RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5055
Malaria parasiteP. yoelii
Genotype
Genetic modification not successful
DisruptedGene model (rodent): PY17X_0916600; Gene model (P.falciparum): PF3D7_1133300; Gene product: LEM3/CDC50 family protein, putative (CDC50c)
PhenotypeNo phenotype has been described
Last modified: 29 July 2021, 15:12
  *RMgm-5055
Successful modificationThe gene/parasite could not be changed/generated by the genetic modification.
The following genetic modifications were attempted Gene disruption
Number of attempts to introduce the genetic modification Unknown
Reference (PubMed-PMID number) Reference 1 (PMID number) : 34301597
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
Attempts to generate the mutant parasite were performed by
Name PI/ResearcherYang Z, Yuan J
Name Group/DepartmentState Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signal Network, School o
Name InstituteXiamen University
CityXiamen, Fujian
CountryChina

  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PY17X_0916600
Gene Model P. falciparum ortholog PF3D7_1133300
Gene productLEM3/CDC50 family protein, putative
Gene product: Alternative nameCDC50c
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 modificationThe unsuccessful attempts to disrupt this gene indicate an essential function during asexual blood-stage growth

CRISPR-Cas9 plasmid pYCm was used for all the parasite genetic modification. To construct vectors for gene editing, we amplified 5′ and 3′ genomic sequence (400 to 500 bp) of target genes as homologous arms using specific primers and inserted the sequences into specific restriction sites in pYCm. Oligonucleotides for single guide RNAs (sgRNAs) were mixed in pairs, denatured at 95°C for 3 min, annealed at room temperature for 5 min, and ligated into pYCm. The sgRNAs were designed to target the coding region of a gene using the online program EuPaGDT. DNA fragments encoding 6HA, 4Myc, 3V5, and GFP or mCherry were inserted between the left and right arms in frame with the gene of interest. For each gene, two sgRNAs were designed to target sites close to the C- or N-terminal part of the coding region. Infected red blood cells (iRBCs) were electroporated with 5 to 10 μg of plasmid DNA using the Lonza Nucleofector . Transfected parasites were immediately intravenously injected into a naïve mouse and were exposed to pyrimethamine (6 mg/ml) 24 hours after transfection.
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