Summary

RMgm-225
Malaria parasiteP. berghei
Genotype
MutatedGene model (rodent): PBANKA_1102200; Gene model (P.falciparum): PF3D7_0502400; Gene product: merozoite surface protein 8 | ring-stage membrane protein 1 (MSP8)
Details mutation: The protein lacks the C-terminal EGF domains and the GPP-anchor sequence.
Phenotype Asexual bloodstage;
Last modified: 4 March 2010, 23:43
  *RMgm-225
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene mutation
Reference (PubMed-PMID number) Reference 1 (PMID number) : 18359107
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone Not applicable
Other information parent line
The mutant parasite was generated by
Name PI/ResearcherT.F. de Koning-Ward, S. Crabb
Name Group/DepartmentThe Walter and Eliza Hall Institute of Medical Research
Name InstituteThe Walter and Eliza Hall Institute of Medical Research
CityParkville, Victoria 3050
CountryAustralia
Name of the mutant parasite
RMgm numberRMgm-225
Principal nameΔmsp8
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNo difference in the overall growth rates of asexual blood stages in Balb/c mice and no significant difference in overall reticulocyte preference at any time point during the infection (see also 'Additional information').
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses a truncated form of merozoite surface protein 8 (MSP8). The truncated gene lacks the C-terminal EGF domains and the GPP-anchor sequence.

Protein (function)
MSP8 is a glycosylphosphatidyl inositol (GPI)-anchored membrane protein. A distinctive feature of this protein is a C-terminal double epidermal growth factor (EGF)-like domain resembling that seen in the dominantly expressed merozoite surface protein, MSP1.
The exact location of the protein is not clear but evidence has been presented for a localisation in the parasitophorous vacuole membrane in the ring stage and in the food vacuole in older trophozoites.

Phenotype
The phenotype analyses indicate that the lack of expression of MSP8 does not affect in vivo blood-stage development.

Additional information
Wild-type P. berghei has a preference for invading reticulocytes, however, at low parasite burdens in mice the majority of parasites can be found inside mature erythrocytes (normocytes), which is reflective of the high normocyte:reticulocyte ratio during the early stages of infection. As the levels of parasitemia increase, there is a marked increase in circulating reticulocytes in response to the decrease in erythrocyte levels, such that reticulocytes account for up to 40% of circulating cells. With its preference for reticulocytes, therefore, the overwhelming majority of parasites can be found inside reticulocytes in the latter stages of infection. A similar finding to the wild-type parent was observed with the ΔMSP8 parasite line, in that there was no difference in the overall growth rates in Balb/c mice, nor was there a significant difference in overall reticulocyte preference at any time point, also not when only normocytes were observed

MSP8 expression has been analysed by Western analysis and IFA. A time course analysis of MSP8 expression in blood stages showed predominant expression in the trophozoite stage, substantially earlier than schizont-specific antigens such as MSP1. MSP8 was also detected in the ring-stages and by immunofluorescence assay (IFA) it was found to localise to the parasitophorous vacuole. By schizont stages, however, MSP8 was present in the food vacuole. For merozoites weak apical reactivity was observed with the anti-MSP8 antibodies in IFA but was indistinguishable in the mutant and wild-type parasites. 

Other mutants


  Mutated: Mutant parasite with a mutated gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1102200
Gene Model P. falciparum ortholog PF3D7_0502400
Gene productmerozoite surface protein 8 | ring-stage membrane protein 1
Gene product: Alternative nameMSP8
Details of the genetic modification
Short description of the mutationThe protein lacks the C-terminal EGF domains and the GPP-anchor sequence.
Inducable system usedNo
Short description of the conditional mutagenesisNot available
Additional remarks inducable system
Type of plasmid/constructPlasmid double cross-over
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid KpnI/BamHI
Selectable marker used to select the mutant parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe integration of the construct into the MSP8 locus results in truncation of the MSP8 prior to the start of the double EGF domains. The truncated gene is under control of its own 5'-UTR region and the 3'-UTR region of hsp86.
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 1CGGtGGTACCtttcttttattagcccactctctatatattatagg
Additional information primer 1Pb8-5′F (KpnI)
Sequence Primer 2CCGCTCGAGttaaattcgatttttttcaataaaatattctgaaatttttttc
Additional information primer 2PB8-5′R (XhoI)
Sequence Primer 3GGGATATCgttaatatgcatatattatgttatttgaaactag
Additional information primer 3PB8-3′F (EcoRV)
Sequence Primer 4CGCGGATccatcgttcgaaaaaaacagacaatgtac
Additional information primer 4PB8-3′R (BamHI)
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6