCCGGAGATGACATTCTTTCAAACAAATCAACTGCTGCGCAGGCTGCAGCTC
CTGCCGAGTCTGTGTTCGTTCCCTTTTGTCCGGAGCTCGGAAGAGAGAAGGACAATGAAG
CGACGTATCGACCCATCTTCATTTCCAAGACCTTCTCAGACAACGGGGTTCCCTACGACT
TTGTGGTTCTCGAGAAGAGAAGGAAGACTGACGACGCAGCCACTGCGGAACCGAGCAACG
CAATGAGCTCCTTGACGTCCACGAGGGAGACAACTCCCGTGCACGGGTTGCAGGCTCCTT
CTTCGGCCGCAGCCATTGCCCCGGTGTTGGCGTGGATGGACGAAGAAGACCGGAAAAAAC
GCGAGCAAAAGGAACTGATTCGGGCCGTTCCGCATGTTCACTTTAGAGGCCATGAAGAGT
TCCAGTACCTTGATCTCATTGCCGACATTATTAACAATGGAAGGACAATGGATGACCGAA
CGGGCGTTGGTGTCATCTCCAAATTCGGCTGCACTATGCGCTACTCGCTGGATCAGGCCT
TTCCACTTCTCACCACAAAGCGTGTGTTCTGGAAAGGGGTCCTCGAAGAGTTGCTGTGGT
TCATTCGCGGCGACACGAACGCAAACCATCTTTCTGAGAAGGGCGTGAAGATCTGGGACA
AGAATGTGACACGCGAGTTCCTCGATTCGCGCAATCTCCCCCACCGAGAGGTCGGAGACA
TCGGCCCGGGCTACGGCTTCCAGTGGAGACACTTCGGCGCGGCATACAAAGACATGCACA
CAGACTACACAGGGCAGGGCGTCGACCAGCTGAAGAATGTGATCCAGATGCTGAGAACGA
ATCCAACAGATCGTCGCATGCTCATGACTGCCTGGAATCCTGCAGCGCTGGACGAAATGG
CGCTGCCGCCTTGTCACTTGTTGTGCCAGTTCTACGTGAACGACCAGAAGGAGCTGTCGT
GCATCATGTATCAGCGGTCGTGCGATGTCGGCCTCGGCGTCCCCTTCAACATCGCTTCCT
ATTCGCTTTTGACGCTCATGGTTGCACACGTCTGCAACCTAAAACCTAAGGAGTTCATTC
ACTTCATGGGGAACACGCATGTCTACACGAACCATGTCGAGGCTTTAAAAGAGCAGCTGC
GGAGAGAACCGAGACCGTTCCCCATTGTGAACATCCTCAACAAGGAACGCATCAAGGAAA
TCGACGATTTCACCGCCGAGGATTTTGAGGTCGTGGGCTACGTCCCGCACGGACGAATCC
AGATGGAGATGGCTGTCTAGCGGAAATACAGAAGCTAGCTTTGATCCCGTTTTTCTTACT
TATATATTTATACCAATTGATTGTATTTATAACTGTAAAAATGTGTATGTTGTGTGCATA
TTTTTTTTTGTGCATGCACATGCATGTAAATAGCTAAAATTATGAACATTTTATTTTTTG
TTCAGAAAAAAAAAACTTTACACACATAAAATGGCTAGTATGAATAGCCATATTTTATAT
AAATTAAATCCTATGAATTTATGACCATATTAAAAATTTAGATATTTATGGAACATAATA
TGTTTGAAACAATAAGACAAAATTATTATTATTATTATTATTTTTACTGTTATAATTATG
TTGTCTCTTCAATGATTCATAAATAGTTGGACTTGATTTTTAAAATGTTTATAATATGAT
TAGCATAGTTAAATAAAAAAAGTTGAAAAATTAAAAAAAAACATATAAACACAAATGATG
TTTTTTCCTTCAATTTCGATATCG
Restriction sites to linearize plasmid ClaI/SpeI double digest
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 modificationThe ccp1/lap2 gene has been disrupted using a construct that contains the tgdhfr selectable marker and that integrates by double cross-over recombination. This mutant line (429cl1) has been described as mutant RMgm-121. The ccp4 (lap6) gene has been disrupted in this line.
