RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-4072
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_0910900; Gene model (P.falciparum): PF3D7_1137800; Gene product: sporozoite surface protein essential for liver stage development, putative (SPELD)
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)
Replacement locus: Gene model: PBANKA_0910900; Gene product: sporozoite surface protein essential for liver stage development, putative (SPELD)
Phenotype Sporozoite; Liver stage;
Last modified: 14 January 2017, 19:38
  *RMgm-4072
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) : 28067322
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/ResearcherAl-Nihmi FM, 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-4072
Principal namepbspeld 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
SporozoiteNormal (salivary gland) sporozoite production. Normal gliding motility. Transmission of pbspeld ko sporozoites through mosquito bite did not initiate blood stage infection. High doses (2 × 104) of pbspeld ko sporozoites delivered through intravenously route led to occasional break through infection with a pre patent period of 9 days versus of 3.5 days for WT sporozoites
Liver stageNormal (salivary gland) sporozoite production. Normal gliding motility. Transmission of pbspeld ko sporozoites through mosquito bite did not initiate blood stage infection. High doses (2 × 104) of pbspeld ko sporozoites delivered through intravenously route led to occasional break through infection with a pre patent period of 9 days versus of 3.5 days for WT sporozoites.
Normal hepatocyte invasion of mutant sporozoites. Mutants showed normal pattern of intracellular differentiation into EEFs at 12 hrs post infection of HepG2 cells and were similar in size to WT EEFs. At 36 hours the mutant EEFs were smaller than WT, and the difference in size was more pronounced at 62 hrs time point that revealed a distinct block in EEF development likely around 36 hr.
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of SPELD and expresses GFP

Protein (function)
SPELD was identified as the most abundantly expressed sequence tag in a sporozoite SAGE transcriptome analysis. Homologs of SPELD were not detected in other apicomplexa. PbSPELD sequence was analysed for the presence of transmembrane regions using multiple programs (TMHMM, TMpred, DAS, TopPred2), which predicted a single transmembrane region that is shared by all Plasmodium SPELD proteins. The SPELD proteins contain a tyrosine-rich domain, and the most abundant amino acid is tyrosine.

Phenotype
Normal oocyst production.  The sporulation pattern inside mutant oocysts was comparable to wild type oocyst. Strongly reduced infectevity of sporozoites. Normal (salivary gland) sporozoite production. Normal gliding motility. Transmission of pbspeld ko sporozoites through mosquito bite did not initiate blood stage infection.  High doses (2 ×  104) of pbspeld ko sporozoites delivered through intravenously route led to occasional break through infection with a pre patent period of 9 days versus of 3.5 days for WT sporozoites.
Normal hepatocyte invasion of mutant sporozoites.  Mutants showed normal pattern of intracellular differentiation into EEFs at 12 hrs post infection of HepG2 cells and were similar in size to WT EEFs. At 36 hours the mutant EEFs were smaller than WT, and the difference in size was more pronounced at 62 hrs time point that revealed a distinct block in EEF development likely around 36 hr. These analyses indicate a role of SPELD in EEF development.

Additional information
Analysis of a mutant expressing a C-terminal tagged mCherry version of SPELD showed expression in oocyst, sporozoites and early EEFs and evidence for a surface location in sporozoites and location at the parasitophorous vacuole membrame of EEFs. (see mutant RMgm-4073).

Other mutants


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0910900
Gene Model P. falciparum ortholog PF3D7_1137800
Gene productsporozoite surface protein essential for liver stage development, putative
Gene product: Alternative nameSPELD
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 markerunknown
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modification
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 / InsertionReplacement locus
Gene Model of Parasite PBANKA_0910900
Gene productsporozoite surface protein essential for liver stage development, putative
Gene product: Alternative nameSPELD
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