SummaryRMgm-1506
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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) : 27427910 |
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 | Kehrer J; Mair GR |
Name Group/Department | Integrative Parasitology, Center for Infectious Diseases |
Name Institute | University of Heidelberg Medical School |
City | Heidelberg |
Country | Germany |
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Name of the mutant parasite | |
RMgm number | RMgm-1506 |
Principal name | patccp.PP |
Alternative name | |
Standardized name | |
Is the mutant parasite cloned after genetic modification | Yes |
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Phenotype | |
Asexual blood stage | Not tested |
Gametocyte/Gamete | Not tested |
Fertilization and ookinete | Not tested |
Oocyst | Normal oocyst formation |
Sporozoite | Normal oocyst formation. Reduced numbers of salivary gland sporozoites. The large number of midgut sporozoites in the patccp.PP mutant did not result into highly infected salivary glands, which was not due to a failure of the mutant to exit the oocysts and enter the hemolymph. patccp.PP sporozoites are defective in adhesion and gliding. Reduced infectivity of sporozoites as determined by less efficient establishment of blood infections and prolonged prepatent period after mosquito bite and i.v. infections of mice. |
Liver stage | Reduced infectivity of sporozoites as determined by less efficient establishment of blood infections and prolonged prepatent period after mosquito bite and i.v. infections of mice. |
Additional remarks phenotype | Mutant/mutation A mutant lacking expression of PAT (Δpat; RMgm-1500); A mutant in which the endogeneous pat gene is replaced with the pat gene of P. falciparum 3D7: Δpat;pat(PF3D7)::mcherry; RMgm-1501) A mutant expressing GFP-tagged PAT and mCherry-tagged G377 (pat::gfp;g377:mcherry; RMgm-1503): A mutant lacking expression of PAT and expressing mCherry-tagged G377 (PBANKA_1463000) and a mutant lacking expression of PAT and expressing mCherry-tagged PPLP2 (PBANKA_1463000) A mutant expressing a GFP-tagged version of TRAP (ss::gfp::trap; RMgm-1505) A 'promoter-swap' mutant expressing PAT under the promoter of the ccp1/lap2 promoter (PBANKA_1300700)(patccp.PP; RMgm-1506) |
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Details of the target gene | |||||||||||||||||||||||||||
Gene Model of Rodent Parasite | PBANKA_0303900 | ||||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_0206200 | ||||||||||||||||||||||||||
Gene product | pantothenate transporter | ||||||||||||||||||||||||||
Gene product: Alternative name | PAT | ||||||||||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||||||||||
Short description of the mutation | 'promoter-swap' mutant; the promoter of PAT replaced by the promoter of PBANKA_1300700 (ccp1/lap) | ||||||||||||||||||||||||||
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 | tgdhfr | ||||||||||||||||||||||||||
Promoter of the selectable marker | pbdhfr | ||||||||||||||||||||||||||
Selection (positive) procedure | pyrimethamine | ||||||||||||||||||||||||||
Selection (negative) procedure | No | ||||||||||||||||||||||||||
Additional remarks genetic modification | To change time and sex-specific PAT expression a generic plasmid was first generated that contains the following parts: 348 bp of 5’ UTR of PBANKA_030390 (PCR-amplified with primers 1144 and 1145), the Toxoplasma dhfr/ts selection cassette, followed by 543 bp of the 5’end of the ORF of pat (PCR-amplified with primer 1148 and 149). The promoter region from ccp (1273 bp) was then ligated immediately 5’ to the PAT ORF using the EcoRV and NdeI restriction sites from pL1260 [46] by AleI and NdeI digestion. The plasmid was digested overnight with KpnI and KspI prior to transfection, precipitated and resuspended in AMAXA solution for nucleofector transfection of P. berghei schizonts; transfection, selection of mutant (drug-resistant) parasites and limiting dilution of clonal lines was as described. | ||||||||||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||||||||||
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