RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5520
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
TaggedGene model (rodent): PBANKA_1202000; Gene model (P.falciparum): PF3D7_1003600; Gene product: inner membrane complex protein 1c, putative (IMC1c; ALV2)
Name tag: mCherry
Phenotype Oocyst;
Last modified: 18 June 2024, 16:48
  *RMgm-5520
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene tagging, 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-5520
Principal nameCap380/GFP-Strep - IMC1c/mCherry
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot tested
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystOocysts were observed with red fluorescence on the outside of the capsule near a (single) small opening.
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses a C-terminal GFP-Strep tagged version of Cap380 and a C-terminal mCherry-tagged version of IMC1c (inner membrane complex protein 1c, putative).

This mutant was obtained by crossing the following (published) mutant lines, RMgm-5519 that expresses a C-terminal GFP-Strep tagged version of Cap380 and RMgm-1122 that expresses a C-terminal mCherry-tagged version of IMC1c (inner membrane complex protein 1c, putative).

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 fluorescence microscopy, we used our Cap380/GFP-Strep parasite line (RMgm-5519) to visualise the oocyst capsule. To also visualise sporozoites, we used our parasite line IMC1c/mCherry that stably expresses the sporozoite-expressed alveolin IMC1c fused to mCherry and displays red fluorescence in mature sporozoites (RMgm-1122). To combine these two fluorescent markers in the same oocyst, the two lines were genetically crossed

Additional information
From the paper: 'We found oocysts with red fluorescence on the outside of the capsule near a small opening. It is difficult to determine conclusively if oocyst undergoing excystation have one, or multiple, holes in their capsule, as their detection relies on their positioning relative to the focal plane of the microscope objective, and additional holes may be forming at different times. Nonetheless, in the egress events we observed, both using Vaseline and conventional cover slips, we only ever observed a single opening (n = 20), indicating this is the most common scenario. Egress of merozoites from the red blood cell has also been reported to take place via a single opening in the host plasma membrane and underlying cytoskeleton. It is noticeable that the oocyst shown in the Figure still possesses a substantial sporoblast body, indicating that the oocyst is not yet fully sporulated (i.e. has used up all sporoblast for sporozoite formation) and thus implying that sporulation can continue whilst excystation is in progress.'

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

  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1202000
Gene Model P. falciparum ortholog PF3D7_1003600
Gene productinner membrane complex protein 1c, putative
Gene product: Alternative nameIMC1c; ALV2
Details of the genetic modification
Name of the tagmCherry
Details of taggingC-terminal
Additional remarks: tagging
Commercial source of tag-antibodies
Type of plasmid/construct(Linear) plasmid double 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 markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modification
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