SummaryRMgm-5098
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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) : 36657610 |
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 | Tanneru N, Sijwali PS |
Name Group/Department | CSIR-Centre for Cellular and Molecular Biology |
Name Institute | CSIR-Centre for Cellular and Molecular Biology |
City | Hyderabad |
Country | India |
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Name of the mutant parasite | |
RMgm number | RMgm-5098 |
Principal name | PbKD |
Alternative name | |
Standardized name | |
Is the mutant parasite cloned after genetic modification | Yes |
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Phenotype | |
Asexual blood stage | PbKD parasites grew similar to wild type parasites in the presence of trimethoprim, but showed drastically reduced growth in the absence of trimethoprim and eventually disappeared. In the presence of trimethoprim, PbKD-infected mice had to be euthanized, whereas in the absence of trimethoprim, the parasitemia barely reached to 0.5% and the infection was self-limiting. Withdrawal of trimethoprim from PbKD-infected mice about 5% parasitemia also resulted in complete clearance of parasites. |
Gametocyte/Gamete | Not tested |
Fertilization and ookinete | Not tested |
Oocyst | Not tested |
Sporozoite | Not tested |
Liver stage | Not tested |
Additional remarks phenotype | Mutant/mutation Additional information Other mutants |
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Details of the target gene | |||||||||||||||||||||||||||
Gene Model of Rodent Parasite | PBANKA_1033200 | ||||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_1409300 | ||||||||||||||||||||||||||
Gene product | DNA damage-inducible protein 1, putative | ||||||||||||||||||||||||||
Gene product: Alternative name | DDI1 | ||||||||||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||||||||||
Short description of the mutation | a fusion of P. falciparum DDIMyc with HA-tagged mutant E. coli DHFR (cDDHA) | ||||||||||||||||||||||||||
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 | The PbDDI1 gene was targeted for knock-out and knock-down using double cross-over homologous recombination approach. The 5'-UTR (flank 1) and 3’-UTR (flank 2) of PbDDI1 were amplified from P. berghei genomic DNA using PbDdi1- Fl1F/ PbDdi1-Fl1R and PbDdi1-Fl2F/PbDdi1-Fl2R primer sets, respectively. The flank 1 and flank 2 were cloned into the HB-DJ1KO plasmid at NotI-KpnI and AvrII-KasI sites, respectively, to obtain HB-PbDDI-(FL1+FL2) plasmid. The GFP coding sequence was excised from pGTGFPbsc with KpnI-XhoI and subcloned into the similarly digested HB-PbDDI-(FL1+FL2) to obtain HB-pbDDIKO plasmid. For construction of knock-down plasmid, the PfDDIMyc coding sequence was amplified from the P. falciparum genomic DNA using DDiexp-F/DDimyc Rep-R primers, digested with KpnI-XhoI and subcloned into the similarly digested HB-PbDDIKO plasmid in place of GFP to obtain HB-PfDDIKI plasmid. The E. coli mutant DHFR coding sequence with HA-tag (cDDHA) was amplified from the pPM2GDBvm plasmid (a kind gift from Dr. Praveen Balabaskaran Nina) using cDD-F/cDD-R primers, and cloned into the pGT-GFPbsc plasmid at KpnI/XhoI sites to obtain pGT-cDDHA plasmid. The PfDDIMyc coding sequence was amplified from HB-PfDDIKI plasmid using the PfDdi-reF/PfDdi-cDDR primers and cloned into the pGT-cDDHA plasmid at BglII-BamHI site to obtain the pGT-PfDDI-cDDHA plasmid. The pGT-PfDDIMyc/cDDHA plasmid was digested with BglII-XhoI to release the PfDDIMyc/cDDHA insert, which was cloned into the similarly digested HB-DDKI plasmid to obtain HB-pbDDIKD vector. The HB-pbDDIKO and HB-pbDDIKD plasmids were purified using the NucleoBond® Xtra Midi plasmid DNA purification kit (MACHEREY-NAGEL), linearized with NotI-KasI, gel purified to obtain pbDDIKO and pbDDIKD transfection constructs, and used for transfection of P. berghei. | ||||||||||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||||||||||
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