RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5543
Malaria parasiteP. yoelii
Genotype
Genetic modification not successful
DisruptedGene model (rodent): PY17X_0210200; Gene model (P.falciparum): PF3D7_0104300; Gene product: ubiquitin carboxyl-terminal hydrolase 1, putative (UBP1)
PhenotypeNo phenotype has been described
Last modified: 12 September 2024, 11:11
  *RMgm-5543
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 ≥ 5
Reference (PubMed-PMID number) Reference 1 (PMID number) : 38413566
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. yoelii
Parent strain/lineNSM (NSS or NSR strains)
Name parent line/clone Not applicable
Other information parent lineTwo isogenic clones NSR and NSS, derived from P. yoelii NSM, were established after MFQ selective pressure. NSS grew faster than NSR without drug pressure. The NSR and NSS are isogenic parasites with potential mutations in the NSR to confer high-level MFQ resistance
Attempts to generate the mutant parasite were performed by
Name PI/ResearcherXu R, Li J
Name Group/DepartmentState Key Laboratory of Cellular Stress Biology, School of Life Sciences, Faculty of Medicine and Li
Name InstituteXiamen University
CityXiamen
CountryChina

  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PY17X_0210200
Gene Model P. falciparum ortholog PF3D7_0104300
Gene productubiquitin carboxyl-terminal hydrolase 1, putative
Gene product: Alternative nameUBP1
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 CRISPR/Cas9 plasmid pYCm was utilized for gene editing. To construct plasmids for ubp1 allelic replacement at amino acid codon 1560 and 2874 between P. yoelii lines NSS and NSR, four DNA fragments from two regions (R1, from 4393 to 5036; and R2, from 8368 to 9034) were amplified and inserted into the NcoI/XhoI sites of the pYCm vector individually. Three silent nucleotide substitutions were introduced into the single guide RNA (sgRNA) binding sites in the donor templates using synthetic oligonucleotides and PCR amplification to yield the editing plasmids.

To construct plasmids for gene tagging or tag insertion, a fragment (450–800 bp) from the C- or N-terminal of coding regions as left or right homologous arm and a fragment (450–800 bp) from 3’UTR or 5’UTR regions as right or left homologous arm were amplified, respectively. DNA sequence encoding 6HA, 4Myc, mCherry, GFP, APEX2, or 3HA was inserted between the left and right arms in the frame and cloned into the pYCm vector.

To construct the plasmid to delete the coding region of the P. yoelii ubp1 gene, 5’- and 3’- genomic fragments (400–600 bp) were amplified as left and right arms and cloned them into restriction sites of HindIII/NcoI and XhoI/EcoRI in the pYCm vector, respectively. Synthesized sgRNAs were annealed and ligated into the restriction site of BsmBI in the vector.
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