The ccp4 gene has been disrupted using a construct that integrates by single cross-over integration (insertion vector) that contains the hdhfr 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.
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 15'-AAATCGATTAAAGCTATTAAAATTAATACG
Additional information primer 1primer 1795
Sequence Primer 25'-TTAAGCTTAAAATATAAATATATGTATGTG
Additional information primer 2primer 1796
Sequence Primer 33'-AAGAATTCGGAATTACTGAATAATGAAAAGC
Additional information primer 3primer 1797
Sequence Primer 43'-TTACTAGTTAATGTATGTATATTATACGC
Additional information primer 4primer 1798
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6

  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0417600
Gene Model P. falciparum ortholog PF3D7_0903800
Gene productLCCL domain-containing protein
Gene product: Alternative nameLAP6; LCCL/lectin adhesive-like protein 6; CCP4
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct usedPlasmid 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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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
ATTGTGAGCGGTAAACAATTTCACACAGGAAACAGCTTGACCATGATTACGCCAAGCTTG
ATTATGAATTTAACTCCGGTTCATTTGTAGTAAAAAGAATAAAATCATCTGATCTAAAAA
ATGAACAAACTAGTATAGTAATAGACACATTTGCATTGTTTGATAAATATAGTATCAACA
ATACTGATGAACAAAATTCGAATATCCCTATAAATTTTTCTGGATGGACACGAAAAATTT
GTAAAAATATTCAATTATATATTAAATATGGGAAGATAAATAATGTTATGGATTACCCTT
CTTTATTATTATCATGTAATTATACGTTTGAAAATATTACTCTCGATAATAATCAAACCA
TAGTTGCACGATGCTTACCAAATTGTTTTAATCAAAAAAATGATATCTTTGGTTCTTATA
TCTATGCGCCACATTCCCCAATATGTAAATCTGCTATACATTCTGGTATTATTACAAACT
TAGGAGGTTTGATTGAAATAACAAAACTTACAAATATTACTCAGTCCTTTAGTACTACAA
TTGATATCACTAGAAATGGCGTTAAAGCTATTATAACGGATAATAAAAACAAGGATTCCT
ATTATTTAACAAAACCAAATGGATCCATATGCAATTATCCAACTACTTATTATAATAGAA
ATCAATCCATATCATCACAAAAAAATAATGACCTAAATAAAACATTTCCTTTTTTTATCC
AATTAAATTCCAATACATTCAAAATACCTCCAGTAGTTTTAGTTAATAAAAATCACGCTG
CTTTACAAAACCAGTTGTATAATATGCACAATTGGTATAACACCGAAAATTATAAAAAAG
AAAATAAATTCATACCGCCTGAAAATGGGAAAGAAATACCAATTCAGAAAAAAATGAAAG
AAAACAAAATTAATATAATAAGCCCCAGTTCAGACATAAACAATAATTTGAAAAATGAAA
TAAAGACATTTGAGCAAAATTACAATAGAGACCAAGCTTGATAATTCCTGCAGCCCAGCT
TAATTCTTTTCGAGCTCTTTATGCTTAAGTTTACAATTTAATATTCATACTTTAAGTATT
TTTTGTAGTATCCTAGATATTGTGCTTTAAATGCTCACCCCTCAAAGCACCAGTAATATT
TTCATCCACTGAAATACCATTAAATTTTCAAAAAAATACTATGCATATAATGTTATACAT
ATAAACATAAAACGCCATGTAAATCAAAAAATATATAAAAATATGTATAAAAATAAATAT
GCACTAAATATAAGCTAATTATGCATAAAAATTAAAGTGCCCTTTATTAACTAGTCGTAA
TTATTTATATTTCTATGTTATAAAAAAATCCTCATATAATAATATAATTAATATATGTAA
