RMgmDB - Rodent Malaria genetically modified Parasites
SummaryRMgm-1305
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*RMgm-1305| Successful modification | The parasite was generated by the genetic modification |
| The mutant contains the following genetic modification(s) | Introduction of a transgene |
| Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 26118994 |
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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 cl15cy1 |
| Other information parent line | |
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| The mutant parasite was generated by | |
| Name PI/Researcher | Philip N; Waters AP |
| Name Group/Department | Wellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity and Inflammation |
| Name Institute | College of Medical Veterinary & Life Sciences, University of Glasgow |
| City | Glasgow |
| Country | UK |
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| Name of the mutant parasite | |
| RMgm number | RMgm-1305 |
| Principal name | pG204 |
| Alternative name | |
| Standardized name | |
| Is the mutant parasite cloned after genetic modification | Yes |
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| Phenotype | |
| Asexual blood stage | Expression of (myc-tagged) Os-TIR1 protein in blood stages |
| Gametocyte/Gamete | Not tested |
| Fertilization and ookinete | Not tested |
| Oocyst | Not tested |
| Sporozoite | Not tested |
| Liver stage | Not tested |
| Additional remarks phenotype | Mutant/mutation See RMgm-1304 for a similar mutant (pG230) expressing Os-TIR1. In this mutant the gene is under control of the 3'UTR of p48/45. This line produces less ookinetes compared to the pG204 line. These mutants ('transgenic acceptor lines') are used to specifically and rapidly and selectively degrade Plasmodium proteins that have been tagged with an auxin-inducible degron (AID). This requires tagging genes of interest (GOI) in the Os-TIR1-expressing mutants with the aid sequence. Subsequently, these parasites expressing both Os-TIR1 and a AID-tagged GOI need to be treated with auxin for degradation of the AID-tagged protein. The system relies on the highly conserved Skp1, Cullin1, F box protein ubiquitin ligase (SCF) complex where the F box protein recruits specific substrates for degradation. Auxin functions as a molecular glue that promotes and stabilizes physical interaction between the auxin receptor, TIR1 (an F box protein), and proteins containing an auxin-inducible degron (AID) motif. Plasmodium falciparum homologs: SKp1 - PF3D7_1367000; Cul1 - PF3D7_0811000; Rbx - PF3D7_0319100. These mutants have been used for conditional degradation of the calcium-regulated protein phosphatase, calcineurin (PBANKA_122740) in multiple life cycle stages. In addition, it has been used for conditional degradation of CDPK1 (PBANKA_0314200), PPKL (PBANKA_1329500) and DOZI (PBANKA_1217700) in blood stages and gametocytes. For these studies the calcineurin (and cdpk1 and ppkl) gene was tagged (C-terminal) with the AID-2xHA degron (CnA-AID) in the Os-TIR expressing parsite lines (RMgm-1306, RMgm-1307). Incubation of parasites with auxin (500µM) resulted in the degradation of the tagged protein. See mutants RMgm-1306 and RMgm-1307 for phenotype analyses of mutant parasites in which calcineurin was conditionally degraded using this system. Other mutants: RMgm-1306 and RMgm-1307: Os-TIR1 expressing parasites in which the the calcineurin gene was tagged with the AID-2xHA degron (CnA-AID) |
Transgene: Mutant parasite expressing a transgene| top of page | |||||||||||||||||||
| Type and details of transgene | |||||||||||||||||||
| Is the transgene Plasmodium derived | Transgene: not Plasmodium | ||||||||||||||||||
| Transgene name | (myc-tagged) Os-TIR1 protein | ||||||||||||||||||
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| Details of the genetic modification | |||||||||||||||||||
| Inducable system used | No | ||||||||||||||||||
| Additional remarks inducable system | |||||||||||||||||||
| Type of plasmid/construct | (Linear) plasmid double cross-over | ||||||||||||||||||
| PlasmoGEM (Sanger) construct/vector used | No | ||||||||||||||||||
| Modified PlasmoGEM construct/vector used | No | ||||||||||||||||||
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| Plasmid/construct sequence | |||||||||||||||||||
| Restriction sites to linearize plasmid | |||||||||||||||||||
| Selectable marker used to select the mutant parasite | No selectable marker | ||||||||||||||||||
| Promoter of the selectable marker | N.A. (see 'Additional remarks selection procedure') | ||||||||||||||||||
| Selection (positive) procedure | N.A. (see 'Additional remarks selection procedure') | ||||||||||||||||||
| Selection (negative) procedure | N.A. (see 'Additional remarks selection procedure') | ||||||||||||||||||
| Additional remarks genetic modification | The plasmid (pG230) used to generate the OsTIR1 expressing parent line pG230 (in P. berghei ANKA HP background; see RMgm-1304) was derived from the pG0148 vector (Sinha et al., 2014). The cfp gene in pG0148 was replaced by ostir1-9myc [amplified from BYP6743 plasmid (Yeast Genetic Resource Center, Osaka, Japan) with primer pair GU2102/GU2063] using the XhoI and SmaI restriction sites between the hsp70 (PBANKA_071190) promoter and p45/48 3′ UTR. The pG230 plasmid contains target regions for double crossover integration into the locus for the non-essential gene p230p and also a negative selection cassette to generate a marker-free line. To generate the pG204 construct the p45/48 3′ UTR was replaced by the p28 3′ UTR | ||||||||||||||||||
| Additional remarks selection procedure | The selectable marker cassette hdhfr-yfcu used to integrate the construct into the 230p locus with positive selection (pyrimethamine) has been removed by negative selection (5-FC) | ||||||||||||||||||
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| Other details transgene | |||||||||||||||||||
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| Promoter | |||||||||||||||||||
| Gene Model of Parasite | PBANKA_0711900 | ||||||||||||||||||
| Gene Model P. falciparum ortholog | PF3D7_0818900 | ||||||||||||||||||
| Gene product | heat shock protein 70 | ||||||||||||||||||
| Gene product: Alternative name | HSP70 | ||||||||||||||||||
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| 3'-UTR | |||||||||||||||||||
| Gene Model of Parasite | PBANKA_0514900 | ||||||||||||||||||
| Gene product | 28 kDa ookinete surface protein | ||||||||||||||||||
| Gene product: Alternative name | P28 | ||||||||||||||||||
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| Insertion/Replacement locus | |||||||||||||||||||
| Replacement / Insertion | Replacement locus | ||||||||||||||||||
| Gene Model of Parasite | PBANKA_0306000 | ||||||||||||||||||
| Gene product | 6-cysteine protein | ||||||||||||||||||
| Gene product: Alternative name | 230p | ||||||||||||||||||
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