RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-762
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_1114700; Gene model (P.falciparum): PF3D7_0515100; Gene product: rhomboid protease ROM9 (ROM9)
Transgene
 Transgene not Plasmodium: A fusion of GFP (gfp-mu3) and Luciferase Firefly (LucIAV)
Promoter: Gene model: PBANKA_0915000; Gene model (P.falciparum): PF3D7_1133400; Gene product: apical membrane antigen 1 (ama1)
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)
PhenotypeNo phenotype has been described
Last modified: 16 March 2013, 12:19
  *RMgm-762
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) Reference 1 (PMID number) : 23490234
MR4 number
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Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone P. berghei ANKA 1037m1f1mocl1 (RMgm-32)
Other information parent lineP. berghei ANKA 1037m1f1mocl1 (1037cl1; RMgm-32) is a reference ANKA mutant line which expresses GFP-luciferase under control of a schizont-specific promoter. This reference line does not contain a drug-selectable marker (PubMed: PMID: 20019192).
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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
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Name of the mutant parasite
RMgm numberRMgm-762
Principal name2124cl1; 2125cl1
Alternative nameΔrom9-a; Δrom9-b
Standardized name
Is the mutant parasite cloned after genetic modificationYes
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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 stageNot different from wild type
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of the rhomboid protease ROM9.

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). 

Phenotype
Phenotype analyses indicate that ROM9 is not essential throughout the complete life cycle.

Additional information
∆rom9-a parasites were analysed during development in mosquitoes and in hepatocytes. ∆rom9-a parasites produced wild type levels of oocysts and salivary gland sporozoites. Gliding motility, in vitro hepatocyte traversal and invasion of ∆rom9-a sporozoites and prepatent period were not significantly different from those characteristics of wild type parasites. Immunofluorescence analyses of liver stage parasites stained with antibodies against markers for parasite development (HSP70), parasitophorous vacuole membrane (UIS4 and EXP1) and merozoite formation (MSP1), also revealed no distinct differences in morphology between ∆rom9-a and wild type liver stage parasites at 24h or 48h after sporozoite invasion. However, ∆rom9 parasite loads between 53 and 57 hours after infection (measured by RT-PCR, FACS and luciferase assay) were consistently lower in both in vitro and in vivo  which may indicate that this enzyme plays a (minor) role during liver stage development.

