RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-1030
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_0902100; Gene model (P.falciparum): PF3D7_1147000; Gene product: sporozoite and liver stage asparagine-rich protein | sporozoite asparagine-rich protein (SLARP; S22; SAP1)
Transgene
Transgene not Plasmodium: GFP
Promoter: Gene model: PBANKA_0711900; Gene model (P.falciparum): PF3D7_0818900; Gene product: heat shock protein 70 (HSP70)
3'UTR: Gene model: PBANKA_0719300; Gene product: bifunctional dihydrofolate reductase-thymidylate synthase, putative (dhfr/ts)
Replacement locus: Gene model: PBANKA_0902100; Gene product: sporozoite and liver stage asparagine-rich protein sporozoite asparagine-rich protein (SLARP; S22; SAP1)
Phenotype Liver stage;
Last modified: 27 April 2014, 10:56
  *RMgm-1030
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) : 24755823
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/ResearcherManzoni G; Silvie O
Name Group/DepartmentINSERM
Name InstituteINSERM
CityParis
CountryFrance
Name of the mutant parasite
RMgm numberRMgm-1030
Principal nameΔslarp-GFP
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot tested
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageTo analyse in more details the fate of Δslarp sporozoites, time-lapse fluorescence microscopy was used to image control Δp230p-GFP (RMgm-1026) and Δslarp-GFP parasites in HepG2 cultures from 7 to 24 hours postinfection. Transformation of elongated sporozoites into round forms was observed with both Δp230p-GFP and Δslarp-GFP. However, whilst Δp230p-GFP persisted in the culture and started growing over the observation period, Δslarp-GFP failed to develop and died, as evidence by disappearance of the GFP signal from infected cells. No detectable sign of death of infected host cells was observed by microscopy, suggesting that elimination of the Δslarp mutant parasites, unlike other liver stage-arresting mutants19, does not result from host cell apoptosis.
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of SLARP and expresses GFP under the control of the HSP70 promoter.  The parasites have been generated and selected using the GOMO method of transfection (see RMgm-1026 for more details for this transfection method and the construct used)

Protein (function)
SLARP is a conserved Plasmodium gene that is specifically expressed at pre-erythrocytic stages and is required for parasite development in the liver. SLARP-deficient sporozoites injected into susceptible mice cannot convert into blood stages in vivo.The SLARP protein is expressed in sporozoites and in early liver stages and is involved in the regulation of transcription.

Phenotype
The phenotype has not been analysed in detail. See also the  link slarp for other mutants lacking expression of SLARP

To analyse in more details the fate of Δslarp sporozoites, time-lapse fluorescence microscopy was used to image control Δp230p-GFP (RMgm-1026) and Δslarp-GFP parasites in HepG2 cultures from 7 to 24 hours postinfection. Transformation of elongated sporozoites into round forms was observed with both Δp230p-GFP and Δslarp-GFP. However, whilst Δp230p-GFP persisted in the culture and started growing over the observation period, Δslarp-GFP failed to develop and died, as evidence by disappearance of the GFP signal from infected cells. No detectable sign of death of infected host cells was observed by microscopy, suggesting that elimination of the Δslarp mutant parasites, unlike other liver stage-arresting mutants19, does not result from host cell apoptosis.

Additional information
The parasites are selected by a combination of positive selection (pyrimethamine), negative selection (5-FC) and FACS sorting (GOMO transfection; 'Gene Out Marker Out'; see mutant RMgm-1026 for more details for this transfection method and the construct used).

Other mutants
See the link slarp


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0902100
Gene Model P. falciparum ortholog PF3D7_1147000
Gene productsporozoite and liver stage asparagine-rich protein | sporozoite asparagine-rich protein
Gene product: Alternative nameSLARP; S22; SAP1
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
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 parasitehdhfr/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modificationThe parasites have been generated and selected using the GOMO method of transfection (see RMgm-1026 for more details for this transfection method)
Additional remarks selection procedureThe parasites are selected by a combination of positive selection (pyrimethamine), negative selection (5-FC) and FACS sorting.
1) Transfected parasites are first selected in a mouse by pyrimethamine treatment
2) GFP+mCherry+ parasites are selected by FACS sorting and used to infect a mice
3) This mouse is treated with 5-FC to select for parasites that have the selectable marker removed
4) GFP+mCherry- and marker free parasites are selected by FAC sorting and used to infect a mouse
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 1TCCCCGCGGACCTATATTTAATACATTTACACCTCCACG
Additional information primer 1PbSLARP5’For
Sequence Primer 2ATAAGAATGCGGCCGCtcaTCTCAAAAACATAGGAACGTGCCTTGG
Additional information primer 2PbSLARP5’rev
Sequence Primer 3CCGCTCGAGCTACTCAAGTATGTTATATTTATGCATGCC
Additional information primer 3PbSLARP3’For
Sequence Primer 4GGGGTACCGAAAATATCTAAGAAAATCATGTAACGACG
Additional information primer 4PbSLARP3’rev
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
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 parasitehdhfr/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modification
Additional remarks selection procedureThe parasites are selected by a combination of positive selection (pyrimethamine), negative selection (5-FC) and FACS sorting.
1) Transfected parasites are first selected in a mouse by pyrimethamine treatment
2) GFP+mCherry+ parasites are selected by FACS sorting and used to infect a mice
3) This mouse is treated with 5-FC to select for parasites that have the selectable marker removed
4) GFP+mCherry- and marker free parasites are selected by FAC sorting and used to infect a mouse
Other details transgene
Promoter
Gene Model of Parasite PBANKA_0711900
Gene Model P. falciparum ortholog PF3D7_0818900
Gene productheat shock protein 70
Gene product: Alternative nameHSP70
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_0902100
Gene productsporozoite and liver stage asparagine-rich protein sporozoite asparagine-rich protein
Gene product: Alternative nameSLARP; S22; SAP1
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