RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-1128
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_0603900; Gene model (P.falciparum): PF3D7_1205200; Gene product: HAD domain ookinete protein, putative | MORN1-associated HAD phosphatase, putative (HADO)
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; Oocyst; Sporozoite; Liver stage;
Last modified: 24 September 2014, 12:09
  *RMgm-1128
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) : 25225164
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 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. This reference line does not contain a drug-selectable marker (PubMed: PMID: 16242190).
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The mutant parasite was generated by
Name PI/ResearcherAkinosoglou KA, Vlachou D.
Name Group/DepartmentDepartment of Life Sciences
Name InstituteImperial College London
CityLondon
CountryUK
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Name of the mutant parasite
RMgm numberRMgm-1128
Principal nameΔpbhado
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
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Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNormal numbers of gametocytes.
Fertilization and ookineteReduced ookinete formation (42% compared to 72% in wild type).
OocystReduced ookinete formation (42% compared to 72% in wild type). Reduced oocyst formation.
SporozoiteReduced oocyst formation. Reduced sporozoite formation.
Liver stageReduced sporozoite formation. However, sporozoites are infectious to mice (by mosquito-bite infection)
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of HADO (HAD domain ookinete protein, putative) and expresses GFP under control of the constitutive eef1a promoter

Protein (function)
HADO encodes a 379 amino acid protein (44.7 kD). Manual annotation and homology modeling analysis revealed that HADO encodes a haloacid dehalogenase (HAD) domain protein with structural similarity to the human magnesium dependent phosphatase Mdp-1.

Phenotype
Normal numbers of gametocytes. Reduced ookinete formation (42% compared to 72% in wild type). Reduced oocyst formation. Reduced sporozoite formation. However, sporozoites are infectious to mice (by mosquito-bite infection).

Additional information
In this study an independent mutant lacking expression of HADO has been generated in the 2.34 wild type line of P. berghei ANKA.

RT-PCR analysis showed: expression in gametocytes and up-regulation in zygotes/ookinetes.
Western blot analysis using a rabbit polyclonal antibody raised against the C-terminal peptide VFPINFKDRNSIKNL (240-254 aa) of HADO identified a ~45 kD protein expressed in ookinetes. IFA of in vitro purified ookinetes using the P. berghei HADO antibody revealed a distinctive cortical localization along the concave ookinete periphery.

Other mutants

  Disrupted: Mutant parasite with a disrupted gene
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Details of the target gene
Gene Model of Rodent Parasite PBANKA_0603900
Gene Model P. falciparum ortholog PF3D7_1205200
Gene productHAD domain ookinete protein, putative | MORN1-associated HAD phosphatase, putative
Gene product: Alternative nameHADO
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Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct used(Linear) plasmid double cross-over
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid
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 modificationBriefly, 500-1000 bp regions upstream and downstream of each target gene were amplified from P. berghei genomic DNA using oligonucleotide primers carrying restriction enzyme sites: ApaI/HindIII for upstream and EcorI/BamHI for downstream regions (se below for primer information). PCR products were purified using a PCR purification kit (Qiagen) and cloned into the pBS-TgDHFR vector that encompasses polylinker sites flanking the modified Toxoplasma gondii dihydrofolate gene (tgdhfr/ts) conferring resistance to pyrimethamine (Dessens et al., 1999).
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 1TT-GGGCCC-CTGTTGTCTTTTGTTTGCCC
Additional information primer 1PbHADO a (P13); Disruption upstream target ApaI
Sequence Primer 2CC-AAGCTT-TTTGGTATGGTTGCTCATTTTATTTA
Additional information primer 2PbHADO b (P14); Disruption upstream target HindIII
Sequence Primer 3T-GAATTC-TCCAATTAATTTCAAGGATCGAAATTC
Additional information primer 3PbHADO c (P15); Disruption downstream target EcoRI
Sequence Primer 4TT-GGATCC-ACTATACCATGAATTAAACCCGATTG
Additional information primer 4PbHADO d (P16); Disruption downstream target BamHI
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6
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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 nameGFP (gfp-mu3)
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Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct(Linear) plasmid 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 markereef1a
Selection (positive) procedureFACS (flowsorting)
Selection (negative) procedureNo
Additional remarks genetic modificationThe GFP gene (1 copy) has been inserted into the 230p locus (PBANKA_030600) 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.
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Other details transgene
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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
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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