RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-1284
Malaria parasiteP. berghei
Genotype
Transgene
 Transgene Plasmodium: Gene model: PBANKA_0819800; Gene model (P.falciparum): PF3D7_0918900; Gene product: gamma-glutamylcysteine synthetase (γ-GCS; ggcs)
Promoter: Gene model: PBANKA_1133300; Gene model (P.falciparum): PF3D7_1357100; Gene product: elongation factor 1-alpha (eef1a)
3'UTR: Gene model: Not available; Gene product: Not available
Insertion locus: Gene model: Not available; Gene product: Not available (small subunit ribosomal rna gene (c-type unit))
Phenotype Asexual bloodstage;
Last modified: 9 June 2015, 19:43
  *RMgm-1284
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 26010448
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 2.34
Other information parent lineP. berghei ANKA 2.34 is a cloned, gametocyte producer line of the ANKA strain (PubMed: PMID: 15137943).
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The mutant parasite was generated by
Name PI/ResearcherVega-Rodríguez J; Serrano AE
Name Group/DepartmentDepartment of Microbiology and Medical Zoology
Name InstituteUniversity of Puerto Rico, School of Medicine
CitySan Juan
CountryPuerto Rico
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Name of the mutant parasite
RMgm numberRMgm-1284
Principal namepbggcs-oe
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
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Phenotype
Asexual blood stageEvidence is presented that increased γ-GCS levels do not alter the sensitivity of blood stages to the antimalarials chloroquine and artemisinin
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant contains 2-3 addtional copies of the ggcs gene (encoding γ-GCS) , introduced into the silent c/d-rRNA gene unit and under control of the constitutive eef1a promoter. These copies are introduced by single cross-over integration

Protein (function)
γ-GCS catalyzes the rate limiting step during GSH biosynthesis. GSH is a thiol-based tripeptide implicated in a variety of cellular processes, including detoxification of xenobiotics and protection against reactive oxygen species

Phenotype
Evidence is presented that increased γ-GCS levels do not alter the sensitivity of blood stages to the antimalarials chloroquine and artemisinin

See also RMgm-204 for a P. berghei mutant lacking expression of gamma-glutamylcysteine synthetase (γ-GCS).

Additional information
Overexpression of pbggcs mRNA in blood stages of the pbggcs-oe parasites was demonstrated by RNase Protection Assay. The pbggcs mRNA levels in blood stages of pbggcs-oe parasites were 4-5 times higher relative to wild type parasites.
To investigate whether overexpression of the pbggcs gene results in increased parasite GSH levels, total GSH was determined in pbggcs-oe and wild type parasites by HPLC. Total GSH levels were significantly higher in pbggcs-oe (17 - 22 nmol/109 parasites, SD ±15.2, P<0.05) parasites when compared to wild type (7.4 nmol/109 parasites, SD ±1.7).

Other mutants
RMgm-204: A mutant lacking expression of gamma-glutamylcysteine synthetase (γ-GCS).

  Transgene: Mutant parasite expressing a transgene
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Type and details of transgene
Is the transgene Plasmodium derived Transgene: Plasmodium
Gene Model of Parasite PBANKA_0819800
Gene Model P. falciparum ortholog PF3D7_0918900
Gene productgamma-glutamylcysteine synthetase
Gene product: Alternative nameγ-GCS; ggcs
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Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct(Linear) plasmid single 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 parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe pL1136 vector containing the Toxoplasma gondii dihydrofolate reductase—thymidylate synthase (tgdhfr/ts) selectable marker was used as a backbone for the creation of a pbggcs overexpressing plasmid. The complete pbggcs ORF, including 465 bp of the 3’UTR, was amplified from ANKA 2.34 genomic DNA using primers 2562 (5’-CATGCCATGGATGGGTTTTCTAAAAATTGGAACTCC-3’; KpnI site is underlined) and 2563 (5’- CGGGGTACCTGGTGTGTATATACCAAACCGTTTC-3’; KpnI site is underlined), cloned into the TOPO TA vector (Invitrogene) and sequenced. The pbggcs coding sequence containing the 3’UTR was excised from the pbggcs-TOPO plasmid using the NcoI and the KpnI restriction enzymes and subsequently cloned into the pL0017 after removing the GFP coding sequence from the plasmid. The resulting pL1136 plasmid was linearized using the SacII restriction enzyme and transfected into P. berghei (ANKA 2.34) purified schizonts. Transfection, selection of transformed parasites with pyrimethamine, and cloning of pbggcs-oe parasites were carried out as previously described. Clonal parasites (pbggcs-oe1; pbggcs-oe2) obtained by limiting dilution were analyzed for correct integration of the pbggcs over-expression plasmid into the c/dssurrna on chromosome 5/6 by Southern analysis of chromosomes separated by Field Inverted Gel Electrophoresis (FIGE).
Additional remarks selection procedure
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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 Not available
Gene productNot available
Gene product: Alternative name
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 / InsertionInsertion locus
Gene Model of Parasite Not available
Gene productNot available
Gene product: Alternative namesmall subunit ribosomal rna gene (c-type unit)
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