Summary

RMgm-96
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_1353400; Gene model (P.falciparum): PF3D7_1340000; Gene product: secreted ookinete protein, putative (PSOP7; putative secreted ookinete protein 7)
Phenotype Fertilization and ookinete; Oocyst; Sporozoite; Liver stage;
Last modified: 18 February 2009, 01:55
  *RMgm-96
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) : 18761621
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/ResearcherA. Ecker; R.E. Sinden
Name Group/DepartmentDivision of Cell and Molecular Biology
Name InstituteImperial College
CityLondon
CountryUnited Kingdom
Name of the mutant parasite
RMgm numberRMgm-96
Principal name∆psop7
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNot different from wild type
Fertilization and ookineteNormal production of ookinetes. Reduced ookinete invasion and traversal of the midgut wall (see also 'Additional remarks phenotype').
OocystStrong reduction of oocyst formation (0% of wild type). Only two oocysts were observed in a total of 130 mosquitoes.
SporozoiteNo sporozoites are produced. No tramission to C57BL/6 mice by mosquito bite.
When ookinetes were injected directly into the mosquito hemocoel to bypass the midgut barrier mosquito infectivity was rescued as assayed by the quantification of salivary gland sporozoites. The development of oocysts in the hemocoel is thus not affected. The formed sporozoites were also fully infectious to mice by mosquito-bite.
Liver stageWhen ookinetes were injected directly into the mosquito hemocoel to bypass the midgut barrier mosquito infectivity was rescued as assayed by the quantification of salivary gland sporozoites. The development of oocysts in the hemocoel is thus not affected. The formed sporozoites were also fully infectious to mice by mosquito-bite.
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of PSOP7 (putative secreted ookinete protein).

Protein (function)
Unknown. The protein is detected in a proteome analysis of ookinetes and contains a predicted signal sequence and a CBM_5_12 (carbohydrate binding) domain (see also below, additional information). 

Phenotype
The phenotype analyzes indicate a role during invasion/traversal of the ookinetes throug the midgut wall.

Additional information
When ookinetes were injected directly into the mosquito hemocoel to bypass the midgut barrier mosquito infectivity was rescued as assayed by the quantification of salivary gland sporozoites. The development of oocysts in the hemocoel is thus not affected. The formed sporozoites were also fully infectious to mice by mosquito-bite.

A mutant has been genereated with a C-terminal c-myc tag of PSOP7 (RMgm-112). IFA-analysis using c-myc antibodies showed an apical location in the ookinetes.
The tagged protein PSOP7-myc migrates significantly faster in SDS-PAGE than its predicted molecular weight  and thus may undergo proteolytic processing. The mutant expressing tagged PSOP7 show normal mosquito infectivity, indicating that the myc-tagged protein is functional. 


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1353400
Gene Model P. falciparum ortholog PF3D7_1340000
Gene productsecreted ookinete protein, putative
Gene product: Alternative namePSOP7; putative secreted ookinete protein 7
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
Sequence Primer 1TTGGGCCCAATGTTCACATGAACATATAAGG
Additional information primer 1AE07a (ApaI); 5'
Sequence Primer 2CCAAGCTTCGTATATGTATAGGTGGTGCTAA
Additional information primer 2AE07b (HindIII); 5'
Sequence Primer 3TGAATTCTAGCATATACATAATAATGCTGC
Additional information primer 3AE07c (EcoRI); 3'
Sequence Primer 4GGGGATCCTGAGTATAAAAACTCATCTTTCC
Additional information primer 4AE07d (BamHI); 3'
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6