RMgmDB - Rodent Malaria genetically modified Parasites


Malaria parasiteP. berghei
DisruptedGene model (rodent): PBANKA_0820200; Gene model (P.falciparum): PF3D7_0919300; Gene product: thioredoxin-like protein 1 (TrxL1)
PhenotypeNo phenotype has been described
Last modified: 28 March 2024, 11:48
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/ResearcherHentzschel F, Frischknecht F
Name Group/DepartmentHeidelberg University Medical Faculty, University Hospital Heidelberg
Name InstituteCenter for Infectious Diseases
Name of the mutant parasite
RMgm numberRMgm-5430
Principal nameTrxL1(-)
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Asexual blood stageNot different from wild type
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot different from wild type
SporozoiteNot different from wild type
Liver stageNot different from wild type
Additional remarks phenotype

The mutant lacks expression of TrxL1 (and does not contain a drug-selectable marker cassette that has been removed by negative selection).

Published in: bioRxiv preprint doi: https://doi.org/10.1101/2023.10.19.562943

Protein (function)
Sporozoites and ookinetes are motile, elongated forms with a dense subpellicular microtubules (SPMT) scaffold  underneath the inner membrane complex (IMC). SPMT's of these stages consist of 13-protofilament microtubules with twice-interrupted luminal helices (ILH). The T. gondii ILH consists of thioredoxin-like proteins 1 and 2 (TrxL1, TrxL2) and subpellicular microtubule protein 1 (SPM1). Plasmodium spp. have homologues of TrxL1.

No phenotype different from wild type was detected throughout the complete life cycle. Sporozoite production and gliding motility of sporozoites also similar to wild type parasites.

Additional information
 From the paper: 'Microtubule inner proteins, MIPs, are microtubule associated proteins that bind to tubulin from the luminal side. Here we investigate the role of four MIPs in a rodent malaria parasite for their role in transmission to and from the mosquito. We show by single and double gene deletions that SPM1 and TrxL1, MIPs associated with the subpellicular microtubules are dispensable for transmission from the vertebrate host to the mosquito and back. In contrast, FAP20 and FAP52, MIPs associated with the axonemes of gametes, are essential for transmission to mosquitoes but only if both genes are deleted. In the absence of both, FAP20 and FAP52 the B-tubule of the axoneme partly detaches from the A-tubule resulting in the deficiency of axonemal beating and hence gamete formation and egress. Our data suggest that a high level of redundancy ensures microtubule stability in the transmissive stages of Plasmodium'.

Other mutants

  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0820200
Gene Model P. falciparum ortholog PF3D7_0919300
Gene productthioredoxin-like protein 1
Gene product: Alternative nameTrxL1
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/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) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modificationFor gene deletion of PbSPM1 (PBANKA_0810700), PbTrxL1 (PBANKA_0820200), PbFAP20 (PBANKA_1108000) and PbFAP52 (PBANKA_1361500), the respective transfection vectors were obtained from PlasmoGEM. Prior to transfection, 3-5 µg of each vector were linearized using NotI followed by ethanol precipitation.
The linearized vectors were transfected into the parental P. berghei strain (PbANKA wildtype for SPM1 and TrxL1, and Pb820 wildtype for FAP20 and FAP50).
In order to be able to reuse pyrimethamine as a selective (positive) pressure in subsequent mutant generations, 5-fluorocytosine (5-FC) selection (negative) was performed. Here, trxL1(-) and fap20(-) parasites that recycled the selection cassette were negatively selected for via 5-FC (1 mg/ml) administered via the mice’s drinking water and isogenic clones were isolated by limited dilution and verified via genotyping PCR accordingly. After obtaining marker-free clones, the second knockout (spm1 or fap52) was generated in these lines.
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