SummaryRMgm-5253
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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) : 36928686 |
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 | RMgm-4900 |
Other information parent line | The mutant RMgm-4900 contains a DiCre expression cassette. The Dicre gene (the N-terminal Cre 59 (residues Thr19-Asn59) and C-terminal Cre 60 (Asn60-Asp343) portions of the Cre fused at their N-terminus to FKBP12 and FRB, respectively) is under control of the constitutive hsp70 promoter. The DiCre expression cassette is introduced into the silent p230p locus. In addition, it expresses mCherry and does not contain a drug-selectable marker |
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The mutant parasite was generated by | |
Name PI/Researcher | Loubens M, Silvie O |
Name Group/Department | Sorbonne Université, INSERM, CNRS, Centre d’Immunologie et des Maladies Infectieuses |
Name Institute | CIMI-Paris |
City | Paris |
Country | France |
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Name of the mutant parasite | |
RMgm number | RMgm-5253 |
Principal name | clampcKO |
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 | Not different from wild type |
Additional remarks phenotype | Mutant/mutation Phenotype Additional information Evidence is presented that: Other mutants |
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Details of the target gene | |||||||||||||||||||||||||||
Gene Model of Rodent Parasite | PBANKA_0514200 | ||||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_1030200 | ||||||||||||||||||||||||||
Gene product | claudin-like apicomplexan microneme protein, putative | ||||||||||||||||||||||||||
Gene product: Alternative name | CLAMP | ||||||||||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||||||||||
Short description of the mutation | a mutated clamp gene with two LoxN sites (up- and downstream of the gene) | ||||||||||||||||||||||||||
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 | unknown | ||||||||||||||||||||||||||
Selection (positive) procedure | pyrimethamine | ||||||||||||||||||||||||||
Selection (negative) procedure | No | ||||||||||||||||||||||||||
Additional remarks genetic modification | Two plasmids, P1 and P2, were generated for insertion of LoxN sites upstream and downstream of clamp, respectively. The P1 plasmid was assembled by inserting two homologous sequences, 5’HR1 and 5’HR2, both localized upstream 463 of clamp promoter region, in the pUpstream2Lox plasmid (Addgene #164573), which contains a GFP-2A-hDHFR cassette flanked by two LoxN sites. Both 5’HR1 (895 bp) and 5’HR2 (1014 bp) were amplified by PCR from WT PbANKA genomic DNA, and inserted into KpnI/XhoI and NheI sites, respectively, of the pUpstream2Lox plasmid. The P2 plasmid was assembled by inserting two homologous sequences, 3’HR1 and 3’HR2, corresponding to the end of clamp ORF and to clamp 3’UTR, respectively, in the pDownstream1Lox plasmid (Addgene #164574), which contains a GFP-2A-hDHFR cassette followed by a single LoxN site. Both 3’HR1 (862 bp) and 3’HR2 (999 bp) were amplified by PCR from WT PbANKA genomic DNA, and inserted into KpnI/XhoI and NheI sites, respectively, of the pDownstream1Lox plasmid. A triple Flag epitope tag was inserted in frame with clamp ORF immediately before the stop codon. In addition, a 559 bp fragment corresponding to the 3’ UTR sequence from P. yoelii clamp gene (PY17X_0515300) was inserted immediately downstream of 3’HR1, to allow proper gene expression and avoid spontaneous recombination with the 3’ UTR of P. berghei clamp, which was used as 3’HR2. Plasmids P1 and P2 were verified by Sanger DNA sequencing (Eurofins Genomics) and linearized with KpnI and NheI before transfection. Parental DiCre parasites were transfected with the P1 plasmid to generate clamp-P1 parasites, which were then exposed to rapamycin and transfected with the P2 plasmid to generate the final clampcKO line For the first transfection, schizonts purified from an overnight culture of PbDiCre blood stage parasites were transfected with 10 μg of linearized P1 plasmid using the AMAXA Nucleofector device (program U033), and immediately injected intravenously into the tail vein of SWISS mice. To permit the selection of resistant transgenic parasites, pyrimethamine (35 mg/L) and 5-flurocytosine (0.5 mg/ml) were added to the mouse drinking water, starting one day after transfection. The parasitaemia was monitored daily by flow cytometry and the mice sacrificed at a parasitaemia of 2-3%, allowing preparation of frozen parasite stocks and isolation of parasites for genomic DNA extraction. After drug selection, GFP+/mCherry+ clamp-P1 parasites were sorted by flow cytometry. Mice were injected intraperitoneally 491 with frozen 492 parasite stocks and monitored until the parasitaemia was between 0.1 and 1%. On the day of sorting, one drop of tail blood was collected in 1ml PBS and used for sorting of 100 iRBCs on a FACSAria II (Becton-Dickinson). Sorted parasites were recovered in 200 μl RPMI containing 20% FBS and injected intravenously into two mice. clamp-P1 parasites were exposed to a single dose of rapamycin to induce excision of the GFP-2A-hDHFR cassette. The resulting GFP-/mCherry+ clamp-P1 parasites, which retained a single LoxN site inserted upstream of clamp, were sorted by flow cytometry and amplified in mice. Rapamycin-exposed clamp-P1 parasites were then transfected with the P2 plasmid to generate the clampcKO line. GFP+/mCherry+ clampcKO parasites were sorted by flow cytometry and cloned by limiting dilution and injection into mice to generate the final population. | ||||||||||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||||||||||
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