Summary

RMgm-963
Malaria parasiteP. berghei
Genotype
Genetic modification not successful
DisruptedGene model (rodent): PBANKA_1209400; Gene model (P.falciparum): PF3D7_1011000; Gene product: inner membrane complex sub-compartment protein 1 (ISP1)
PhenotypeNo phenotype has been described
Last modified: 23 November 2013, 21:56
  *RMgm-963
Successful modificationThe gene/parasite could not be changed/generated by the genetic modification.
The following genetic modifications were attempted Gene disruption
Number of attempts to introduce the genetic modification ≥ 5
Reference (PubMed-PMID number) Reference 1 (PMID number) : 24244852
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).
Attempts to generate the mutant parasite were performed 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
CityNottingham
CountryUK

  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1209400
Gene Model P. falciparum ortholog PF3D7_1011000
Gene productinner membrane complex sub-compartment protein 1
Gene product: Alternative nameISP1
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 modificationAttempts to knock-out isp1 were unsuccessful indicating an essential role of ISP1 for asexual blood stage growth/multiplication.

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.

RMgm-962: A mutant lacking expression of inner membrane complex sub-compartment protein 3, putative (ISP3; PBANKA_132430)

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)
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 15'-CCCCGGGCCCGTAAATTAAAGATCAAATTAGAGAAG-3'
Additional information primer 1
Sequence Primer 25'-GGGGAAGCTTGATATAAATATACATACACACATAC-3'
Additional information primer 2
Sequence Primer 35'-CCCCGAATTCGGAGAAGAACAATTAAAAAGAGTAG-3'
Additional information primer 3
Sequence Primer 45'-GGGGTCTAGAGGTGAATGAAATAAGATCATGGGGG-3
Additional information primer 4
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6