RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-4818
Malaria parasiteP. yoelii
Genotype
DisruptedGene model (rodent): PY17X_0617900; Gene model (P.falciparum): PF3D7_0717500; Gene product: calcium-dependent protein kinase 4 (CDPK4)
Phenotype Gametocyte/Gamete;
Last modified: 16 June 2020, 18:50
  *RMgm-4818
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption
Reference (PubMed-PMID number) Reference 1 (PMID number) : 32273496
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 line
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The mutant parasite was generated by
Name PI/ResearcherJiang Y, Yuan J
Name Group/DepartmentState Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network
Name InstituteSchool of Life Sciences, Xiamen University
CityXiamen, Fujian
CountryChina
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Name of the mutant parasite
RMgm numberRMgm-4818
Principal nameΔ0617900
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
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Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNormal gametocyte production; fertile female gametes are formed; no formation of male gametes
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of PY17X_0619400 (CDPK4)

Protein (function)
CDPK4 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. In plants, CDPKs translate Ca2+ signals generated by external stimuli into cellular responses, thereby regulating cell division and differentiation, the development of tolerance to stress stimuli and the specific defense responses to pathogens.

Phenotype
Normal asexual blood stage growth/multiplication;
Normal gametocyte production; fertile female gametes are formed; no formation of male gametes.
The phenotype has not been analysed in great detail. The P. yoelii gene-deletion mutant has been used to study the protein GEP1. See mutant RMgm-4813 for a P. yoelli gene-deletion mutant lacking GEP1 for detailed analyses (see also below)

Additional information
From the Abstract (based on analyses of mutant RMgm-4813)
GEP1 disruption abolishes XA-stimulated cGMP synthesis and the subsequent signaling and cellular events, such as Ca2+ mobilization, gamete formation, and gametes egress out of erythrocytes. GEP1 interacts with GCα, a cGMP synthesizing enzyme in gametocytes. Both GEP1 and GCα are expressed in cytoplasmic puncta of both male and female gametocytes. Depletion of GCα impairs XA-stimulated gametogenesis, mimicking the defect of GEP1 disruption.

Other mutants
See other mutants generated in this study

  Disrupted: Mutant parasite with a disrupted gene
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Details of the target gene
Gene Model of Rodent Parasite PY17X_0617900
Gene Model P. falciparum ortholog PF3D7_0717500
Gene productcalcium-dependent protein kinase 4
Gene product: Alternative nameCDPK4
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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) procedureNo
Additional remarks genetic modificationCRISPR/Cas9 plasmid pYCm was used for all the genetic modifications. For gene deleting, 5’-genomic and 3’-genomic segments (400 to 700 bp) of the target genes were amplified as left and right homologous arms, respectively, using gene specific primers. The PCR products were digested with appropriate restriction enzymes and the digested products were inserted into matched restriction sites of pYCm.
Oligonucleotides for sgRNAs were annealed and ligated into pYCm17. For each deletion modification, two sgRNAs were designed to disrupt the coding region of a target gene using the online program ZiFit47. For gene tagging, a 400 to 800 bp segment from N-terminal or C-terminal of the coding region and 400 to 800 bp sequences from 5’UTR or 3’UTR of a target gene were amplified and fused with a DNA fragment encoding 6HA or 4Myc in frame at N-terminal or C-terminal of the gene. For each tagging modification, two sgRNAs were designed to target sites close to the C-terminal or N-terminal of the gene coding region.
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