RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-641
Malaria parasiteP. yoelii
Genotype
TaggedGene model (rodent): PY17X_0946500; Gene model (P.falciparum): Not available; Gene product: hypothetical protein (ETRAMP)
Name tag: c-myc
Phenotype Asexual bloodstage; Sporozoite; Liver stage;
Last modified: 31 August 2011, 21:42
  *RMgm-641
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) : 21819513
MR4 number
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Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. yoelii
Parent strain/lineP. y. yoelii 17XNL
Name parent line/clone Not applicable
Other information parent line17XNL is a non-lethal strain of P. yoelii
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The mutant parasite was generated by
Name PI/ResearcherD.C. MacKellar; S.H.I. Kappe
Name Group/DepartmentMolecular and Cellular Biology Program
Name InstituteUniversity of Washington
CitySeattle, Washington
CountryUSA
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Name of the mutant parasite
RMgm numberRMgm-641
Principal namePY03652myc
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
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Phenotype
Asexual blood stagec-myc tagged protein detected in rings, trophozoites and mature schizonts. No evidence for expression in immature schizonts.
Gametocyte/GameteNot tested
Fertilization and ookineteNot different from wild type
OocystNot different from wild type
SporozoiteNo evidence for expression of c-myc tagged protein in sporozoites
Liver stagec-myc tagged protein detected in liver stages at 24, 40 and 46 hours after infection
Additional remarks phenotype

Mutant/mutation
The mutant expresses a C-terminal c-myc tagged version of PY03652

Protein (function)
Early transcribed membrane protein (ETRAMP) family member. Plasmodium conserved family with greater than ten members in P. falciparum. ETRAMPs are abundantly expressed early in the intraerythrocytic cycle and are small (frequently less than 200 aa) integral membrane proteins that are localized within the parasitophorous vacuolar membrane (PVM). All members have signal peptides plus a transmembrane domain. The ETRAMP/SEP proteins of P. yoelii and P. berghei (8-11 genes) show homology to members of the ETRAMP family of proteins of P. falciparum (14 genes) but the orthologous relationship of the different members is not completely resolved.

Phenotype
c-myc tagged protein detected in rings, trophozoites and mature schizonts. No evidence for expression in immature schizonts. Fluorescence microscopy indicates a location at the parasitophorous vacuole membrane in rings and merozoites (PVM). In mature schizonts/merozoites a punctuate staining patterns is observed, suggesting a location in secretory organelles. No evidence for export into host erythrocyte. c-myc tagged protein detected in liver stages at 24, 40 and 46 hours after infection (not at 12h). In contrast to PY03652myc labeling of structures within erythrocytic merozoites, no localization of PY03652myc was seen within exo‐erythrocytic merozoites of the late liver stage parasite.

Additional information
PY03652 has been successfully targeted for deletion by double-homologous recombination (RMgm-643).

The ETRAMP/SEP proteins of P. yoelii and P. berghei (8-11 genes) show homology to members of the ETRAMP family of proteins of P. falciparum (14 genes) but the orthologous relationship of the different members is not completely resolved.

P. falciparum P. yoelii P. berghei Syntenic

PFB0120w (ETRAMP2)
PF11_0040 (ETRAMP11.2)

 PY00205 PBANKA_050110 (PbSEP3) Py-Pb
PFD1120c (ETRAMP4) PY03869 PBANKA_052420 (PbSEP2) Py-Pb
PF10_0323 (ETRAP10.2) PY07506 PBANKA_051700 Py-Pb
PF10_0164 (ETRAMP10.3) PY00204 (PyUIS4) PBANKA_050120 (UIS4) Py-Pb
PF11_0039 (ETRAMP11.1) PY04799 PBANKA_052480 (PbSEP1)  
PF13_0012 (ETRAMP13) PY03011 (PyUIS3) PBANKA_140080(UIS3) Py-Pb
- Py02667 PBANKA_020160 Py-Pb
- PY03365
PY05433
PY06488		 -  
- PY03652 -  


Other mutants
RMgm-640: mutant expressing a c-myc tagged version of PY02667 under control of the endogenous promoter

RMgm-643: A mutant lacking expression of PY03652. The mutant was cloned and the entire life cycle analyzed
RMgm-644: A mutant lacking expression of PY04799. The mutant was cloned and the entire life cycle analyzed

RMgm-646: A mutant lacking expression of PY00205. The mutant was analyzed as an enriched population of transgenic parasites containing residual P. yoelii WT
RMgm-647: A mutant lacking expression of PY03869. The mutant was analyzed as an enriched population of transgenic parasites containing residual P. yoelii WT
RMgm-648: A mutant lacking expression of PY03365. The mutant was analyzed as an enriched population of transgenic parasites containing residual P. yoelii WT
RMgm-649: A mutant lacking expression of PY05433. The mutant was analyzed as an enriched population of transgenic parasites containing residual P. yoelii WT
RMgm-650: A mutant lacking expression of PY06488. The mutant was analyzed as an enriched population of transgenic parasites containing residual P. yoelii WT

RMgm-642: unsuccessful attempt to disrupt PY02667
RMgm-645: unsuccessful attempt to disrupt PY07506

  Tagged: Mutant parasite with a tagged gene
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Details of the target gene
Gene Model of Rodent Parasite PY17X_0946500
Gene Model P. falciparum ortholog Not available
Gene producthypothetical protein
Gene product: Alternative nameETRAMP
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Details of the genetic modification
Name of the tagc-myc
Details of taggingC-terminal
Additional remarks: taggingFour tandem copies of the c-myc epitope fused to the c-terminus, expressed
under control of the endogenous 5’ non-coding region.
Commercial source of tag-antibodies
Type of plasmid/constructPlasmid 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 parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationFor PY03652myc, the construct encoding the epitope‐tagged protein was integrated into the genome upstream of the endogenous ORF, resulting in two copies of the gene
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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Conceptual design of the database: Dr. C.J. Janse (Leiden Malaria Research Group, Leiden, The Netherlands).
Technical design and realization: S. Mededovic and A. van Wigcheren