Summary

RMgm-4071
Malaria parasiteP. yoelii
Genotype
TaggedGene model (rodent): PY17X_0515900; Gene model (P.falciparum): PF3D7_1030900; Gene product: ookinete surface protein P28 (P28, Pfs28)
Name tag: mCherry
Phenotype Fertilization and ookinete; Oocyst;
Last modified: 14 January 2017, 18:47
  *RMgm-4071
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) : 28034675
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 line
The mutant parasite was generated by
Name PI/ResearcherZhang, C; Yuan, J
Name Group/DepartmentState Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Biology, School of Life
Name InstituteXiamen University
CityXiamen, Fujian
CountryChina
Name of the mutant parasite
RMgm numberRMgm-4071
Principal nameP28::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 ookineteP28::mCherry expression in female gametes, zygotes, ookinetes and very young (1 day) oocysts. No expression in 4-day oocysts.
OocystP28::mCherry expression in female gametes, zygotes, ookinetes and very young (1 day) oocysts. No expression in 4-day oocysts.
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses a C-terminal mCherry tagged P28 protein.

For tagging P28 the CRISPR/Cas9 method was used as described for mutant RMgm-1095  with a slight modification of the pYCm vector. In this vector the original hdhfr slectable marker was replaced by the hdhfr-yfcu selectable marker. This allows for killing mutated parasites that still contain remaining episomal plasmids by 5-FC treatment of mice. Removal of the episomal plasmid is essential for subsequent mutations in the same parasite using the CRISPR/Cas9 method (see also mutants RMgm-4069 and RMgm-4070.

Protein (function)
P28 is one of two major surface proteins (P25 and P28) of the plasma membrane of ookinetes. The proteins are conserved between different Plasmodium species. The proteins are characterized by a secretory N-terminal signal sequence followed by three or four epidermal growth factor (EGF) domains and a glycosylphosphatidylinositol (GPI) anchor.

Phenotype
Normal development of blood and mosquito stages indicating an negative effect of tagging on the function of P28 (however also parasites lacking expression of P28 show only slight reduction of ookinete/oocyst development; see mutant RMgm-1)
No expression of P28::mCherry in asexual blood stages and gametocytes.
P28::mCherry expression in female gametes, zygotes, ookinetes and very young (1 day) oocysts. No expression in 4-day oocysts.

Additional information
See mutant RMgm-1095 for a detailed description of the CRISPR/Cas9 method.
Both for tagging P28 and for disruption of CTRP the CRISPR/Cas9 method was used as described for mutant RMgm-1095  with a slight modification of the pYCm vector. In this vector the original hdhfr slectable marker was replaced by the hdhfr-yfcu selectable marker. This allows for killing mutated parasites that still contain remaining episomal plasmids by 5-FC treatment of mice. Removal of the episomal plasmid is essential for subsequent mutations in the same parasite using the CRISPR/Cas9 method.

Other mutants


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PY17X_0515900
Gene Model P. falciparum ortholog PF3D7_1030900
Gene productookinete surface protein P28
Gene product: Alternative nameP28, Pfs28
Details of the genetic modification
Name of the tagmCherry
Details of taggingC-terminal
Additional remarks: tagging
Commercial source of tag-antibodies
Type of plasmid/constructCRISPR/Cas9 construct: integration through double strand break repair
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/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modificationFor tagging P28 the CRISPR/Cas9 method was used as described for mutant RMgm-1095 with a slight modification of the pYCm vector. In this vector the original hdhfr slectable marker was replaced by the hdhfr-yfcu selectable marker. This allows for killing mutated parasites that still contain remaining episomal plasmids by 5-FC treatment of mice. Removal of the episomal plasmid is essential for subsequent mutations in the same parasite using the CRISPR/Cas9 method.
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