Other mutants
RMgm-176: A P. berghei mutant lacking expression of rhomboid protease ROM1 (partial disruption)
RMgm-177: 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-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
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Details of the target gene
Gene Model of Rodent Parasite PBANKA_1114700
Gene Model P. falciparum ortholog PF3D7_0515100
Gene productrhomboid protease ROM9
Gene product: Alternative nameROM9
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Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct usedPCR construct
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
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Plasmid/construct sequence
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GAACTCGTACTCCTTGGTGACGTCGCGAATAAGCGCATGTCATTTGTTGTATTATATGAT
TTGGAATTTTTATTTATTATATTTTTTTTCATATGTATATGCATATTTCAGTTTGATAAG
TTACTTTCAGATTGAAAAAGTTGTCGTAATATTTCTTATGGAGTAAAAAAGATATAAATA
TTTTTCGTATATAAGTTGGGGCGATTTTATGTACAAATTTTTAAAAAGTCGATAAACAAA
TAAATAATAATATAGAAGAAACAAAAATAAAACAAATAAATAATAATATAAAATAGAAGA
GGAAGCAAAAAAAAATGATATTATTTAATAAAGTAATATTTTTTTGAAAAAGAATAGTTA
CATGTATTATTTTCCCAGTTTACAAAATCCAATAAGTAATGATGAAGTAATTTAACTGAG
CACATTATAATATTAGATAATTTCCATAAATATATGACATATACATGTAAACTAAATGGC
TAATCCTTTAATGCTATTTTTTACGATGGAACATAAGAAATAAGGTATTTTGTTTTATTG
TTATATGCAAAAAAAAATGTCCTTTTAACAATGAAACAAGAAAAAAAAAAGACAAAAAAA
TGTTAACATATTGTGGTTATACTGCTAAATCGAATGCAACTTGTAAAAGAGTATATTTTT
TTAAGATATTATTTTTATAATAGATTTTCTTAAAGCAATTTTTTTTTCGATATTTTGGTT
ATTTCCCATTACTCGTATATATACACACAAATAAAATTCATACCCCAATTCCAGCATATT
CTTACGAGGTCGACGATGCTTGTAGATGGCCCAGCTTAATTCTTTTCGAGCTCTTTATGC
TTAAGTTTACAATTTAATATTCATACTTTAAGTATTTTTTGTAGTATCCTAGATATTGTG
CTTTAAATGCTCACCCCTCAAAGCACCAGTAATATTTTCATCCACTGAAATACCATTAAA
TTTTCAAAAAAATACTATGCATATAATGTTATACATATAAACATAAAACGCCATGTAAAT
CAAAAAATATATAAAAATATGTATAAAAATAAATATGCACTAAATATAAGCTAATTATGC
ATAAAAATTAAAGTGCCCTTTATTAACTAGTCGTAATTATTTATATTTCTATGTTATAAA
AAAATCCTCATATAATAATATAATTAATATATGTAATGTTTTTTTTATTTTATAATTTTA
ATATAAAATAATATGTAAATTAATTCAAAAAATAAATATAATTGTTGTGAAACAAAAAAC
GTAATTTTTTCATTTGCCTTCAAAATTTAAATTTATTTTAATATTTCCTAAAATATATAT
ACTTTGTGTATAAATATATAAAAATATATATTTGCTTATAAATAAATAAAAAATTTTATA
AAACATAGGGGGATCCATGGTTGGTTCGCTAAACTGCATCGTCGCTGTGTCCCAGAACAT
GGGCATCGGCAAGAACGGGGACCTGCCCTGGCCACCGCTCAGGAACGAATTTAGATATTT
CCAGAGAATGACCACAACCTCTTCAGTAGAAGGTAAACAGAATCTGGTGATTATGGGTAA
GAAGACCTGGTTCTCCATTCCTGAGAAGAATCGACCTTTAAAGGGTAGAATTAATTTAGT
TCTCAGCAGAGAACTCAAGGAACCTCCACAAGGAGCTCATTTTCTTTCCAGAAGTCTAGA
TGATGCCTTAAAACTTACTGAACAACCAGAATTAGCAAATAAAGTAGACATGGTCTGGAT
AGTTGGTGGCAGTTCTGTTTATAAGGAAGCCATGAATCACCCAGGCCATCTTAAACTATT
TGTGACAAGGATCATGCAAGACTTTGAAAGTGACACGTTTTTTCCAGAAATTGATTTGGA
GAAATATAAACTTCTGCCAGAATACCCAGGTGTTCTCTCTGATGTCCAGGAGGAGAAAGG
CATTAAGTACAAATTTGAAGTATATGAGAAGAATGATGCTAGCGGAGGAGGTGGATCTGG
