RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5331
Malaria parasiteP. berghei
Genotype
Genetic modification not successful
DisruptedGene model (rodent): PBANKA_1426500; Gene model (P.falciparum): PF3D7_0811000; Gene product: cullin-1, putative (Cullin1, CUL1)
PhenotypeNo phenotype has been described
Last modified: 29 March 2023, 16:06
  *RMgm-5331
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 4
Reference (PubMed-PMID number) Reference 1 (PMID number) : 36898988
top of page
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone P. berghei ANKA 2.34
Other information parent lineP. berghei ANKA 2.34 is a cloned, gametocyte producer line of the ANKA strain (PubMed: PMID: 15137943).
top of page
Attempts to generate the mutant parasite were performed by
Name PI/ResearcherRashpa R, Brochet M
Name Group/DepartmentFaculty of Medicine, Department of Microbiology and Molecular Medicine
Name InstituteUniversity of Geneva
CityGeneva
CountrySwitzerland
  Disrupted: Mutant parasite with a disrupted gene
top of page
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1426500
Gene Model P. falciparum ortholog PF3D7_0811000
Gene productcullin-1, putative
Gene product: Alternative nameCullin1, CUL1
top of page
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 usedYes
See "Additional remarks genetic modification"
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

3xHA, KO (disruption) or AID/HA targeting vectors were generated using phage recombineering in Escherichia coli TSA strain with PlasmoGEM vectors (https://plasmogem.umu.se/pbgem/). For final targeting vectors not available in the PlasmoGEM repository, generation of knockout and tagging constructs were performed using sequential recombineering and gateway steps. For each gene of interest (goi), the Zeocin-resistance/Phe-sensitivity cassette was introduced using oligonucleotides goi HA-F x goi HA-R and goi KO-F x goi KO-R for 3xHA, AID/HA tagging and KO targeting vectors, respectively. Insertion of the GWcassette following the gateway reaction was confirmed using primer pairs GW1 x goiQCR1 and GW2 x goiQCR2.The modified library inserts were then released from the plasmid backbone using NotI. The AID/HA targeting vectors were transfected into the 615 parasite line, while the KO/GD and triple HA targeting vectors were transfected into the 2.34 line unless otherwise specified.
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
top of page
Conceptual design of the database: Dr. C.J. Janse (Leiden Malaria Research Group, Leiden, The Netherlands).
Technical design and realization: S. Mededovic and A. van Wigcheren