Back to search resultsSummaryRMgm-1138
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Successful modification | The parasite was generated by the genetic modification |
The mutant contains the following genetic modification(s) | Gene tagging |
Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 25257508 |
MR4 number | |
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Parent parasite used to introduce the genetic modification | |
Rodent Malaria Parasite | P. berghei |
Parent strain/line | P. berghei ANKA |
Name parent line/clone | P. berghei ANKA 507cl1 (RMgm-7) |
Other information parent line | P.berghei ANKA 507cl1 (RMgm-7) is a reference ANKA mutant line which expresses GFP under control of a constitutive promoter. This reference line does not contain a drug-selectable marker (PubMed: PMID: 16242190). |
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The mutant parasite was generated by | |
Name PI/Researcher | Sahu T; Menard R; Baldacci P |
Name Group/Department | Unité de Biologie et Génétique du Paludisme |
Name Institute | Institut Pasteur |
City | Paris |
Country | France |
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Name of the mutant parasite | |
RMgm number | RMgm-1138 |
Principal name | ZIPCO-HA |
Alternative name | |
Standardized name | |
Is the mutant parasite cloned after genetic modification | Yes |
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Phenotype | |
Asexual blood stage | Not different from wild type |
Gametocyte/Gamete | Not tested |
Fertilization and ookinete | Not tested |
Oocyst | Not tested |
Sporozoite | Not different from wild type |
Liver stage | To assess the expression of ZIPCO-HA in sporozoites, Western blot analysis was performed using anti-HA monoclonal antibodies. No specific band was detected at the expected 35 kDa in ZIPCO-HA sporozoites. To investigate ZIPCO-HA production by and localization in liver stages, ZIPCO-HA infected HepG2 cells were stained using a monoclonal anti-HA antibody. A specific signal was detected in EEFs at 48 and 63 hpi. At 48 hpi, there was a punctuated labeling within and at the periphery of the developing parasite. Double labeling with MSP-1, a parasite protein located at the plasma membrane, showed regions of overlap with the HA signal. In more advanced EEFs at the cytomere stage, the HA labeling was detected around groups of nuclei and again there was overlap with the MSP1 signal. These data strongly suggest that ZIPCOHA is mainly located at the parasite plasma membrane. |
Additional remarks phenotype | Mutant/mutation
Phenotype analysys of the mutant expressing HA-tagged ZIPCO showed peak expression of ZIPCO in late liver stages and evidence is presented for localisation of ZIPCO in the parasite plasma membrane. |
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Details of the target gene | |||||||||||||||||||||||||||
Gene Model of Rodent Parasite | PBANKA_0506500 | ||||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_1022300 | ||||||||||||||||||||||||||
Gene product | ZIP domain-containing protein, putative | ||||||||||||||||||||||||||
Gene product: Alternative name | ZIP domain-containing protein, ZIPCO | ||||||||||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||||||||||
Name of the tag | HA | ||||||||||||||||||||||||||
Details of tagging | C-terminal | ||||||||||||||||||||||||||
Additional remarks: tagging | |||||||||||||||||||||||||||
Commercial source of tag-antibodies | |||||||||||||||||||||||||||
Type of plasmid/construct | (Linear) plasmid double cross-over | ||||||||||||||||||||||||||
PlasmoGEM (Sanger) construct/vector used | No | ||||||||||||||||||||||||||
Modified PlasmoGEM construct/vector used | No | ||||||||||||||||||||||||||
Plasmid/construct map | |||||||||||||||||||||||||||
Plasmid/construct sequence | |||||||||||||||||||||||||||
Restriction sites to linearize plasmid | |||||||||||||||||||||||||||
Selectable marker used to select the mutant parasite | hdhfr | ||||||||||||||||||||||||||
Promoter of the selectable marker | pbdhfr | ||||||||||||||||||||||||||
Selection (positive) procedure | pyrimethamine | ||||||||||||||||||||||||||
Selection (negative) procedure | No | ||||||||||||||||||||||||||
Additional remarks genetic modification | The plasmid pBC-zipco-HA-hDHFR contains a HA-FLAG tag fused in frame to the carboxy terminus of the ZIPCO coding sequence, followed by the 3’UTR of Pbhsp70 and the hDHFR cassette. The entire zipco gene (1.4 kb) was cloned between ApaI and ClaI sites, upstream of the HA-FLAG sequence in the pBC-HA-hDHFR plasmid described in Boisson et al (2011). The 3' UTR of zipco (1 kb) was introduced between NotI and AscI sites. The resulting plasmid pBC-zipco-HA was sequenced, and the HA sequence was found to be in phase with the carboxy terminus of ZIPCO. The plasmid was digested with ApaI and AscI and transfected into ANKA-GFP (WT-F) parasites | ||||||||||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||||||||||
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