RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-1110
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_0106200; Gene model (P.falciparum): PF3D7_0607600; Gene product: spindle assembly abnormal protein 6, putative (SAS6)
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;
Last modified: 1 September 2014, 16:58
  *RMgm-1110
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) : 25154861
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 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).
The mutant parasite was generated by
Name PI/ResearcherMarques, SR; Sinden RE
Name Group/DepartmentDepartment of Life Sciences
Name InstituteImperial College of London
CityLondon
CountryUK
Name of the mutant parasite
RMgm numberRMgm-1110
Principal nameΔsas6-gfp
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNormal production of male and female gametocytes. No motile gametes are formed after activation of male gametocytes.
Fertilization and ookineteNormal production of male and female gametocytes. No motile gametes are formed after activation of male gametocytes. Strongly reduced numbers of ookinetes (the few ookinetes formed have a normal morphology).
OocystStrongly reduced ookinete formation. Strongly reduced numbers of oocyst. Oocyst have a reduced size and aberrant DNA synthesis .
SporozoiteStrongly reduced ookinete formation. Strongly reduced numbers of oocyst. Oocyst have a reduced size. No sporozoite formation
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of SAS6 (spindle assembly abnormal protein 6) and expresses GFP under the constitutive eef1 promoter.

Protein (function)
Each male gametocyte forms 8 male gametes, which are simple flagellate cells.  For successful gamete formation, nucleus and cytoplasm of parental gametocytes have to be  exquisitely coordinated. In the nuclear compartment, 3 endomitotic divisions produce 8  newly replicated genomes. Simultaneously, in the cytoplasmic compartment, an  amorphous microtubule organizing centre develops into two planar tetrads of basal bodies (BB),  which separate into 8 individual BBs. Each BB serves as a template for one axoneme  and remains connected with the genome trough a nuclear pore.  The pairing of a single haploid genome/nucleus with each flagellum is critical for the formation  of fully functional male gametes. In a process termed exflagellation,  the newly assembled individual haploid flagellate gametes are released, BB first,  from the residual gametocyte body.BBs are established platforms for eukaryotic flagella/cilia assembly.  Plasmodium genomes contain few conserved BB gene orthologues one of which encodes SAS6 which belongs to  an ancestral conserved module of proteins that correlates with presence of centrioles/BBs.  SAS-6 family members are required for the earliest steps of centriole formation in a range of organisms  and its depletion often results in failure to form centrioles or produces other centriole abnormalities leading to severe flagellar/ciliary anomalies. These anomalies include flagellar absence,  loss of flagellar 9-fold symmetry and cilia length reduction.

Phenotype
Phenotype analyses show the absence of formation of motile male gametes (see also 'Additional information' for an explanation). However, a few ookinetes can be formed. These ookinetes form oocysts but these oocysts lack the formation of viable sporozoites, indicating that SAS6 plays also a role during oocyst formation

Additional information
Analysis of a mutant expressing a cMyc-tagged form of SAS6 (RMgm-1111) showed expression in male gametocytes and gametes (and absence in females).

Flagellum formation with an anti a-tubulin antibody was examined and simultaneously  nuclear organization by DAPI staining. At 10 min. after activation (mpa),  wt and Δsas6 male gametocytes are indistinguishable from each other:  tubulin containing microtubule structures are visible in the cytoplasm and  DAPI measurements suggest that DNA replication is normal. At 15 mpa, wt parasites undergo  exflagellation and tubulin stained wt microgametes can be seen either in the process of release  from the gametocyte body or already detached from it.  Wt flagella usually exhibit wave-like shapes reflecting motility of male gametes.  Δsas6 form tubulin-containing structures that protrude from gametocyte bodies but rarely  detach from them. These tubulin structures, which do not appear to move, display abnormal and linear morphology.  Malformed microgametes projecting out of Δsas6 male gametocytes rarely contain DNA (3%),  as opposed to wt, in which DNA is associated with most flagella detaching or detached  from the gametocyte body (92%). The canonical ‘‘9+2’’ microtubule structure of flagella is  severely disrupted and basal bodies are rare in Δsas6. Taken together these results indicate that depletion of SAS-6 results in the formation of fewer BBs,  which is most certainly responsible for disruption of canonical axonemal structures and lack of  axoneme nucleation.

Other mutants
A independent mutant lacking expressing SAS6 (and expressing GFP) (RMgm-1110)
A mutant expressing a cMyc-tagged form of SAS6 (RMgm-1111)


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0106200
Gene Model P. falciparum ortholog PF3D7_0607600
Gene productspindle assembly abnormal protein 6, putative
Gene product: Alternative nameSAS6
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 modificationSee primers below used for amplifying the 3' and 5' target regions
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 1GTG GTA CCA CAC TTT TAA GGC TAC TTC TG
Additional information primer 15'targetting sequence
Sequence Primer 2CGC GGG CCC ACA TAT GCT TTT CTT TTC TAA TTA CTG
Additional information primer 25'targetting sequence
Sequence Primer 3GTT A GGATCC GTG CTT ATT ATA TCT GCA CTT CCC
Additional information primer 33'targetting sequence
Sequence Primer 4CCT CTC TAGA T TTA TTG TTA CAT ATC CAC
Additional information primer 43'targetting sequence
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6

  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameGFP (gfp-mu3)
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct(Linear) PCR construct 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 procedure
Other details transgene
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
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