SummaryRMgm-1102
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Successful modification | The 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) : 25043043 |
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 cl15cy1 |
Other information parent line | A reference wild type clone from the ANKA strain of P. berghei (PubMed: PMID: 17406255). |
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The mutant parasite was generated by | |
Name PI/Researcher | Elsworth, B.; de Koning-Ward, T.F. |
Name Group/Department | Deakin University |
Name Institute | Deakin University |
City | Waurn Ponds |
Country | Australia |
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Name of the mutant parasite | |
RMgm number | RMgm-1102 |
Principal name | Pbi101KD |
Alternative name | |
Standardized name | |
Is the mutant parasite cloned after genetic modification | Yes |
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Phenotype | |
Asexual blood stage | Strongly reduced growth (parasitemia) in mice treated with ATc. Evidence is presented that treated parasites are able to invade and develop into ring forms but are affected in the development of trophozoite and schizont stage. Evidence is presented that export of proteins into the host RBC is affected in ATc-treated Pbi101KD 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 |
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Details of the target gene | |||||||||||||||||||||||||||
Gene Model of Rodent Parasite | PBANKA_0931200 | ||||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_1116800 | ||||||||||||||||||||||||||
Gene product | heat shock protein 101 | chaperone protein ClpB2 | ||||||||||||||||||||||||||
Gene product: Alternative name | HSP101 | ||||||||||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||||||||||
Short description of the mutation | The hsp101 promoter modified to contain a tet-inducible promoter and TRAD4 activating domain | ||||||||||||||||||||||||||
Inducable system used | TET-based (TRAD4) | ||||||||||||||||||||||||||
Short description of the conditional mutagenesis | Downregulation of HSP101 expression by addition of the tetracycline derivative ATc | ||||||||||||||||||||||||||
Additional remarks inducable system |
Conditional expression system for stage-specific, tetracycline-dependent (ATc) gene regulation. Tetracycline derivative anhydrotetracycline(ATc). For tetracyclin (ATc)-dependent gene regulation the hsp101 locus was mutated using construct pPRF-TRAD4-Tet07-HAPRFhDHFR (RMgm-797) for introducing a tet-inducible promoter and TRAD4 activating domain | ||||||||||||||||||||||||||
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 | eef1a | ||||||||||||||||||||||||||
Selection (positive) procedure | pyrimethamine | ||||||||||||||||||||||||||
Selection (negative) procedure | No | ||||||||||||||||||||||||||
Additional remarks genetic modification | For tetracyclin (ATc)-dependent gene regulation the hsp101 locus was mutated using construct pPRF-TRAD4-Tet07-HAPRFhDHFR (RMgm-797) for introducing a tet-inducible promoter and TRAD4 activating domain The constructpTg-ranTRAD4-iHSP101 was used to generate the P. berghei i101 KD. This plasmid was based on pPRF-TRAD4-Tet07-HAPRFhDHFR but modified to include a BsiWI restriction site downstream of the profilin coding sequence and a BssHII restriction enzyme site between the profilin 5' untranslated region and the TRAD4 sequence. This enabled cloning of the first 1.7 kilobases (kb) of theHSP101 coding sequence (PbANKA_09312;amplified with DO390F, 5'-caccctgcagATGGTACGGAACATTGCTAAAAATT-3', and DO414R, 5'-gtatcgtacgccatggCTATAACTCTTGGTTTACCCG-3', the latter containing an internal NcoI site) into the PstI and BsiWI cloning sites and 0.85 kb of the HSP101 5' untranslated region (amplified using oligonucleotides DO392F, 5'-gtaccatggCGTACGGTATGCAATTGCTCTTAATGCATTTGC-3', and DO394R, 5'-tatgcgcgc TTTCTACTAAATTTATAGTAAATATAGATATA-3') into the NcoI and BssHII sites. Before transfection, DNA was linearized with NcoI. | ||||||||||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||||||||||
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