Summary

RMgm-4704
Malaria parasiteP. berghei
Genotype
Genetic modification not successful
DisruptedGene model (rodent): PBANKA_0309500; Gene model (P.falciparum): PF3D7_0212600; Gene product: secreted protein with altered thrombospondin repeat domain (SPATR)
PhenotypeNo phenotype has been described
Last modified: 23 January 2020, 12:03
  *RMgm-4704
Successful modificationThe gene/parasite could not be changed/generated by the genetic modification.
The following genetic modifications were attempted Gene disruption
Number of attempts to introduce the genetic modification 3
Reference (PubMed-PMID number) Reference 1 (PMID number) : 31755154
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
Attempts to generate the mutant parasite were performed by
Name PI/ResearcherGupta R, Mishra S
Name Group/DepartmentDivision of Molecular Parasitology and Immunology
Name InstituteCSIR-Central Drug Research Institute
CityLucknow
CountryIndia

  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0309500
Gene Model P. falciparum ortholog PF3D7_0212600
Gene productsecreted protein with altered thrombospondin repeat domain
Gene product: Alternative nameSPATR
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/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationMultiple attempts to delete the spatr gene were unsuccessful indicating an essential role for multiplication/growth of asexual blood stages

For the generation of Spatr knockout, two independent constructs were generated. For this, two fragments F1 (0.672 Kb) and F2 (0.564 Kb) were amplified using primer pairs 1239/1240 and 1241/1242 respectively. Both fragments were cloned in pBC‐GFP‐hDHFR vector at XhoI/ClaI for F1 and NotI/AscI for F2. Similar gene disruption vector was prepared with long homology regions (F3 and F4 with the size of 1.12 and 1.02 Kb, respectively) for double‐crossover recombination. F3 and F4 were amplified using primer pairs 1245/1240 and 1241/1246 respectively. Integration fragment was separated from vector backbone by digesting with XhoI/AscI and transfected into P. berghei ANKA WT line

Secreted protein with altered thrombospondin repeat (SPATR) is a protein conserved in apicomplexan parasites and shown to be a micronemal protein in T. gondii. Plasmodium SPATR is a 30 kDa protein and is highly expressed during merozoite, gametocyte and sporozoite stages

See RMgm-4703 for conditional knock-out of spatr in sporozoites. The following was found:

Spatr cKO sporozoites glide and invade hepatocyte normally.
Spatr cKO parasites develop normally into hepatic merozoites.
SPATR is not required for hepatic merozoite egress from the parasitophorous vacuole while being essential for blood stage infection.
Spatr cKO hepatic merozoites do not establish blood infection
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