RMgmDB - Rodent Malaria genetically modified Parasites


Malaria parasiteP. berghei
DisruptedGene model (rodent): PBANKA_1324300; Gene model (P.falciparum): PF3D7_1460600; Gene product: inner membrane complex sub-compartment protein 3 (ISP3)
PhenotypeNo phenotype has been described
Last modified: 23 November 2013, 21:42
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) : 24244852
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/ResearcherB. Poulin; R. Tewari
Name Group/DepartmentCentre for Genetics and Genomics
Name InstituteSchool of Life Sciences, Queens Medical Centre, University of Nottingham
Name of the mutant parasite
RMgm numberRMgm-962
Principal nameΔisp3
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Asexual blood stageNot different from wild type
Gametocyte/GameteNot different from wild type
Fertilization and ookineteNot different from wild type
OocystNot different from wild type
SporozoiteNot different from wild type
Liver stageNot different from wild type
Additional remarks phenotype

The mutant lacks expression of inner membrane complex sub-compartment protein 3, putative (ISP3)

Protein (function)
Four IMC sub-compartment proteins (ISPs) were described in T. gondii coded by genes restricted to the Apicomplexa. ISPs are small proteins of approximately 150 amino acids and usually characterized by a MetGly(Xaa)2–5CysCys sequence motif at the N-terminus (except for ISP4), but are otherwise relatively divergent and without either obvious domains, low complexity sequence or homology to other known proteins. To identify ISPs in Plasmodium,  an ISP-specific hidden Markov model was constructed from the four ISPs previously identified in T. gondii. Plasmodium species were shown to contain only genes for ISP1 (PF3D7_1011000) and ISP3 (PF3D7_1460600). These families are the most conserved in terms of protein sequence and domain architecture and generally have strong predictions for N-myristoylation and palmitoylation.

The similar phenotypes of the mutant to wild type parasites indicate that ISP3 has no essential role throughout the complete life cycle (although not all stages have been analysed in detail)

Additional information
Localisation of both ISP1 and ISP3 have been analysed in mutants expressing GFP-tagged version of the proteins (RMgm-964, RMgm-965).

ISP1-GFP was only detected at low levels by fluorescence microscopy in live asexual blood stages, and by IFA. ISP1-GFP showed faint cytosolic fluorescence in activatedmale and female gametocytes, and in addition there was strong peripheral membrane localisation in activated male gametocytes and a strong peripheral and polarised localisation in activated female gametocytes. In zygotes, this polarised area expanded to a crescent shape and the ISP1-GFP signal was reduced in the parasite cytosol. In contrast, ISP3-GFP was clearly evident in schizonts and merozoites, while in female gametocytes and zygotes it showed a pattern similar to ISP1-GFP with both a diffuse and localised distribution. The localisation of ISP3-GFP in developing schizonts resembles an IMC-like pattern. A striking difference between ISP1-GFP and ISP3-GFP was observed in ookinetes: while ISP1-GFP localisation was strong and largely limited to the apical tip of the ookinete, ISP3-GFP was distributed along the parasite periphery. Similarly, sporozoites isolated from mid-gut oocysts showed strong ISP1-GFP fluorescence at their apical tip, while ISP3-GFP was distributed throughout the cell but concentrated at the periphery of the parasite. Intriguingly, in sporozoites isolated from mosquito salivary glands, the distinctive apical localisation of ISP1-GFP was lost and instead the protein was distributed throughout the cell and concentrated at the periphery of the sporozoite, reminiscent of ISP3-GFP. While ISP1-GFP and ISP3-GFP fluorescence was distributed continuously along the length of extracellular sporozoites, it was disrupted at 5 h post-infection in the central protruding region of transforming intracellular sporozoites. At later stages of liver stage development (24 and 48 h), ISP1-GFP and ISP3-GFP appeared to be restricted to a small area at the parasite periphery.ISP1-GFP and ISP3- GFP expression increased towards the end of liver stage development, where both proteins were localised in internal structures inside the parasite. Liver merozoites released in culture appeared to display a circumferential distribution of ISP1 and ISP3.

Evidence is presented that in the absence of ISP3, the apical localisation and polarity of ISP1 (PBANKA_120940) is altered.
Attempts to knock-out isp1 (PBANKA_120940; RMgm-963) were unsuccessful indicating an essential role of ISP1 for asexual blood stage growth/multiplication.

Evidence is presented that ISP1-GFP and ISP3-GFP are myristoylated proteins and are are phosphorylated in both asexual (schizont) and sexual stages (activated gametocytes)

Other mutants
Attempts to knock-out isp1 (PBANKA_120940; RMgm-963) were unsuccessful indicating an essential role of ISP1 for asexual blood stage growth/multiplication.
RMgm-964: A mutant expressing a C-terminal GFP tagged version of ISP3
RMgm-965: A mutant expressing a C-terminal GFP tagged version of ISP1 (PBANKA_120940)

  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1324300
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 usedPlasmid double cross-over
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 parasitetgdhfr
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
Additional information primer 1
Additional information primer 2
Additional information primer 3
Additional information primer 4
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6