RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-4817
Malaria parasiteP. yoelii
Genotype
DisruptedGene model (rodent): PY17X_0619400; Gene model (P.falciparum): PF3D7_0719200; Gene product: NIMA related kinase 4 (NEK4)
Phenotype Gametocyte/Gamete;
Last modified: 16 June 2020, 18:45
  *RMgm-4817
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
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/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
Name of the mutant parasite
RMgm numberRMgm-4817
Principal nameΔ0619400
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNormal gametocyte production; fertile male gametes are formed; female gametes are infertile
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of PY17X_0619400 (NEK4)

Protein (function)
NEK4 is a serine/threonine NIMA-related kinase; these kinases are mostly described as being involved in controlling/coordinating signalling processes in mitosis. NEK4 gene deletion in (rodent) malaria parasites results in defective (infertile) female gametes. Male gametes are fertile.

Phenotype

Normal asexual blood stage growth/multiplication;
Normal gametocyte production; defective (infertile) female gametes are produced. Male gametes are fertile.
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
Details of the target gene
Gene Model of Rodent Parasite PY17X_0619400
Gene Model P. falciparum ortholog PF3D7_0719200
Gene productNIMA related kinase 4
Gene product: Alternative nameNEK4
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