Back to search resultsSummaryRMgm-5007
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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) | Not published (yet) |
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 that expresses GFP under the 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 | Balaban AE, Sinnis P |
Name Group/Department | Johns Hopkins Malaria Research Institute and Department of Molecular Microbiology & Immunology |
Name Institute | Johns Hopkins Bloomberg School of Public Health |
City | Baltimore |
Country | USA |
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Name of the mutant parasite | |
RMgm number | RMgm-5007 |
Principal name | ΔRep2 |
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 | Normal numbers of hemolymph (midgut) sporozoites. Reduced numbers of salivary gland sporozoites. |
Liver stage | Reduced infectivity of salivary gland sporozoites to infect hepatocytes |
Additional remarks phenotype | Mutant/mutation Published in bioRxiv preprint doi: https://doi.org/10.1101/2021.05.12.443759 Protein (function) ΔRep1 and ΔRep3 had comparable salivary gland infections to controls. In contrast, ΔRep2 and Scr parasites were inhibited in their capacity to enter the salivary glands, with fewer salivary gland associated sporozoites. We confirmed that ΔRep2 and Scr salivary gland associated sporozoites were inside the salivary glands, as opposed to attached to the external surface of the glands by live confocal imaging of salivary glands Additional information ΔRep1: lacking orange part Scr Repeat Sequence KLKQP PPPP PNNAPPRD GPPAAPPD PDQNPDPD PQPPQPQN PPNPPPNP From the Abstract: Other mutants |
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Details of the target gene | |||||||||||||||||||||||||||
Gene Model of Rodent Parasite | PBANKA_0403200 | ||||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_0304600 | ||||||||||||||||||||||||||
Gene product | circumsporozoite (CS) protein | ||||||||||||||||||||||||||
Gene product: Alternative name | CSP | ||||||||||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||||||||||
Short description of the mutation | Deletion of of internally located repeats of the central repeat sequence of CSP | ||||||||||||||||||||||||||
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 | ||||||||||||||||||||||||||
Promoter of the selectable marker | eef1a | ||||||||||||||||||||||||||
Selection (positive) procedure | pyrimethamine | ||||||||||||||||||||||||||
Selection (negative) procedure | No | ||||||||||||||||||||||||||
Additional remarks genetic modification | Mutant csp repeat region oligonucleotides were commercially synthesized by Genscript (Piscataway, NJ) and provided in the pUC57 plasmid. Repeat sequences were digested from the pUC57 plasmid using HindIII-HF and SexAI and ligated into an intermediate CSP vector in Bluescript SK, containing the KpnI to PacI sequence from the csp locus which includes 500 bp of 5’utr and the full length csp sequence. The csp sequence in this vector was engineered to have HindIII-HF and SexAI restriction sites flanking the repeats. Once the mutant repeat sequences were ligated into the intermediate CSP vector, a KpnI and PacI digest was performed and this fragment was ligated into the previously described transfection vector, pCSRep, altered to have an additional 1 kb of 3’ UTR in order to minimize correction of the repeat mutations. Recombinant parasites were generated by double homologous recombination in which the native csp locus was replaced by a mutant copy of csp with its control elements and an upstream selection cassette. pCSRep was digested with XhoI and KasI | ||||||||||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||||||||||
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Type and details of transgene | |||||||||||||||||||
Is the transgene Plasmodium derived | Transgene: not Plasmodium | ||||||||||||||||||
Transgene name | GFP (gfp-mu3) | ||||||||||||||||||
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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 | gfp (FACS) | ||||||||||||||||||
Promoter of the selectable marker | eef1a | ||||||||||||||||||
Selection (positive) procedure | FACS (flowsorting) | ||||||||||||||||||
Selection (negative) procedure | No | ||||||||||||||||||
Additional remarks genetic modification | |||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||
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Other details transgene | |||||||||||||||||||
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Promoter | |||||||||||||||||||
Gene Model of Parasite | PBANKA_1133300 | ||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_1357100 | ||||||||||||||||||
Gene product | elongation factor 1-alpha | ||||||||||||||||||
Gene product: Alternative name | eef1a | ||||||||||||||||||
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3'-UTR | |||||||||||||||||||
Gene Model of Parasite | PBANKA_0719300 | ||||||||||||||||||
Gene product | bifunctional dihydrofolate reductase-thymidylate synthase, putative | ||||||||||||||||||
Gene product: Alternative name | dhfr/ts | ||||||||||||||||||
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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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