RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5589
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PBANKA_1411000; Gene model (P.falciparum): PF3D7_1312500; Gene product: conserved Plasmodium protein, unknown function (Scot1; Sporozoite Conserved Orthologous Transcript 1)
Name tag: 3XHA-mCherry
Phenotype Oocyst; Sporozoite; Liver stage;
Last modified: 7 January 2025, 18:51
  *RMgm-5589
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene tagging
Reference (PubMed-PMID number) Reference 1 (PMID number) : 39037752
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/ResearcherGhosh A, Mishra S
Name Group/DepartmentDivision of Molecular Microbiology and Immunology
Name InstituteCSIR-Central Drug Research Institute
CityLucknow
CountryIndia
Name of the mutant parasite
RMgm numberRMgm-5589
Principal nameScot1-3XHA-mCherry
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
OocystExpression of Scot1 in oocysts. Expression was not detected in the blood, gamete, ookinete.
SporozoiteExpression of Scot1 in oocysts and sporozoites. Expression was not detected in the blood, gamete, ookinete.
Sporozoites were stained with anti-mCherry and anti-TRAP antibodies. A granular localization pattern of Scot1-3XHA-mCherry was observed, which colocalized with the TRAP signal in sporozoites, indicating that Scot1 is a micronemal protein.
Liver stageExpression of Scot1 in liver stages (at 12, 24 and 62 hpi). Expression was not detected in liver stages at 36 or 48 hpi.
Additional remarks phenotype

Mutant/mutation
The mutant expresses a C-terminal 3xHA-mCherry-tagged version of Scot1

Protein (function)
The Scot1 (micronemal) protein lacks a signal sequence and transmembrane domain

Phenotype
Expression of Scot1 in oocysts, sporozoites and liver stages (at 12, 24 and 62 hpi). Expression was not detected in the blood, gamete, ookinete, or liver stages (at 36 or 48 hpi). The localization of Scot1-3XHA-mCherry was analyzed in sporozoites and liver stages. Sporozoites were stained with anti-mCherry and anti-TRAP antibodies. A granular localization pattern of Scot1-3XHA-mCherry was observed, which colocalized with the TRAP signal in sporozoites, indicating that Scot1 is a micronemal protein.

Analysis of a mutant lacking expression of Scot1 (RMgm-5587) showed the following: Normal blood, oocyst and sporozoite production and normal invasion of hepatocytes by sporozoites. No blood stage infection in C57BL/6after after intravenous injection of salivary gland sporozoites or infected by mosquito bites. Normal invasion of hepatocytes. In livers of infected mice collected at 40 and 55 hpi, the parasite burden (quantified by amplifying 18S rRNA using real-time PCR), no difference in the 18S rRNA copy number was found at 40 hpi, but it was significantly lower at 55 hpi. Normal growth, numbers and sizes of liver stages. Reduced nuclear division at 62 hpi. No detached liver stages detected.

Additional information

Other mutants


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1411000
Gene Model P. falciparum ortholog PF3D7_1312500
Gene productconserved Plasmodium protein, unknown function
Gene product: Alternative nameScot1; Sporozoite Conserved Orthologous Transcript 1
Details of the genetic modification
Name of the tag3XHA-mCherry
Details of taggingC-terminal
Additional remarks: tagging
Commercial source of tag-antibodies
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 modificationFor the endogenous tagging of Scot1 (PBANKA_1411000) with 3XHA-mCherry, two fragments, F1 (0.84 kb) and F2 (0.52 kb), were amplified using primers 1165/1171 and 1167/1168 and cloned into the pBC-3XHA-mCherry-hDHFR vector at XhoI/BglII and NotI/AscI, respectively.
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