RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-4890
Malaria parasiteP. yoelii
Genotype
DisruptedGene model (rodent): PY17X_1328100; Gene model (P.falciparum): PF3D7_1460600; Gene product: inner membrane complex sub-compartment protein 3 (ISP3)
Phenotype Fertilization and ookinete;
Last modified: 29 October 2020, 17:44
  *RMgm-4890
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) : 32395856
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/ResearcherWang X, Yuan J
Name Group/DepartmentState Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signal Network
Name InstituteSchool of Life Sciences, Xiamen University
CityXiamen
CountryChina
Name of the mutant parasite
RMgm numberRMgm-4890
Principal nameΔisp3
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationNo
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNot different from wild type
Fertilization and ookineteBoth Δisp1 and Δisp3 mutants showed normal development of asexual blood stages and gametocytes in vivo as well as gamete formation and zygote-to-retort differentiation in vitro. However, the mature ookinete conversion in vitro was significantly reduced in Δisp1 (conversion rate: 34%) but not in Δisp3 (57%) compared with wild type (WT, 64%)
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of ISP3

Protein (function)
The inner membrane complex (IMC)-residing protein ISP (IMC subcompartment protein) is restricted to the phylum apicomplexa and plays key roles in parasite biology. Two ISP members, ISP1 and ISP3, are found in Plasmodium parasites, and display clear apical polarity of expression in zygotes, suggesting possible role in zygote protrusion or elongation. However, parasites with disruption of ISP1 display a modest decrease in zygote-to-ookinete differentiation, while ISP3 depletion has no significant effect on this process.

Phenotype
Both Δisp1 and Δisp3 mutants showed normal development of asexual blood stages and gametocytes in vivo as well as gamete formation and zygote-to-retort differentiation in vitro. However, the mature ookinete conversion in vitro was significantly reduced in Δisp1 (conversion rate: 34%) but not in Δisp3 (57%) compared with wild type (WT, 64%).

Additional information
From the Abstract:
'Here, we show that palmitoylation of N-terminal cysteines of two inner membrane complex (IMC) proteins (ISP1/ISP3) regulates the IMC localization of ISP1/ISP3 and zygote-to-ookinete differentiation. Palmitoylation of ISP1/ISP3 is catalyzed by an the IMC-residing palmitoyl-S-acyl-transferase (PAT) DHHC2. IMC-anchored ISP1 and ISP3 interact with microtubule component b-tubulin, serving as tethers to maintain the proper structure of subpellicular microtubules (SPMs) during zygote elongation.

'we further disrupted isp3 in the Δisp1 parasite and generated a double knockout Δisp1/3 mutant (see RMgm-4891). The mature ookinete conversion rate of this Δisp1/3 parasite (9%) was significantly lower than that of Δisp1 or Δisp3. Measurement of DNA content after Hoechst 33342 stain indicated the P28-positive gametes of the Δisp1/3 parasite could be fertilized and further develop from diploid to tetraploid, suggesting normal DNA replication. However, time-course (stages I–V) analysis of ookinete differentiation in vitro  revealed developmental arrest at early stages (I and II) for the Δisp1/3 cell. We also isolated parasites from infected mosquito midguts and observed a similar defect of Δisp1/3 in vivo. Consequently, the Δisp1/3 parasite showed significantly reduced number of day 7 midgut oocysts and day 14 salivary gland sporozoites compared with the WT in the infected mosquitoes. Together, these results demonstrate that loss of both ISP1 and ISP3 causes a severe defect in early ookinete differentiation and therefore mosquito transmission of the parasite'. 

Other mutants


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PY17X_1328100
Gene Model P. falciparum ortholog PF3D7_1460600
Gene productinner membrane complex sub-compartment protein 3
Gene product: Alternative nameISP3
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 parasite genomic modification. To construct plasmid vectors for gene editing, we amplified 50 and 30 genomic sequence (400–500 bp) of target genes as homologous arms using specific primers and inserted the sequences into specific restriction sites in pYCm. The sgRNAs were designed to target the coding region of a gene using the online program EuPaGDT. Oligonucleotides for 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. DNA fragments encoding 6HA, 4Myc, 3V5, and Flag tags or BFP were inserted between the left and right arms in frame with 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 5–10 lg plasmid DNA using Lonza Nucleofector described previously (Zhang et al, 2014). Transfected parasites were immediately intravenously injected into a naı¨ve mouse and were exposed to pyrimethamine (7 mg/ml) 24 h post-transfection. Parasites with transfected plasmids usually appear after 5–6 days under drug pressure. Some modified parasites subjected for sequential modification were negatively selected to remove pYCm plasmid.
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