Summary

RMgm-4647
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_1425200; Gene model (P.falciparum): PF3D7_0812300; Gene product: sporozoite surface protein 3, putative (PbS23/SSP3)
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_0711900; Gene product: heat shock protein 70 (HSP70)
Insertion locus: Gene model: PBANKA_1425200; Gene product: sporozoite surface protein 3, putative (PbS23/SSP3)
Phenotype Sporozoite; Liver stage;
Last modified: 3 July 2019, 12:16
  *RMgm-4647
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) : 31251952
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone Not applicable
Other information parent line
The mutant parasite was generated by
Name PI/ResearcherTogiri J, Kumar KA
Name Group/DepartmentDepartment of Animal Biology, School of Life Sciences
Name InstituteUniversity of Hyderabad
CityHyderabad
CountryIndia
Name of the mutant parasite
RMgm numberRMgm-4647
Principal namePbS23/SSP3 KO
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
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
SporozoiteProduction of salivary gland sporozoites comparable to wild type. Normal sporozoite gliding motility, cell traversal and hepatocyte infection in vitro.
Liver stageNormal sporozoite gliding motility, cell traversal and hepatocyte infection in vitro. Normal growth (size) of liver stages in vitro at 12, 36 and 62 hour after infection of hepatocytes. Evidence is presented for a significant reduction in the formation of hepatic merozoites in PbS23/SSP3 KO mutants as revealed by DAPI staining. (Strongly) reduced infectivity in vivo (no blood infections or prolonged period after intravenous injection of sporozoites).
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of PbS23/SSP3 and expresses GFP under control of the constitutive hsp70 promoter

Protein (function)
PbS23/SSP3 was identified in SSH screen between salivary gland sporozoites versus merozoites and encode secretory protein having a predicted molecular mass of 50,4 kDa and a putative signal peptide (N-terminus) and transmembrane domain (C-terminus).

Phenotype
Production of salivary gland sporozoites comparable to wild type. Normal sporozoite gliding motility, cell traversal and hepatocyte infection in vitro. Normal growth (size) of liver stages in vitro at 12, 36 and 62 hour after infection of hepatocytes. Evidence is presented for a significant reduction in the formation of hepatic merozoites in PbS23/SSP3 KO mutants as revealed by DAPI staining. (Strongly) reduced infectivity in vivo (no blood infections or prolonged period after intravenous injection of sporozoites or after mosquito bite).

See also a P. yoelii mutant RMgm-1107 lacking expression of SSP3: this mutant shows a defect in sporozoite motility whereas no defect was detected in sporozoite infectivity

Additional information
Quantitative real-time PCR analysis revealed maximal expression of PbS23/SSP3 in salivary gland sporozoite stage followed by midgut oocyst stages. The expression of PbS23/SSP3 was minimal during different time points of liver stage development and in mixed blood stages.

Other mutants


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1425200
Gene Model P. falciparum ortholog PF3D7_0812300
Gene productsporozoite surface protein 3, putative
Gene product: Alternative namePbS23/SSP3
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 parasitehdhfr
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationPbS23/SSP3 KO construct was generated by amplifying 573 bp for 5’ and 531 bp for 3’ fragments using primers FP6/RP6 and FP7/RP7 respectively. 5’ and 3’ fragments were cloned into pBC-GFP-hDHFR vector at XhoI/ClaI and NotI/AscI respectively. The targeting construct was released by XhoI and AscI digestion and gel purified.
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 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
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/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 parasitehdhfr
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modification
Additional remarks selection procedure
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_0711900
Gene productheat shock protein 70
Gene product: Alternative nameHSP70
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 PBANKA_1425200
Gene productsporozoite surface protein 3, putative
Gene product: Alternative namePbS23/SSP3
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