RMgmDB - Rodent Malaria genetically modified Parasites


Malaria parasiteP. berghei
Genetic modification not successful
DisruptedGene model (rodent): PBANKA_0715600; Gene model (P.falciparum): PF3D7_0413600; Gene product: 26S protease regulatory subunit 6B, putative (Rpt3; PbRpt3)
PhenotypeNo phenotype has been described
Last modified: 5 January 2024, 18:43
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 ≥ 5
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/ResearcherLaine C, Khalife J, Pierrot C
Name Group/DepartmentCIIL - Center for Infection and Immunity of Lille
Name InstituteUniv. Lille, CNRS, Inserm, CHU Lille, Institute Pasteur de Lille

  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0715600
Gene Model P. falciparum ortholog PF3D7_0413600
Gene product26S protease regulatory subunit 6B, putative
Gene product: Alternative nameRpt3; PbRpt3
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 usedYes
Name of PlasmoGEM construct/vectorPbGEM-022521
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/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe unsuccessful attempts to disrupt this gene indicate an essential function during asexual blood stage growth/multiplication.

To further study whether the lack of PbRpt3 could affect P. berghei blood stages life cycle, we attempted to generate knock-out (KO) lines by taking advantage of the available PlasmoGEM plasmid (PbGEM-022521; 93 % deletion of the PbRpt3 gene). Transfections were performed in two different strains of P. berghei ANKA (pG230 and PbGFP) with this KOgem construct. After selection with pyrimethamine, resistant parasites were genotyped by PCR. A total of 4 independent transfections were performed, allowing the detection of parasites 7 to 9 days after transfectionon under pyrimethamine selection. In two of these transfections, the diagnosis genotyping showed either no integration of the resistance cassette at the PbRpt3 locus, or an integration only of the 5’ side. A[er two other transfections, we detected both the integration of the construct on 5’ and 3’ sides, and the presence of the wild-type PbRpt3 gene. In order to increase the ratio of transgenic vs wild-type parasites, we first performed up to 7 successive passages of the parasites in mice under pyrimethamine regimen. However, this did not lead to any enrichment in transgenic parasites. Next, a total of five aNempts were performed to clone the parasites by limiting dilutoon either from the newly resistant parasites (first appearance a[er transfection and drug selection) or from different passages under pyrimethamine pression. Depending on the quantity of parasites injected per mouse, we either obtained no parasites in the recipient mice (3 experiments with a total of 26 recipient mice), or parasites showing both wild-type and KO construct-integrated genotype (for 1 experiment, 2/10 mice have shown parasites). In the fifth cloning experiment (20 parasites/mouse, 10 recipient mice), 2/10 mice showed blood parasites, and after passage on 3 mice, the parasites were genotyped as pure wildtype parasites. In summary, all attempts to select or clone parasites which have integrated the dhfr resistance cassette at the PbRpt3 locus were either unsuccessful or only allowed to clone wild-type parasites. These results are supportive of the essentiality of PbRpt3 during the erythrocytic stage of P. berghei. This could be interpreted as differing from the results previously obtained by Bushell et al showing a slow growing rate of PbRpt3-KO parasites at erythrocytic stages
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