RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5097
Malaria parasiteP. berghei
Genotype
Genetic modification not successful
DisruptedGene model (rodent): PBANKA_1033200; Gene model (P.falciparum): PF3D7_1409300; Gene product: DNA damage-inducible protein 1, putative (DDI1)
PhenotypeNo phenotype has been described
Last modified: 4 November 2021, 12:42
  *RMgm-5097
Successful modificationThe gene/parasite could not be changed/generated by the genetic modification.
The following genetic modifications were attempted Gene disruption
Number of attempts to introduce the genetic modification Unknown
Reference (PubMed-PMID number) Not published (yet)
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone Not applicable
Other information parent line
Attempts to generate the mutant parasite were performed by
Name PI/ResearcherTanneru N, Sijwali PS
Name Group/DepartmentCSIR-Centre for Cellular and Molecular Biology
Name InstituteCSIR-Centre for Cellular and Molecular Biology
CityHyderabad
CountryIndia

  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1033200
Gene Model P. falciparum ortholog PF3D7_1409300
Gene productDNA damage-inducible protein 1, putative
Gene product: Alternative nameDDI1
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct used(Linear) plasmid double cross-over
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid
Partial or complete disruption of the geneComplete
Additional remarks partial/complete disruption
Selectable marker used to select the mutant parasitehdhfr
Promoter of the selectable markerunknown
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationPublished in: bioRxiv preprint doi: https://doi.org/10.1101/2021.10.29.466443.

The unsuccessful attempts to disrupt this gene indicate an essential function during asexual blood stage growth.

Plasmodium DDI1 is expressed in all major parasite stages.

PF3D7_1409300 DDI1 was first discovered as one of the over expressed proteins upon treatment of Saccharomyces cerevisiae with DNA damaging agents, hence, it was named as the DNA damage inducible 1 protein (DDI1). Deletion analysis demonstrated that both UBL and RVP domains of S. cerevisiae DDI1 (ScDDI1) are necessary for inhibition of protein secretion. DDI1 proteins are conserved in eukaryotes and involved in a variety of cellular processes, including proteasomal degradation of specific proteins and DNA-protein crosslink repair. All DDI1 proteins contain ubiquitin-like (UBL) and retroviral aspartyl protease (RVP) domains, and some also contain ubiquitin-associated (UBA) domain, which mediate distinct activities of these proteins. The DDI1 proteins of Plasmodium and other Apicomplexan parasites vary in domain architecture, share UBL and VP domains, and the majority of proteins contain the UBA domain.

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
Primer information: Primers used for amplification of the target sequences  Click to view information
Primer information: Primers used for amplification of the target sequences  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
Sequence Primer 3
Additional information primer 3
Sequence Primer 4
Additional information primer 4
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6