Summary

RMgm-4070
Malaria parasiteP. yoelii
Genotype
DisruptedGene model (rodent): PY17X_0410700; Gene model (P.falciparum): PF3D7_0310100; Gene product: calcium-dependent protein kinase 3 (CDPK3)
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, 10:53
  *RMgm-4070
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption, 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-4070
Principal nameΔCDPK3
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 ookineteOokinetes show strongly reduced forward gliding
OocystOokinetes show strongly reduced forward gliding. No oocyst are formed.
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of CDPK3 ( and expresses a C-terminal mCherry tagged P28 protein.

The cdpk3 gene was disrupted in mutant RMgm-4071 that expresses a C-terminal mCherry tagged P28 protein.

Both for tagging P28 and for disruption of CDPK3 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.

Protein (function)
CDPK3 belongs to an expanded family of Ca2+ dependent protein kinases (CDPKs). CDPKs combine an amino-terminal serine/threonine kinase domain and a carboxy-terminal calmodulin-like domain, composed of four EF hands, in the same molecule. The protein plays a role in ookinetes. Ookinetes lacking expression of CDPK3 fail to bind and traverse the cell of the midgut wall.

Phenotype
Ookinetes show strongly reduced forward gliding. No oocyst are formed.

Additional information
See mutant RMgm-1095 for a detailed description of the CRISPR/Cas9 method.
Both for tagging P28 and for disruption of CDPK3 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


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PY17X_0410700
Gene Model P. falciparum ortholog PF3D7_0310100
Gene productcalcium-dependent protein kinase 3
Gene product: Alternative nameCDPK3
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct usedCRISPR/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
Partial or complete disruption of the geneComplete
Additional remarks partial/complete disruption
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 modificationTo disrupt CDPK3 the CRISPR/Cas9 method as described for mutant RMgm-1095 was used 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

  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 modificationBoth for tagging P28 and for disruption of CDPK3 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