SummaryRMgm-5220
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Successful modification | The parasite was generated by the genetic modification |
The mutant contains the following genetic modification(s) | Gene disruption, Introduction of a transgene |
Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 35804459 |
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 | Not applicable |
Other information parent line | |
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The mutant parasite was generated by | |
Name PI/Researcher | Yoshikawa Y, Ikadai H |
Name Group/Department | Laboratory of Veterinary Parasitology, School of Veterinary Medicine |
Name Institute | Kitasato University |
City | Towada, Aomori |
Country | Japan |
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Name of the mutant parasite | |
RMgm number | RMgm-5220 |
Principal name | PbBrca2‑KO |
Alternative name | |
Standardized name | |
Is the mutant parasite cloned after genetic modification | Yes |
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Phenotype | |
Asexual blood stage | (Slightly) reduced growth/multiplication of asexual blood-stage parasites in mice. |
Gametocyte/Gamete | (Slightly) reduced formation of female gametocytes |
Fertilization and ookinete | Reduced ookinete production. Mature ookinetes formed had a similar (light-microscope) morphology as wild-type ookinetes. |
Oocyst | Reduced oocyst production. Presence of degenerated, vacuolated oocysts. No sporozoite formation inside oocysts. |
Sporozoite | No salivary gland sporozoites |
Liver stage | No infection of mice by mosquito bite. |
Additional remarks phenotype | Mutant/mutation Other mutants |
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Details of the target gene | |||||||||||||||||||||||||
Gene Model of Rodent Parasite | PbANKA_1343400 | ||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_1328200 | ||||||||||||||||||||||||
Gene product | conserved Plasmodium protein, unknown function | ||||||||||||||||||||||||
Gene product: Alternative name | BRCA2; breast cancer susceptibility protein 2 | ||||||||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||||||||
Inducable system used | No | ||||||||||||||||||||||||
Additional remarks inducable system | |||||||||||||||||||||||||
Type of plasmid/construct used | (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 | |||||||||||||||||||||||||
Partial or complete disruption of the gene | Complete | ||||||||||||||||||||||||
Additional remarks partial/complete disruption | |||||||||||||||||||||||||
Selectable marker used to select the mutant parasite | puromycin-N-acetyltransferase (pac) | ||||||||||||||||||||||||
Promoter of the selectable marker | eef1a | ||||||||||||||||||||||||
Selection (positive) procedure | pyrimethamine | ||||||||||||||||||||||||
Selection (negative) procedure | No | ||||||||||||||||||||||||
Additional remarks genetic modification | See mutant RMgm-4180 for the transfection and selection method (selection with pac). PbBrca2 was knocked out by double-crossover HR technology using a pBluescript II-based plasmid containing a puromycin resistance gene inserted upstream (5′) of the PbEf1α gene and downstream (3′) of the PbDhfr-ts gene. A donor plasmid with 1000-bp homology arms was cloned upstream with the primers 5′-PbBrca2 forward (F) (5′-CGGGGTACCTTTTATTGTATCCCTATAA-3′), and 5′-PbBrca2 reverse (R) (5′-GGCGGGCCCCTTTATTTAATTATTAAGATTTTTTTGTTAC-3′); and cloned downstream with the primers 3′ PbBrca2 F (5′-CCGTCTAGATATCGTTTTAAGGTTGTTTC-3′), and 3′-PbBrca2 R (5′-CCGGCGGCCGCTATGTTGTATTGTTTGTTTT-3′). To clone the homology arms, the plasmids were treated with the restriction enzymes KpnI and ApaI for upstream of the PbBrca2 gene and XbaI and NotI for downstream of the PbBrca2 gene . Ten micrograms of donor vector was linearized using ScaI and used for transfection | ||||||||||||||||||||||||
Additional remarks selection procedure | See mutant RMgm-4180 for the transfection and selection method (selection with pac). | ||||||||||||||||||||||||
Primer information: Primers used for amplification of the target sequences
Primer information: Primers used for amplification of the target sequences
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Type and details of transgene | |||||||||||||||||||
Is the transgene Plasmodium derived | Transgene: not Plasmodium | ||||||||||||||||||
Transgene name | GFP | ||||||||||||||||||
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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 | pbdhfr | ||||||||||||||||||
Promoter of the selectable marker | pbdhfr | ||||||||||||||||||
Selection (positive) procedure | pyrimethamine | ||||||||||||||||||
Selection (negative) procedure | No | ||||||||||||||||||
Additional remarks genetic modification | To make a GFP expression cassette driven by the hsp70 promoter, the 1.2 kb upstream region and 1.0 kb downstream region of hsp70 were ligated to either side of the GFP coding sequence. To make a targeted insertion vector, this expression cassette was ligated into the downstream region of the pyrimethamine-resistant DHFR-TS gene. A transgenic GFP-expressing parasite clone (named WT:GFP) was obtained by inserting this construct downstream of the dhfr/ts locus by homologous recombination. | ||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||
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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_0711900 | ||||||||||||||||||
Gene product | heat shock protein 70 | ||||||||||||||||||
Gene product: Alternative name | HSP70 | ||||||||||||||||||
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Insertion/Replacement locus | |||||||||||||||||||
Replacement / Insertion | Insertion locus | ||||||||||||||||||
Gene Model of Parasite | PBANKA_0719300 | ||||||||||||||||||
Gene product | bifunctional dihydrofolate reductase-thymidylate synthase, putative | ||||||||||||||||||
Gene product: Alternative name | dhfr/ts | ||||||||||||||||||
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