RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5519
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PBANKA_1218100; Gene model (P.falciparum): PF3D7_0320400; Gene product: oocyst capsule protein Cap380, putative
Name tag: GFP-Strep
Phenotype Oocyst;
Last modified: 18 June 2024, 16:29
  *RMgm-5519
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) : 38097730
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone P. berghei ANKA 2.34
Other information parent lineP. berghei ANKA 2.34 is a cloned, gametocyte producer line of the ANKA strain (PubMed: PMID: 15137943).
The mutant parasite was generated by
Name PI/ResearcherSaeed S, Dessens JT
Name Group/DepartmentDepartment of Infection Biology, Faculty of Infectious and Tropical Diseases
Name InstituteLondon School of Hygiene & Tropical Medicine
CityLondon
CountryUK
Name of the mutant parasite
RMgm numberRMgm-5519
Principal nameCap380/GFP-Strep
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot tested
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystAssessment of GFP expression by confocal fluorescence microscopy of live Cap380/GFP parasites revealed its expression in oocysts from young oocyst through to sporulation. Fluorescence localised predominantly at the oocyst periphery.
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses a C-terminal GFP-Strep tagged version of Cap380

Protein (function)
Cap380 (Plasmodium oocyst capsule protein 380) localises to the P. berghei oocyst capsule, and its depletion leads to oocysts that are gradually eliminated

Phenotype
To investigate sporozoite egress in P. berghei by fuorescence microscopy, we used our Cap380/GFP-Strep parasite line to visualise the capsule.
Assessment of GFP expression by confocal fluorescence microscopy of live Cap380/GFP parasites revealed its expression in oocysts from young oocyst through to sporulation. Fluorescence localised predominantly at the oocyst periphery.

To also visualise sporozoites, we used our parasite line IMC1c/mCherry (RMgm-1122) that stably expresses the sporozoite-expressed alveolin IMC1c fused to mCherry and displays red fuorescence in mature sporozoites. To combine these two fuorescent markers in the same oocyst, the two lines were genetically crossed and the resulting ookinete cultures presented to naïve mosquitoes in membrane feeders, as described resulting in the selection of mutant RMgm-5519.

Additional information
From the paper: 'To study the oocyst capsule during sporozoite egress, we examined Cap380/GFP-Strep oocyst-infected midguts at two weeks post-infected blood feed, when oocyst sporulation is considered to be at its peak. In addition to intact oocysts, live fluorescence microscopy revealed the presence of flattened capsules presumably resulting from oocysts vacated, or being vacated, by their sporozoites. Notably, these empty capsule structures were not visible when midguts were examined by phase contrast (i.e. without capturing GFP fluorescence). This demonstrates that the oocyst does not need to be intact or alive for the capsule to fluoresce, consistent with the capsule constituting a three-dimensional, supramolecular, extracellular matrix that retains water and homeostatic balance.


Other mutants


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1218100
Gene Model P. falciparum ortholog PF3D7_0320400
Gene productoocyst capsule protein Cap380, putative
Gene product: Alternative name
Details of the genetic modification
Name of the tagGFP-Strep
Details of taggingC-terminal
Additional remarks: tagging
Commercial source of tag-antibodies
Type of plasmid/construct(Linear) plasmid single cross-over
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
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationPlasmid pBS-EGFP-hDHFR was PCR-amplified with primers GFP-StrepII-F (GGAGGAGGAAGTGGAGGAGGATCCGCATGGAGTCATCCTCAATTTGAAAAATAAATTCTAGAAGATCCCGTTTTTCTTACTTATATA) and GFP-StrepII-R (CTCCACTTCCTCCTCCTTTGTATAGTTCATCCATGCCATGTGTAAT), followed by circularization of the PCR product by In-Fusion to give pBS-GFP-Strep. The 3’-proximal 2.2kb of the CAP380 coding sequence was PCR amplified with primers CAP380-F (TTGGGCTGCAGTCGATTCGAATGTGAATCCAGGG) and CAP380-R (ATGAGGGCCCCTAAGCTTGCTCTTCGCAACACATTTAG) and cloned into SalI/HindIII-digested pBS-GFP-Strep by in-fusion cloning to give plasmid pBS-Cap380/GFP-Strep. Plasmid pBS-Cap380/GFP-Strep was linearized with BglII prior to transfection of purified schizonts to generate parasite line Cap380/GFP-Strep by single crossover homologous recombination.
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