Summary

RMgm-640
Malaria parasiteP. yoelii
Genotype
TaggedGene model (rodent): PY17X_0203000; Gene model (P.falciparum): Not available; Gene product: integral membrane protein
Name tag: c-myc
Phenotype Asexual bloodstage; Sporozoite; Liver stage;
Last modified: 26 December 2011, 16:05
  *RMgm-640
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
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
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
Name of the mutant parasite
RMgm numberRMgm-640
Principal namePY02667myc
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stagec-myc tagged protein detected in asexual blood stages
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNo evidence for expression of c-myc tagged protein in sporozoites
Liver stagec-myc tagged protein detected in liver stages at 18, 24, 40 and 46 hours after infection
Additional remarks phenotype

Mutant/mutation
The mutant expresses a C-terminal c-myc tagged version of PY02667

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 asexual blood stages. Fluorescence microscopy indicates a location at the parasitophorous vacuole membrane (PVM). No evidence for export into host erythrocyte. c-myc tagged protein detected in liver stages at 18, 24, 40 and 46 hours after infection (not at 12 h). Fluorescence microscopy indicates a location at the parasitophorous vacuole membrane (PVM)

Additional information
PY02667 is refractory to deletion and thus likely essential for blood stage replication (RMgm-642). Importantly, the replacement of PY02667 with an epitope-tagged copy as described here suggests this failure was not due to inaccessibility of the genomic locus.

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-641: A mutant expressing a second, c-myc tagged copy of PY03652 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
Details of the target gene
Gene Model of Rodent Parasite PY17X_0203000
Gene Model P. falciparum ortholog Not available
Gene productintegral membrane protein
Gene product: Alternative name
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 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 parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationFor PY02667myc, the construct was targeted to replace the endogenous ORF by double crossover homologous recombination.
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