RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-4658

Malaria parasite	P. berghei
Genotype
Tagged	Gene 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 modification	The 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
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Parent parasite used to introduce the genetic modification
Rodent Malaria Parasite	P. berghei
Parent strain/line	P. berghei ANKA
Name parent line/clone	Not applicable
Other information parent line
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The mutant parasite was generated by
Name PI/Researcher	Yeoh LM, McFadden GI, Ralph SA
Name Group/Department	Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institut
Name Institute	The University of Melbourne
City	Parkville
Country	Australia
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Name of the mutant parasite
RMgm number	RMgm-4658
Principal name	PbSR-MG HA-tagged
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modification	Yes
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Phenotype
Asexual blood stage	Localizing an integrated, hemagglutinin-tagged (HA-tagged) PbSR-MG confirmed its expected nuclear localization
Gametocyte/Gamete	Localizing an integrated, hemagglutinin-tagged (HA-tagged) PbSR-MG confirmed its expected nuclear localization
Fertilization and ookinete	Not tested
Oocyst	Not tested
Sporozoite	Not tested
Liver stage	Not 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

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Details of the target gene

Gene Model of Rodent Parasite

PBANKA_0921600

Gene Model P. falciparum ortholog

PF3D7_1126800

Gene product

RNA-binding protein, putative

Gene product: Alternative name

PbSR-MG

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Details of the genetic modification

Name of the tag

triple-HA

Details of tagging

C-terminal

Additional remarks: tagging

Commercial source of tag-antibodies

Type of plasmid/construct

(Linear) plasmid single cross-over

PlasmoGEM (Sanger) construct/vector used

Modified PlasmoGEM construct/vector used

Plasmid/construct map

Plasmid/construct sequence

Restriction sites to linearize plasmid

Selectable marker used to select the mutant parasite

hdhfr

Promoter of the selectable marker

eef1a

Selection (positive) procedure

pyrimethamine

Selection (negative) procedure

Additional remarks genetic modification

The 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

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