TGGAGGTGGAAGTGCTAGCGTGACAGGGGGAATGGCAAGCAAGTGGGATCAGAAGGGTAT
GGACATTGCCTATGAGGAGGCGGCCTTAGGTTACAAAGAGGGTGGTGTTCCTATTGGCGG
ATGTCTTATCAATAACAAAGACGGAAGTGTTCTCGGTCGTGGTCACAACATGAGATTTCA
AAAGGGATCCGCCACACTACATGGTGAGATCTCCACTTTGGAAAACTGTGGGAGATTAGA
GGGCAAAGTGTACAAAGATACCACTTTGTATACGACGCTGTCTCCATGCGACATGTGTAC
AGGTGCCATCATCATGTATGGTATTCCACGCTGTGTTGTCGGTGAGAACGTTAATTTCAA
AAGTAAGGGCGAGAAATATTTACAAACTAGAGGTCACGAGGTTGTTGTTGTTGACGATGA
GAGGTGTAAAAAGATCATGAAACAATTTATCGATGAAAGACCTCAGGATTGGTTTGAAGA
TATTGGTGAGGCTTCGGAACCATTTAAGAACGTCTACTTGCTACCTCAAACAAACCAATT
GCTGGGTTTGTACACCATCATCAGAAATAAGAATACAACTAGACCTGATTTCATTTTCTA
CTCCGATAGAATCATCAGATTGTTGGTTGAAGAAGGTTTGAACCATCTACCTGTGCAAAA
GCAAATTGTGGAAACTGACACCAACGAAAACTTCGAAGGTGTCTCATTCATGGGTAAAAT
CTGTGGTGTTTCCATTGTCAGAGCTGGTGAATCGATGGAGCAAGGATTAAGAGACTGTTG
TAGGTCTGTGCGTATCGGTAAAATTTTAATTCAAAGGGACGAGGAGACTGCTTTACCAAA
GTTATTCTACGAAAAATTACCAGAGGATATATCTGAAAGGTATGTCTTCCTATTAGACCC
AATGCTGGCCACCGGTGGTAGTGCTATCATGGCTACAGAAGTCTTGATTAAGAGAGGTGT
TAAGCCAGAGAGAATTTACTTCTTAAACCTAATCTGTAGTAAGGAAGGGATTGAAAAATA
CCATGCCGCCTTCCCAGAGGTCAGAATTGTTACTGGTGCCCTCGACAGAGGTCTAGATGA
AAACAAGTATCTAGTTCCAGGGTTGGGTGACTTTGGTGACAGATACTACTGTGTTTAACT
CGATCCCGTTTTTCTTACTTATATATTTATACCAATTGATTGTATTTATAACTGTAAAAA
TGTGTATGTTGTGTGCATATTTTTTTTTGTGCATGCACATGCATGTAAATAGCTAAAATT
ATGAACATTTTATTTTTTGTTCAGAAAAAAAAAACTTTACACACATAAAATGGCTAGTAT
GAATAGCCATATTTTATATAAATTAAATCCTATGAATTTATGACCATATTAAAAATTTAG
ATATTTATGGAACATAATATGTTTGAAACAATAAGACAAAATTATTATTATTATTATTAT
TTTTACTGTTATAATTATGTTGTCTCTTCAATGATTCATAAATAGTTGGACTTGATTTTT
AAAATGTTTATAATATGATTAGCATAGTTAAATAAAAAAAGTTGAAAAATTAAAAAAAAA
CATATAAACACAAATGATGTTTTTTCCTTCAACCTTCAATTTCGGATCCACTAGCTCTAG
TCTATATGAACATAAAACTCCTTTTTTTTGCTTAATTGTAGAAAATAACGAATAATAAAT
TGTATATACCATTATAATATGTTGAAATATATACATAAATAATATATTTTATGTAACATG
ATAGAACAAAAAATTATACATTATTGGCTAGCTATAAAATAAATATGAAATATACACATT
TTTTCAGGATACTAAAAAATAATTTTTTTTTGAATAAATTTATATATTTAATTAAATGAT
ATTTATATTCGCATAGTTTTGCCTAGTTTTTGTCAGAGCGAAATACTCTATAATATTTTT
TTTATTGTAGCAAATGCTGTACATAAAATACATTTTTTTTATATTTTATGTTTTTTATTT
ATATTATATGTATCCATTTTTTGCTTTATTTTTATGTTTAAACTTTTACGAAATTATAAA
AATTAAGAAGTGAAAATAAATGAATAATCGATGGGCATACAAAAGACAATGCTAATAAAA
ATTTCAATCCAGTTTATGCTATTCTGGTTAATACTACATTGTATATAAAAAAAAATATAT
ATATATATAATATAATAAGCTGAAAATGTTATACAGTTTTAGGAATAAGAAACAATATAA
AGTTTAAAAAAAATAATATAAAAAATAATAAATTAGCAAAAGTTAAATATATTCATTTGT
ACATATTTATAAATAAATTTTTTTAATTCACAATTTGCTTGTTTCTTCTATTGCTGAAAT
CGCGAGCTGAGTGTCAATGACCAACCT
Restriction sites to linearize plasmid NruI
Partial or complete disruption of the geneComplete
Additional remarks partial/complete disruption
Selectable marker used to select the mutant parasitehdhfr/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modificationThe mutant was generated by disruption of the gene using a linear construct that was generated using a 2-step, anchor tagging PCR method (for primer details see below).

