RMgmDB - Rodent Malaria genetically modified Parasites


Malaria parasiteP. yoelii
TaggedGene model (rodent): PY17X_1328100; Gene model (P.falciparum): PF3D7_1460600; Gene product: inner membrane complex sub-compartment protein 3 (ISP3)
Name tag: sextuple HA epitope (6HA)
Phenotype Asexual bloodstage; Gametocyte/Gamete; Fertilization and ookinete; Oocyst;
Last modified: 30 October 2020, 10:44
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene tagging
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
Name of the mutant parasite
RMgm numberRMgm-4894
Principal nameisp3::6HA
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationNo
Asexual blood stageISP3::6HA expressed in asexual blood stages, gametocytes, ookinetes, and oocysts
Gametocyte/GameteISP3::6HA expressed in asexual blood stages, gametocytes, ookinetes, and oocysts
Fertilization and ookineteISP3::6HA expressed in asexual blood stages, gametocytes, ookinetes, and oocysts (see also below)
OocystISP3::6HA expressed in asexual blood stages, gametocytes, ookinetes, and oocysts
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

The mutant expresses a C-terminal 6HA-tagged version 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.

To confirm the polarized localization of ISP1 and ISP3 in the zygote, we tagged individual endogenous ISP1 (RMgm-4893) or ISP3 with a sextuple HA epitope (6HA) at the C-terminus, obtaining the isp1::6HA and isp3::6HA parasites. Both proteins were expressed in asexual blood stages, gametocytes, ookinetes, and oocysts. Time-course expression analysis showed that both proteins were concentrated at an apical dot in zygotes (stage I), with residual ISP3 distributed at cytoplasm. In stage III, both proteins  are expressed at the periphery of the protrusion, but not zygote remnant. These results support that ISP1/ISP3 is localized in IMC, but not PPM during this differentiation, in agreement with previous observations in P. berghei. Mature ookinetes (stage V) still had peripheral distribution of both ISP1 and ISP3 proteins in whole cell. We further generated a doubly tagged parasite (DTS), isp1::3V5/isp3::6HA (RMgm-4895), by tagging endogenous ISP1 with a triple V5 epitope (3V5) in the isp3::6HA parasite and observed similar co-localization of ISP1/ISP3 at the periphery of zygote protrusion. The localization patterns of ISP1/ISP3 further suggest critical roles of these two proteins in zygote protrusion and/or elongation.

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.

Other mutants

  Tagged: Mutant parasite with a tagged 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
Name of the tagsextuple HA epitope (6HA)
Details of taggingC-terminal
Additional remarks: tagging
Commercial source of tag-antibodies
Type of plasmid/constructCRISPR/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
Selectable marker used to select the mutant parasitehdhfr/yfcu
Promoter of the selectable markerpbdhfr
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