Back to search resultsSummaryRMgm-5258
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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) : 36445080 |
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 2.34 |
Other information parent line | P. berghei ANKA 2.34 is a cloned, gametocyte producer line of the ANKA strain (PubMed: PMID: 15137943). |
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The mutant parasite was generated by | |
Name PI/Researcher | Hussain T, Beck JR |
Name Group/Department | Department of Biomedical Sciences |
Name Institute | Iowa State University |
City | Ames, IA |
Country | USA |
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Name of the mutant parasite | |
RMgm number | RMgm-5258 |
Principal name | EXP2-glmS |
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 different from wild type |
Fertilization and ookinete | Not different from wild type |
Oocyst | Not different from wild type |
Sporozoite | Not different from wild type |
Liver stage | EXP2(glmS) and EXP2(ctrl) sporozoites were isolated by mosquito dissection and allowed to infect Huh7 cells and develop in the presence or absence of 0.3 mM, 0.5 mM or 1 mM GlcN for 48 hours before cells were fixed and processed for immunofluorescence. In untreated EXP2glmS cultures, EXP2 was readily observed at the parasite periphery where it largely co-localized with the liver-stage integral PVM marker UIS4. In contrast, EXP2glmS parasites grown with GlcN displayed a striking reduction in EXP2 intensity by ⁓73-87% while UIS4 levels were not affected. Importantly, EXP2 levels in the control line were not similarly altered by GlcN, indicating the reduction observed in the EXP2glmS parasites was a result of the ribozyme sequence in the exp2 ’ UTR and not an indirect effect of GlcN treatment. Notably, introduction of 0.5 mM GlcN at the time EXP2glmS sporozoites were added to Huh7 host cells (0 hpi) had no impact on invasion but reduced EXP2 levels by ~80% at 48 hpi when maintained in cultures. Moreover, similar EXP2 knockdown was achieved when GlcN was added after sporozoite invasion had occurred, clearly indicating successful EXP2 depletion after host cell entry without confounding effects from perturbed invasion. Evidence is presented that: - EXP2 knockdown reduces liver-stage parasite size and severely impacts intrahepatic parasite development - EXP2 knockdown decreases expression of the liver-stage developmental marker LISP2 |
Additional remarks phenotype | Mutant/mutation EXP2(glmS) and EXP2(ctrl) sporozoites were isolated by mosquito dissection and allowed to infect Huh7 cells and develop in the presence or absence of 0.3 mM, 0.5 mM or 1 mM GlcN for 48 hours before cells were fixed and processed for immunofluorescence. In untreated EXP2glmS cultures, EXP2 was readily observed at the parasite periphery where it largely co-localized with the liver-stage integral PVM marker UIS4. In contrast, EXP2glmS parasites grown with GlcN displayed a striking reduction in EXP2 intensity by ⁓73-87% while UIS4 levels were not affected. Importantly, EXP2 levels in the control line were not similarly altered by GlcN, indicating the reduction observed in the EXP2glmS parasites was a result of the ribozyme sequence in the exp2 ’ UTR and not an indirect effect of GlcN treatment. Notably, introduction of 0.5 mM GlcN at the time EXP2glmS sporozoites were added to Huh7 host cells (0 hpi) had no impact on invasion but reduced EXP2 levels by ~80% at 48 hpi when maintained in cultures. Moreover, similar EXP2 knockdown was achieved when GlcN was added after sporozoite invasion had occurred, clearly indicating successful EXP2 depletion after host cell entry without confounding effects from perturbed invasion. Evidence is presented that: Additional information To evaluate whether glmS can control P. berghei EXP2 levels, we first introduced an mRuby3-3xHA tag followed by the glmS sequence at the endogenous P. berghei exp2 locus by double homologous recombination (see RMgm-5257). The donor plasmid also contains a cassette for expression of GFP driven by the constitutive Pbhsp70 promoter. Addition of GlcN to ex vivo cultures for 18 hours substantially reduced EXP2 levels relative to untreated controls in the glmS line but did not reduce EXP2 levels in the control parasites lacking the ribozyme. Specifically, treatment with 0.5 mM GlcN reduced EXP2 levels by 23±12% while higher concentrations achieved ~50% knockdown (57±12% or 46±12% at 1 or 2 mM GlcN, respectively), similar to previous reports. To determine the suitability of the glmS system for protein knockdown in cultured liver stage parasites, we first evaluated whether GlcN levels sufficient to mediate knockdown in blood stage parasites had any effect on proliferation of Huh7 or HepG2 hepatoma cell lines commonly used to cultivate the P. berghei liver stage. We assessed viability of hepatoma cells by quantifying metabolic activity using a resazurin assay and found that proliferation of both Huh7 and HepG2 cell types was not reduced at GlcN concentrations up to 1 mM. These results indicate GlcN can be supplied in hepatoma cultures at concentrations suitable for maximal blood-stage knockdown without host cell toxicity. As both HepG2 and Huh7 were similarly tolerant to GlcN levels, Huh7 cells were chosen for subsequent experiments due to their superior qualities for imaging intracellular parasites. To evaluate whether the glmS ribozyme can mediate control of parasite protein levels in the liver stage, we next generated a line bearing a 3xFLAG epitope tag followed by the glmS sequence at the endogenous P. berghei exp2 locus. This plasmid also contains a downstream cassette expressing nanoluciferase (NanoLuc) under the control of the Pbhsp70 promoter to provide a sensitive proxy for monitoring parasite development. Clonal lines were derived and designated EXP2(glmS). Other mutants
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Details of the target gene | |||||||||||||||||||||||||||
Gene Model of Rodent Parasite | PBANKA_1334300 | ||||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_1471100 | ||||||||||||||||||||||||||
Gene product | exported protein 2 | ||||||||||||||||||||||||||
Gene product: Alternative name | EXP2 | ||||||||||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||||||||||
Short description of the mutation | Exp2, C-terminally tagged with the glucosamine-activated glmS ribozyme, 3xHA and nanoluciferase | ||||||||||||||||||||||||||
Inducable system used | No | ||||||||||||||||||||||||||
Short description of the conditional mutagenesis | Not available | ||||||||||||||||||||||||||
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 | hdhfr/yfcu | ||||||||||||||||||||||||||
Promoter of the selectable marker | eef1a | ||||||||||||||||||||||||||
Selection (positive) procedure | pyrimethamine | ||||||||||||||||||||||||||
Selection (negative) procedure | No | ||||||||||||||||||||||||||
Additional remarks genetic modification | |||||||||||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||||||||||
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