RMgmDB - Rodent Malaria genetically modified Parasites
SummaryRMgm-5042
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*RMgm-5042| 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) : 34273314 |
| 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 | Yamauchi M, Mita T |
| Name Group/Department | Department of Tropical Medicine and Parasitology, Faculty of Medicine |
| Name Institute | Juntendo University |
| City | Tokyo |
| Country | Japan |
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| Name of the mutant parasite | |
| RMgm number | RMgm-5042 |
| Principal name | PbDHPS-A394G |
| Alternative name | |
| Standardized name | |
| Is the mutant parasite cloned after genetic modification | Yes |
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| Phenotype | |
| Asexual blood stage | Normal growth rate of asexual blood stages, normal gametocyte production, reduced sensitivity to sulfadoxine. |
| Gametocyte/Gamete | Normal growth rate of asexual blood stages, normal gametocyte production, reduced sensitivity to sulfadoxine of asexual blood stages. |
| Fertilization and ookinete | Not tested |
| Oocyst | Not different from wild type |
| Sporozoite | Not tested |
| Liver stage | Not tested |
| Additional remarks phenotype | Mutant/mutation Additional information Sulfadoxine inhibits the parasite's dihydropteroate synthase (DHPS), an essential coenzyme in the folate synthesis pathway, which acts synergistically with pyrimethamine, an inhibitor of dihydrofolate reductase (DHFR). To assess the fitness costs imposed by sulfadoxine resistance alone, we generated a transgenic rodent malaria parasite, P. berghei clone harboring an A394G mutation in dhps (PbDHPS-A394G), corresponding to the causative mutation for sulfadoxine resistance in P. falciparum (PfDHPS-A437G). |
Mutated: Mutant parasite with a mutated gene| top of page | |||||||||||||||||||||||||||
| Details of the target gene | |||||||||||||||||||||||||||
| Gene Model of Rodent Parasite | PBANKA_1426700 | ||||||||||||||||||||||||||
| Gene Model P. falciparum ortholog | PF3D7_0810800 | ||||||||||||||||||||||||||
| Gene product | hydroxymethyldihydropterin pyrophosphokinase-dihydropteroate synthase putative | ||||||||||||||||||||||||||
| Gene product: Alternative name | PPPK-DHPS, DHPS | ||||||||||||||||||||||||||
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| Details of the genetic modification | |||||||||||||||||||||||||||
| Short description of the mutation | a mutated DHPS with amino acid change A394G | ||||||||||||||||||||||||||
| Inducable system used | No | ||||||||||||||||||||||||||
| Short description of the conditional mutagenesis | Not available | ||||||||||||||||||||||||||
| Additional remarks inducable system | |||||||||||||||||||||||||||
| Type of plasmid/construct | CRISPR/Cas9 construct: integration through double strand break repair | ||||||||||||||||||||||||||
| 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 PbDHPS-A394G mutant was generated by genome editing using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (CRISPR/Cas9). The pYC plasmid was originally generated for the genome editing of P. yoelii and was generously gifted by Dr. Yuan (Zhang et al., 2014). The U6 promoter of P. yoelii in the pYC plasmid was replaced by the corresponding gene of P. berghei, resulting in the pBC-1 plasmid. As a donor, a fragment encoding the A394G mutation and silent mutations of P. berghei dhps (PbDHPS) (PBANKA_1426700) was synthesized by GENEWIZ® and used as a template for PCR using a pair of primers, pbdhps-forward and reverse. The resultant PCR fragment was ligated into pBC-1 using the In-Fusion HD Cloning Kit (Takara Bio Inc.) after digestion with HindIII/AflII (pBC-2). A potential PAM site was searched using CHOPCHOP (http://chopchop.cbu.uib.no) and double-stranded DNA coding for gRNA was ligated into a BsmBI site in pBC-2 using In-Fusion ligation. The final plasmid (pBC-pbdhps) was checked with Sanger sequencing. | ||||||||||||||||||||||||||
| Additional remarks selection procedure | |||||||||||||||||||||||||||
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