The 5’- and 3’ targeting regions of the gene were PCR amplified from genomic DNA using primer pairs 1&2 and 3&4 respectively. Primers 2 and 3 have 5’- extensions homologues to the hdhfr::yfcu selectable marker cassette (CATCTACAAGCATCGTCGACCTC in primer 2 and CCTTCAATTTCGGATCCACTAG in primer 3). This selectable marker cassette was excised by digestion with XhoI and NotI from a plasmid (pL0048) that contains the P. berghei eef1a-hdfhr::yfcu-3’dhfr/ts (i.e. promoter-drug selectable marker-3’ terminator sequence) selection cassette. Primers 1 and 4 have 5’-terminal extensions with an anchor-tag suitable for the second PCR reaction. In the second PCR reaction, the amplified 5’- and 3’- targeting sequences were annealed to either side of the selectable marker cassette, and the joint fragment was amplified by the external anchor-tag primers 5/6, resulting in the PCR-based targeting construct

Before transfection, the PCR-based construct was digested with NruI (in primers 1 and 4) to remove the ‘anchor-tag’ and with DpnI that digests any residual pL0048 plasmid.
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 1GAACTCGTACTCCTTGGTGACGTCGCGAATAAGCGCATGTCATTTGTTG
Additional information primer 17079 (NruI); 5’- rom9 targeting region F
Sequence Primer 2CATCTACAAGCATCGTCGACCTCGTAAGAATATGCTGGAATTGG
Additional information primer 27080; 5’- rom9 targeting region R
Sequence Primer 3CCTTCAATTTCGGATCCACTAGCTCTAGTCTATATGAACATAAAACTC
Additional information primer 37081; 3’- rom9 targeting region F
Sequence Primer 4AGGTTGGTCATTGACACTCAGCTCGCGATTTCAGCAATAGAAGAAACAAG
Additional information primer 47082 (NruI); 3’- rom9 targeting region R
Sequence Primer 5GAACTCGTACTCCTTGGTGACG
Additional information primer 54661; anchor-tag primer, F
Sequence Primer 6AGGTTGGTCATTGACACTCAGC
Additional information primer 64662: anchor-tag primer, R
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  Transgene: Mutant parasite expressing a transgene
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Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameA fusion of GFP (gfp-mu3) and Luciferase Firefly (LucIAV)
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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
Plasmid/construct sequence
Restriction sites to linearize plasmid
Selectable marker used to select the mutant parasitegfp (FACS)
Promoter of the selectable markerama-1
Selection (positive) procedureFACS (flowsorting)
Selection (negative) procedureNo
Additional remarks genetic modificationThe GFP-Luciferase gene (1 copy) has been inserted into the 230p locus by double cross-over integration.
Additional remarks selection procedureThis reporter mutant expressing GFP-Luciferase does not contain a drug-selectable marker. This mutant has been selected by FACS sorting after transfection based on GFP fluorescence.
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Other details transgene
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Promoter
Gene Model of Parasite PBANKA_0915000
Gene Model P. falciparum ortholog PF3D7_1133400
Gene productapical membrane antigen 1
Gene product: Alternative nameama1
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
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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 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
Sequence Primer 3
Additional information primer 3
Sequence Primer 4
Additional information primer 4
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Conceptual design of the database: Dr. C.J. Janse (Leiden Malaria Research Group, Leiden, The Netherlands).
Technical design and realization: S. Mededovic and A. van Wigcheren