RMgmDB - Rodent Malaria genetically modified Parasites
SummaryRMgm-1307
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*RMgm-1307| Successful modification | The parasite was generated by the genetic modification |
| The mutant contains the following genetic modification(s) | Gene mutation, 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 | RMgm-1304 |
| Other information parent line | The mutant (pG230) expresses (myc-tagged) Os-TIR1 protein. The ostir1 gene is under control of the strong and constitutive hsp70 promoter and the 3'UTR of p48/45. The transgene is integrated into the silent 230p locus. The mutant does not express a drug-selectable marker. This mutant ('transgenic acceptor lines') is 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. |
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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-1307 |
| Principal name | PbCnA-AID |
| Alternative name | |
| Standardized name | |
| Is the mutant parasite cloned after genetic modification | Yes |
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| Phenotype | |
| Asexual blood stage | Evidence is presented for a reduced invasion of red blood cells by merozoites after depletion of Calcineurin in blood stages |
| Gametocyte/Gamete | Evidence is presented for a reduced (50%) male gamete formation after depletion of Calcineurin in mature gametocytes. |
| Fertilization and ookinete | Evidence is presented for a reduced (90%) ookinete formation after depletion of Calcineurin in mature gametocytes. |
| Oocyst | Evidence is presented for a reduced oocyst production after depletion of Calcineurin in ookinetes. |
| Sporozoite | Not tested |
| Liver stage | Evidence is presented for a reduced (46%) liver stage development after depletion of Calcineurin in sporozoites. |
| Additional remarks phenotype | Mutant/mutation Protein Schizonts, gametocytes, or sporozoites were incubated with 500 mM auxin for different time periods. Auxin stimulated efficient degradation of CnA-AID fusions at each of these stages within only 45 min. Evidence is presented for a reduced invasion of red blood cells by merozoites after depletion of Calcineurin in blood stages. Evidence is presented for a reduced (50%) male gamete formation after depletion of Calcineurin in mature gametocytes. Evidence is presented for a reduced (90%) ookinete formation after depletion of Calcineurin in mature gametocytes. Evidence is presented for a reduced oocyst production after depletion of Calcineurin in ookinetes. Evidence is presented for a reduced (46%) liver stage development after depletion of Calcineurin in sporozoites. The system of conditional degradation 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. In addition to Calcineurin, the study reports the use of this system for conditional degradation of CDPK1 (PBANKA_0314200), PPKL (PBANKA_1329500) and DOZI (PBANKA_1217700) in blood stages and gametocytes. Other mutants: |
Mutated: Mutant parasite with a mutated gene| top of page | |||||||||||||||||||||||||||
| Details of the target gene | |||||||||||||||||||||||||||
| Gene Model of Rodent Parasite | PBANKA_1227400 | ||||||||||||||||||||||||||
| Gene Model P. falciparum ortholog | PF3D7_0802800 | ||||||||||||||||||||||||||
| Gene product | serine/threonine protein phosphatase 2B catalytic subunit A | ||||||||||||||||||||||||||
| Gene product: Alternative name | CNA; CnA; Calcineurin | ||||||||||||||||||||||||||
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| Details of the genetic modification | |||||||||||||||||||||||||||
| Short description of the mutation | C-terminal tagging of Calcineurin with the AID-2xHA degron (CnA-AID) | ||||||||||||||||||||||||||
| Inducable system used | No | ||||||||||||||||||||||||||
| Short description of the conditional mutagenesis | Not available | ||||||||||||||||||||||||||
| Additional remarks inducable system | |||||||||||||||||||||||||||
| Type of plasmid/construct | (Linear) plasmid single 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 | eef1a | ||||||||||||||||||||||||||
| Selection (positive) procedure | pyrimethamine | ||||||||||||||||||||||||||
| Selection (negative) procedure | No | ||||||||||||||||||||||||||
| Additional remarks genetic modification | The aid degron tagging plasmid (pG390) was generated by amplification of the degron CDS from BYP6739 (Yeast Genetic Resource Center, Osaka, Japan) using primer pair BamHIGU2065/BamHI-GU2066, followed by ligation into the PL31-HA plasmid (Philip et al., 2012). A GFP expressing version of the degron plasmid (pG362) was also generated by replacing the drug selection cassette (tgdhfr promoter-tgdhfr) with the bidirectional pbee1fα promoter driving both gfp and hdhfr expression. The pG362 plasmid was generated as detailed below. The PL0035 vector (Philip et al., 2012) was digested with NheI and re-ligated to remove the yfcu pbdhfr- 3′ UTR (pL0035a). Part of pbee1fα promoter was amplified by (SacII)-GU2578/(SpeI)-GU2579 from P. berghei genomic DNA, digested with SacII and SpeI, and ligated into pL0035a (also digested with SacII/SpeI) resulting in the reconstitution of the complete bidirectional pbee1fα promoter driving expression of the hdhfr selectable marker (pL0035b). Primer GU2578 also introduced a KpnI site upstream of the SacII site at the 5’end of pbeef1α promoter. The 3’UTR from the P. berghei gene cam (PBANKA_101060) was amplified with (PstI)-GU2580/(KpnI)-GU2581 from genomic DNA, digested with KpnI and PstI and ligated into pL0035b (pL0035c). The gfp gene was amplified from pL0031-pbppkl vector (Philip et al., 2012) using (KpnI)-GU2598/(KpnI)-GU2599, digested with KpnI and ligated into pL0035c (also digested with KpnI) between the cam-3’UTR and pbee1fα promoter. Finally, the cassette was digested with PstI and NheI and ligated into the aid degron expressing plasmid pG390, where the drug selection cassette (tgdhfr promoter-tgdhfr) had been removed with a PstI/NheI digest. This resulted in the aid-p48/45 3′ UTR-pbdhfr 3′ UTR -hdhfr- ee1fα-gfp-calmodulin 3′ UTR plasmid (pG362). The two additional fluorescence marker expressing plasmids were generated by replacing the gfp ORF with either cfp (pG363) or mcherry (pG364). The pbcna-aid-2xha tagged construct was generated by cloning 1 kb of the pbcna 3’ end up to, but not including the stop codon, into pG390 and pG362 plasmids, amplified by using primers (SacII)-GU2603 and (BamHI)-GU2604. | ||||||||||||||||||||||||||
| Additional remarks selection procedure | |||||||||||||||||||||||||||
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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 | ||||||||||||||||||
| Plasmid/construct map | |||||||||||||||||||
| 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_1359600 | ||||||||||||||||||
| Gene product | transmission blocking target antigen precursor 6-cysteine protein | ||||||||||||||||||
| Gene product: Alternative name | P48/45 | ||||||||||||||||||
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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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