RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-4817

Malaria parasite	P. yoelii
Genotype
Disrupted	Gene 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 modification	The 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 Parasite	P. yoelii
Parent strain/line	P. 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/Researcher	Jiang Y, Yuan J
Name Group/Department	State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network
Name Institute	School of Life Sciences, Xiamen University
City	Xiamen, Fujian
Country	China
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Name of the mutant parasite
RMgm number	RMgm-4817
Principal name	Δ0619400
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modification	Yes
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Phenotype
Asexual blood stage	Not different from wild type
Gametocyte/Gamete	Normal gametocyte production; fertile male gametes are formed; female gametes are infertile
Fertilization and ookinete	Not tested
Oocyst	Not tested
Sporozoite	Not tested
Liver stage	Not 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

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Details of the target gene

Gene Model of Rodent Parasite

PY17X_0619400

Gene Model P. falciparum ortholog

PF3D7_0719200

Gene product

NIMA related kinase 4

Gene product: Alternative name

NEK4

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Details of the genetic modification

Inducable system used

Additional remarks inducable system

Type of plasmid/construct used

CRISPR/Cas9 construct: integration through double strand break repair

PlasmoGEM (Sanger) construct/vector used

Modified PlasmoGEM construct/vector used

Plasmid/construct map

Plasmid/construct sequence

Restriction sites to linearize plasmid

Partial or complete disruption of the gene

Complete

Additional remarks partial/complete disruption

Selectable marker used to select the mutant parasite

hdhfr/yfcu

Promoter of the selectable marker

eef1a

Selection (positive) procedure

pyrimethamine

Selection (negative) procedure

Additional remarks genetic modification

CRISPR/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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