RMgmDB - Rodent Malaria genetically modified Parasites


Malaria parasiteP. berghei
DisruptedGene model (rodent): PBANKA_133460; Gene model (P.falciparum): PF3D7_1471400; Gene product: diacylglycerol kinase, putative
Phenotype Asexual bloodstage; Gametocyte/Gamete; Oocyst; Sporozoite; Liver stage;
Last modified: 17 April 2024, 23:08
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption
Reference (PubMed-PMID number) Not published (yet)
MR4 number
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
The mutant parasite was generated by
Name PI/ResearcherSattler JM, Frischknecht F
Name Group/DepartmentIntegrative Parasitology, Center for Infectious Diseases
Name InstituteHeidelberg University Medical School, ,
Name of the mutant parasite
RMgm numberRMgm-5449
Principal nameDiacylglycerol kinase(-)
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Asexual blood stageReduced growth/multiplication of asexual blood stages
Gametocyte/GameteNormal gametocyte production and male gamete formation (exflagellation).
Fertilization and ookineteNot tested
OocystNormal oocyst formation. Strongly reduced sporozoite formation
SporozoiteNormal oocyst formation. Strongly reduced sporozoite formation
Liver stageBlood stage infection in C57Bl6 after intravenous injection of 100 sporozoites: delayed
Additional remarks phenotype

The mutant lacks expression of PBANKA_133460 (Diacylglycerol kinase).

Published in: bioRxiv preprint doi: https://doi.org/10.1101/2023.10.24.563045

Protein (function)

Reduced growth/multiplication of asexual blood stages.
Normal gametocyte production and male gamete formation (exflagellation). Normal oocyst formation. Strongly reduced/no sporozoite formation. Blood stage infection in C57Bl6 after intravenous injection of 100 sporozoites: delayed

Additional information
In independent transfection experiments 50 genes were targeted genes for deletion (using PlasmoGem gene-deletion DNA constructs). For 26 genes, populations of mixed wild type and transgenic parasites were selected. From those populations transgenic clonal lines for 14 genes could be isolated (see below).
Around half of these 14 clonal mutant parasite lines showed reduced blood stage growth rates comparable to those observed in the previously published PlasmoGEM screen with narrow confidence intervals of PlasmoGEM growth rates being a predictor for the growth rate of clonal lines. The other half showed growth rates closer to the growth rate of wild type parasites.
Life cycle progression of the 14 gene deletion mutants through mosquitoes was analysed, including 3 existing gene deletion mutants with reduced growth/multiplication of asexual blood stages: Plasmepsin IV (-) (mutant RMgm-314; PBANKA_1034400; PF3D7_1408100); Aminopeptidase P (-) (mutant RMgm-813; PBANKA_1318100; PF3D7_1454400); l-aminopeptidase (-) (mutant RMgm-814; PBANKA_1309900; PF3D7_1446200). 
Eight of the 17 mutants mutants showed defects before sporozoite formation. These  mutants lacked the genes encoding plasmepsin IV (no/block sporozoite formation), profilin (no/block oocyst formation), beta-catenin like protein 1 (no male gamete formation/exflagellation), telomeric DNA-binding protein (no/block oocyst formation), mannose-1-phosphate guanyltransferase (man-1-P GT)(no/block oocyst formation), U2 snRNP-associated SURP motif-containing protein (U2snRNP)(no male gamete formation/exflagellation), diacylglycerol kinase (no salivary gland sporozoites) and hypoxanthine-guanine phosphoribosyl transferase (hypox-guan phosph transf) (no/block gametocyte formation).
The other nine mutant lines were able to complete the life cycle. These mutants lacked the genes encoding dynein heavy chain, autophagy-related protein 23 (autophagy-rel protein 23), tubulin tyrosine ligase, a conserved protein, dipeptidyl aminopeptidase 1 (dipeptidyl aminopept 1), protein kinase, V-type(+) pyrophosphatase (V-type(+) pyrophosph), aminopeptidase P (app) or M17 leucyl aminopeptidase (lap).
For 5 out of the 17 mutants infection of C57Bl6 mice led to a delayed infection that could be controlled and cleared by the mice. These 5 mutants lacked the genes encoding aminopeptidase P (app) or M17 leucyl aminopeptidase (lap), U2 snRNP-associated SURP motif-containing protein (U2snRNP), diacylglycerol kinase and hypoxanthine-guanine phosphoribosyl transferase (hypox-guan phosph transf).

Dynein heavy chain:                                                   PBANKA_0615700, PF3D7_0718000
Autophagy-related protein 23:                                   PBANKA_0921700, PF3D7_1126700
Profilin:                                                                        PBANKA_0833000, PF3D7_0932200
Tubulin tyrosine ligase:                                              PBANKA_0901900, PF3D7_1147200
Telomeric DNA binding protein:                                 PBANKA_1205000, PF3D7_1006800
Beta-catenin like protein:                                            PBANKA_0910100, PF3D7_1138600
Man-1-P-GT:                                                               PBANKA_1022300, PF3D7_1420900
Diacylglycerol kinase:                                                PBANKA_1334600, PF3D7_1471400
U2 snRNP:                                                                  PBANKA_1039300 PF3D7_1402700
Conserved protein:                                                    PBANKA_0519500, No
Dipeptidyl aminopeptidase:                                       PBANKA_0931300, PF3D7_1116700
Hypoxanthine-guanine phosphoribosyl transferase: PBANKA_1210800, PF3D7_1012400
Protein kinase:                                                            PBANKA_0826900, PF3D7_0926100
V-type H(+)-translocating pyrophosphatase:            PBANKA_1320500, PF3D7_1235200

Other mutants

  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_133460
Gene Model P. falciparum ortholog PF3D7_1471400
Gene productdiacylglycerol kinase, putative
Gene product: Alternative name
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct used(Linear) PCR construct double cross-over
PlasmoGEM (Sanger) construct/vector usedYes
Name of PlasmoGEM construct/vector-
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 modification
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