TGTTTTTTTTATTTTATAATTTTAATATAAAATAATATGTAAATTAATTCAAAAAATAAA
TATAATTGTTGTGAAACAAAAAACGTAATTTTTTCATTTGCCTTCAAAATTTAAATTTAT
TTTAATATTTCCTAAAATATATATACTTTGTGTATAAATATATAAAAATATATATTTGCT
TATAAATAAATAAAAAATTTTATAAAACATAGGGGGATCCATGGTTGGTTCGCTAAACTG
CATCGTCGCTGTGTCCCAGAACATGGGCATCGGCAAGAACGGGGACCTGCCCTGGCCACC
GCTCAGGAACGAATTTAGATATTTCCAGAGAATGACCACAACCTCTTCAGTAGAAGGTAA
ACAGAATCTGGTGATTATGGGTAAGAAGACCTGGTTCTCCATTCCTGAGAAGAATCGACC
TTTAAAGGGTAGAATTAATTTAGTTCTCAGCAGAGAACTCAAGGAACCTCCACAAGGAGC
TCATTTTCTTTCCAGAAGTCTAGATGATGCCTTAAAACTTACTGAACAACCAGAATTAGC
AAATAAAGTAGACATGGTCTGGATAGTTGGTGGCAGTTCTGTTTATAAGGAAGCCATGAA
TCACCCAGGCCATCTTAAACTATTTGTGACAAGGATCATGCAAGACTTTGAAAGTGACAC
GTTTTTTCCAGAAATTGATTTGGAGAAATATAAACTTCTGCCAGAATACCCAGGTGTTCT
CTCTGATGTCCAGGAGGAGAAAGGCATTAAGTACAAATTTGAAGTATATGAGAAGAATGA
TTAAGGATCCCGTTTTTCTTACTTATATATTTATACCAATTGATTGTATTTATAACTGTA
AAAATGTGTATGTTGTGTGCATATTTTTTTTTGTGCATGCACATGCATGTAAATAGCTAA
AATTATGAACATTTTATTTTTTGTTCAGAAAAAAAAAACTTTACACACATAAAATGGCTA
GTATGAATAGCCATATTTTATATAAATTAAATCCTATGAATTTATGACCATATTAAAAAT
TTAGATATTTATGGAACATAATATGTTTGAAACAATAAGACAAAATTATTATTATTATTA
TTATTTTTACTGTTATAATTATGTTGTCTCTTCAATGATTCATAAATAGTTGGACTTGAT
TTTTAAAATGTTTATAATATGATTAGCATAGTTAAATAAAAAAAGTTGAAAAATTAAAAA
AAAACATATAAACACAAATGATGTTTTTTCCTTCAATTTCGGGTACCGAGCTCG
Restriction sites to linearize plasmid
Partial or complete disruption of the genePartial
Additional remarks partial/complete disruption
Selectable marker used to select the mutant parasitehdhfr
Promoter of the selectable markereef1a
Selection (positive) procedureWR99210
Selection (negative) procedureNo
Additional remarks genetic modificationThe construct to disrupt the ccp4 (lap6) gene has been introduced into the genome of mutant 429cl1 (RMgm-121) that contains a disrupted ccp1/lap2 gene and the tgdhfr selectable marker.
The ccp4 gene has been disrupted using a construct that integrates by single cross-over integration (insertion vector) that contains the hdhfr 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.
Additional remarks selection procedureBecause the mutant contained the tgdhfr selectable marker in its genome (used to disrupt the ccp1/lap2 locus) the ccp4 genes has been disrupted using a construct that contains the hdhfr selectable marker that provides resistance against WR99210.
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 15′-AAGCTTGATTATGAATTTAACTCCGG
Additional information primer 1
Sequence Primer 25′-CTGCAGTTGGTCTCTATTGTAATTTTG
Additional information primer 2
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