Summary

RMgm-4599
Malaria parasiteP. berghei
Genotype
Genetic modification not successful
DisruptedGene model (rodent): PBANKA_1441400; Gene model (P.falciparum): PF3D7_1226600; Gene product: proliferating cell nuclear antigen 2 (PCNA2)
PhenotypeNo phenotype has been described
Last modified: 18 March 2019, 15:32
  *RMgm-4599
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 3
Reference (PubMed-PMID number) Reference 1 (PMID number) : 30865364
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/ResearcherPradhan S, Dhar SK
Name Group/DepartmentSpecial Centre for Molecular Medicine
Name InstituteJawaharlal Nehru University
CityNew Delhi
CountryIndia

  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1441400
Gene Model P. falciparum ortholog PF3D7_1226600
Gene productproliferating cell nuclear antigen 2
Gene product: Alternative namePCNA2
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 markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationTo pinpoint the significance of PCNA2 in the parasite lifecycle, three consecutive attempts were made to knock out the gene by double homologous recombination at the wild‐type pcna2 locus. However, after resistant parasites were detected, post selection using pyrimethamine drug pressure and insertion was confirmed by diagnostic PCR, attempts to obtain the clonal population (clones) of pcna2 KO parasites were unsuccessful.

Proliferating cell nuclear antigen (PCNA) proteins play a role in DNA replication and DNA repair pathways. The genome of Plasmodium parasites encodes for two PCNA proteins. P. falciparum PCNA1 (PfPCNA1) has been previously implicated in DNA replication as well as DNA repair, whereas role of P. falciparum PCNA2 (PfPCNA2) has been assigned to DNA repair, during the asexual stage of the parasite.

See also RMgm-3947 for evidence of knock-out of the PCNA2 gene (and with normal growth of blood stages)

In order to generate the PCNA2 knockout‐targeting construct, a920 bp fragment was amplified from the 5′UTR region of PbPCNA2 and a 609 bp from the 3′UTR of the gene. Both UTRs were cloned in knockout vector flaking the GFP and DHFR cassette.

For generating the PbPCNA2‐GFP tagging construct, 1898 bp fragment, containing 999 bp of 5′UTR and full length PbPCNA2 coding sequence excluding the stop codon, was amplified from the parasite genomic DNA.
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