RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-4658
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PBANKA_0921600; Gene model (P.falciparum): PF3D7_1126800; Gene product: RNA-binding protein, putative (PbSR-MG)
Name tag: triple-HA
Phenotype Asexual bloodstage; Gametocyte/Gamete;
Last modified: 8 August 2019, 12:03
  *RMgm-4658
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) : 31370870
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/ResearcherYeoh LM, McFadden GI, Ralph SA
Name Group/DepartmentDepartment of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institut
Name InstituteThe University of Melbourne
CityParkville
CountryAustralia
Name of the mutant parasite
RMgm numberRMgm-4658
Principal namePbSR-MG HA-tagged
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageLocalizing an integrated, hemagglutinin-tagged (HA-tagged) PbSR-MG confirmed its expected nuclear localization
Gametocyte/GameteLocalizing an integrated, hemagglutinin-tagged (HA-tagged) PbSR-MG confirmed its expected nuclear localization
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses a C-terminal HA-tagged version of PbSR-MG 

Protein (function)
Serine-arginine–rich (SR) proteins are major regulators of alternative splicing, with regular expression levels necessary for maintaining normal alternative splicing and viability. One SR protein, PBANKA_0921600, is specifically upregulated in male gametocytes of P. berghei and downregulated in female gametocytes, and phylogenetic analysis identifies this gene as an orthologue of a known splicing factor in T. gondii. We named PBANKA_0921600 PbSR-MG (P. berghei SR protein male gametocytes).

Localizing an integrated, hemagglutinin-tagged (HA-tagged) PbSR-MG confirmed its expected nuclear localization

Phenotype
Localizing an integrated, hemagglutinin-tagged (HA-tagged) PbSR-MG confirmed its expected nuclear localization

Phenotype analyses of a mutant lacking expression of PbSR-MG showed the follwing (see mutant RMgm-4657):
Normal gametocyte production. Activation of female gametocytes normal. Male exflagellation, however, strongly reduced. Flagella stained with an anti-tubulin antibody remain curled within the gametocyte body. Motility of male free gametes strongly impaired.
Ablation of PbSR-MG also resulted in fewer oocysts per mosquito host. To test if this difference solely reflected the defect in exflagellation, we repeated the mosquito infection using equal numbers of in vitro generated ookinetes. Despite exposing mosquitoes to equal infective load, we still observed a significant difference in oocyst numbers in PbSR-MG KO compared to wild-type parental parasites.

Additional information


Other mutants


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0921600
Gene Model P. falciparum ortholog PF3D7_1126800
Gene productRNA-binding protein, putative
Gene product: Alternative namePbSR-MG
Details of the genetic modification
Name of the tagtriple-HA
Details of taggingC-terminal
Additional remarks: tagging
Commercial source of tag-antibodies
Type of plasmid/construct(Linear) plasmid single 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 modificationThe hemagglutinin epitope-tagged line was created by
modification of a pL0006-based plasmid. This plasmid was designed for single-site recombination into P. berghei and includes three hemagglutinin (HA) tags, followed by the 3'-UTR of KAHRP, followed by a DHFR selectable marker. We cloned the 3' end of PbSR-MG from genomic DNA with the primers “PbSR-MG 3'-HindIIIApaI SP” and “PbSR-MG 3_-AvrII-BglII ASP” and ligated this into the vector backbone.
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